AU2015223025B2 - Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes - Google Patents

Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes Download PDF

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AU2015223025B2
AU2015223025B2 AU2015223025A AU2015223025A AU2015223025B2 AU 2015223025 B2 AU2015223025 B2 AU 2015223025B2 AU 2015223025 A AU2015223025 A AU 2015223025A AU 2015223025 A AU2015223025 A AU 2015223025A AU 2015223025 B2 AU2015223025 B2 AU 2015223025B2
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Robert Dicosimo
Erwin Columbus HAGO
Susan Marie Hennessey
Ling Hua
Kevin D. Nagy
Rodrigo Ramirez
Jayarama K. Shetty
Zhongmei TANG
Zheyong YU
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Nutrition and Biosciences USA 4 Inc
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    • C12Y302/01115—Branched-dextran exo-1,2-alpha-glucosidase (3.2.1.115)

Abstract

A method is disclosed for hydrolyzing an alpha-1,3 or alpha-1,6 glucosyl-glucose linkage in a saccharide (disaccharide or oligosaccharide). This method comprises contacting the saccharide with an alpha-glucosidase enzyme such as transglucosidase under suitable conditions, during which contacting step the enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the saccharide. This method is useful for reducing the amount of oligosaccharides in a filtrate isolated from a glucan synthesis reaction, for example.

Description

The invention is in the field of enzymatic hydrolysis of small sugar polymers. Specifically, this invention pertains to hydrolyzing disaccharides and oligosaccharides comprising one or more alpha-1,3 or alpha-1,6 glucosyl-glucose linkages with an alpha-glucosidase enzyme.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named CL6220USNP__SequenceListing__ST25.txt created on February 11, 2015, and having a size of 266 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.
BACKGROUND
Transglucosidases (EC.2.4.1.24, 1,4-alpha-glucan 6-alphaglucosyltransferase) are D-glucosyltransferase enzymes that catalyze both hydrolytic and transfer reactions on incubation with alpha-D-glucooligosaccharides (1951, Pazurand French, J. Amer. Chem. Soc. 73:3536). Maltose is the most preferred substrate for transglucosylation reactions with this enzyme. Transfer occurs most frequently to HO-6, producing isomaltose from Dglucose, or panose (6-O-alpha-glucosyl maltose) from maltose.
Transglucosidase can also transfer a glucosyl residue to the HO-2 or HO-3 of
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PCT/US2015/017644 another D-glucosyl unit to form Kojibiose or Nigerose. This enzyme can further transfer a D-glucosyl unit back to HO-4 to reform maltose.
As a result of transgSucosyiation reactions with transglucosidase, maltooligosaccharide residues are converted to isomalto-oligosaccharides (IMO) containing a higher proportion of glucosyl residues linked by alpha-D-1,6 glycosidic linkages from the non-reducing end. IMO sugars are used in many food and beverage formulations in Asia. Brier et al. (U.S. Patent Appl, Publ, No. 2003/0167929) disclosed using transglucosidase to produce IMO from barley wort.
Poulose et al. (U.S. Patent Appl. Publ. No. 2008/0229514) disclosed using transglucosidase to degrade polysaccharides such as xanthan and guar gums. Xanthan gum comprises a cellulosic backbone in which alternate glucoses are 1,3-linked to branches containing mannose and glucuronic acid. The backbone of guar gum comprises beta-1,4-linked mannose residues to which galactose residues are alpha-1,6-linked at every other mannose.
Lantero et al. (U.S. Patent No. 5770437) disclosed using a transglucosidase to degrade sucrose, melezitose and trehalulose. These sugars comprise glucose linked to fructose via 1,2- (sucrose), 1,3- (melezitose), or 1.1(trehalulose) linkages.
Although various hydrolytic activities of transglucosidase have been disclosed, this type of enzyme is generally considered to be an alphaglucosidase, given its ability to hydrolyze alpha-linkages between two glucosyl residues. For example, transglucosidase is associated with having maltase activity (hydrolysis of the alpha-1,4 glycosidic link between the two glucosyl residues of maltose), which is a type of alpha-glucosidase activity.
Notwithstanding the foregoing disclosures, surprisingly, it has now been found that alpha-glucosidases such as transglucosidase (EC 2.4.1.24) can hydrolyze alpha-1,3 and alpha-1,6 glycosidic linkages of glucosyl-glucose. Alpha-glucosidase enzymes are disclosed herein as being useful for degrading disaccharides and oligosaccharides containing glucosyl-alpha-1,3-glucose and glucosyl-alpha-1,6-glucose.
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SUMMARY OF INVENTION
In one embodiment, the Invention concerns a method of hydrolyzing an alpha-1,3 or alpha-1,6 glucosyl-glucose linkage in a saccharide comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage, wherein the saccharide is a disaccharide or oligosaccharide, and wherein the method comprises: contacting the saccharide with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the saccharide, and wherein the amount of the saccharide is reduced compared to the amount of the saccharide that was present prior to the contacting step.
In another embodiment, the alpha-glucosidase enzyme of the hydrolysis method is immobilized.
In another embodiment, the saccharide of the hydrolysis method has a degree of polymerization before hydrolysis of 3 to 7. In anotherembodiment, the concentration of the saccharide after the contacting step is less than 50% of the concentration of the saccharide that was present prior to the contacting step.
In another embodiment, the suitable conditions of the hydrolysis method comprise (I) a glucan synthesis reaction, or (ii) a fraction obtained from the glucan synthesis reaction; wherein the saccharide is a byproduct of the glucan synthesis reaction. The glucan synthesis reaction produces at least one insoluble alpha-glucan product in another embodiment, in another embodiment, the fraction is a filtrate of the glucan synthesis reaction, in another embodiment, the glucan synthesis reaction produces at least one soluble alpha-glucan product that is (i) a product of a glucosyltransferase, or (ii) a product of the concerted action of both a glucosyltransferase and an alpha-glucanohydrolase capable of hydrolyzing glucan polymers having one or more alpha-1,3-glycosidic linkages or one or more alpha-1,6-gSycosidic linkages. The fraction is a chromatographic fraction of the glucan synthesis reaction in another embodiment in which the glucan synthesis reaction produces at least one soluble alpha-glucan product.
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In another embodiment, the alpha-glucosidase enzyme is a transglucosidase. in another embodiment, the transglucosidase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1
In another embodiment, the invention concerns a composition produced by contacting a saccharide with an alpha-glucosidase enzyme, wherein the saccharide is a disaccharide or oligosaccharide and comprises at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the saccharide, and wherein the composition comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to the contacting step.
In another embodiment, the saccharide of the composition has a degree of polymerization before hydrolysis of 3 to 7. The concentration of the saccharide after the contacting step is less than 50% of the concentration of the saccharide that was present prior to the contacting step, for example.
In another embodiment, the saccharide of the composition is in (i) a glucan synthesis reaction, or (ii) a fraction obtained from the glucan synthesis reaction; wherein the saccharide is a byproduct of the glucan synthesis reaction. In another embodiment, the fraction is a filtrate of the glucan synthesis reaction or a chromatographic fraction of the glucan synthesis reaction.
In another embodiment, the invention concerns a method of enriching fructose present in a fraction of a glucan synthesis reaction, comprising: (a) contacting a fraction obtained from a glucan synthesis reaction with an alphaglucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of a disaccharide or oligosaccharide comprised within the fraction; and (b) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition having a higher concentration of fructose compared to the fructose concentration of the fraction of step (a).
In another embodiment, the invention concerns a fermentation method comprising: (a) contacting a fraction obtained from a glucan synthesis reaction
2015223025 22 Aug 2018 with an alpha-glucosidase enzyme under suitable conditions, wherein the alphaglucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosylglucose linkage of a disaccharide or oligosaccharide comprised within the fraction; (b) fermenting the fraction of step (a) with a microbe to yield a product, wherein the fermenting can be performed after step (a) or simultaneously with step (a); and (c) optionally, isolating the product of (b); wherein the yield of the product of (b) is increased compared to the product yield of fermenting a fraction of the glucan synthesis reaction that has not been contacted with the enzyme.
In another embodiment, the invention concerns a method comprising: (a) 10 providing a glucan synthesis reaction; (b) obtaining a fraction of the glucan synthesis reaction that comprises a saccharide byproduct of the glucan synthesis reaction, wherein the saccharide byproduct is a disaccharide or oligosaccharide and comprises at least one alpha-1,3 glucosyl-glucose linkage; and (c) contacting the saccharide byproduct in the fraction with an alpha-glucosidase enzyme, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha1,3 glucosyl-glucose linkage of the saccharide byproduct.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES Figure 1: 1H NMR spectra of glucan reaction filtrate material before (starting material) and after (treated material) hydrolysis treatment with NOVO 188 enzyme (see Examples 2-3).
Figure 2: 1H NMR spectra of glucan reaction filtrate material before (starting material) and after (treated material) hydrolysis treatment with TG L-2000 transglucosidase (see Examples 2-3).
Table 1. Summary of Nucleic Acid and Protein Sequence Identification Numbers
Description Nucleic acid SEQ ID NO. Protein SEQ ID NO.
“TG L-2000”, A. niger transglucosidase (mature form without siqnal peptide) 1 (965 aa)
“GC 321 Glucoamylase”, T. reesei glucoamylase (TrGA) (mature form without signal peptide) 2 (599 aa)
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“gtfJ”, Streptococcus salivarius glucosyltransferase. The first 42 amino acids of the protein are deleted compared to GENBANK Identification No. 47527; a start methionine is included. 3 (1477 aa)
“Aclglu 1 ”, Aspergillus clavatus alphaglucosidase, full-length precursor form including signal peptide. 4 (3147 bases) 5 (990 aa)
“Aclglu 1”, Aspergillus clavatus alphaglucosidase, mature form lacking signal peptide. 6 (971 aa)
“Nfiglul ”, Neosartorya fischeri alphaglucosidase, full-length precursor form including signal peptide. 7 (3158 bases) 8 (988 aa)
“Nfiglul”, Neosartorya fischeri alphaglucosidase, mature form lacking signal peptide. 9 (969 aa)
“Ncrglul”, Neurospora crassa alphaglucosidase, full-length precursor form including signal peptide. 10 (3385 bases) 11 (1044 aa)
“Ncrglul”, Neurospora crassa alphaglucosidase, mature form lacking signal peptide. 12 (1022 aa)
“TauSecO98”, Rasamsonia composticola alphaglucosidase, full-length precursor form including signal peptide. 13 (3293 bases) 14 (1035 aa)
“TauSecO98”, Rasamsonia composticola alphaglucosidase, mature form lacking signal peptide. 15 (1013 aa)
“TauSecO99”, Rasamsonia composticola alphaglucosidase, full-length precursor form including signal peptide. 16 (3162 bases) 17 (990 aa)
“TauSecO99”, Rasamsonia composticola alphaglucosidase, mature form lacking signal peptide. 18 (973 aa)
“BloGlul”, Bifidobacterium longum (subsp. longum JDM301) alpha-glucosidase (wild type). 19 (1815 bases) 20 (604 aa)
“BloGlul”, Bifidobacterium longum (subsp. longum JDM301) alpha-glucosidase, codonoptimized sequence. 21 (1812 bases)
“BloGlu2”, Bifidobacterium longum alphaglucosidase (wild type). 22 (604 aa)
“BloGlu2”, Bifidobacterium longum alpha- 23 24
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glucosidase, codon-optimized sequence encoding amino acid sequence. (1812 bases) (604 aa)
“BloGlu3”, Bifidobacterium longum (subsp. F8) alpha-glucosidase (wild type) 25 (1815 bases) 26 (604 aa)
“BloGlu3”, Bifidobacterium longum (subsp. F8) alpha-glucosidase, codon-optimized sequence encoding amino acid sequence. 27 (1812 bases)
“BpsGlul”, Bifidobacterium pseudolongum alpha-glucosidase (wild type). 28 (585 aa)
“BpsGlul”, Bifidobacterium pseudolongum alpha-glucosidase, codon-optimized sequence encoding amino acid sequence. 29 (1755 bases) 30 (586 aa)
“BthGlul”, Bifidobacterium thermophilum RBL67 alpha-glucosidase (wild type). 31 (1806 bases) 32 (601 aa)
“BthGlul”, Bifidobacterium thermophilum RBL67 alpha-glucosidase, codon-optimized sequence. 33 (1803 bases)
“BbrGlu2”, Bifidobacterium breve alphaglucosidase (wild type). 34 (662 aa)
“BbrGlu2”, Bifidobacterium breve alphaglucosidase, codon-optimized sequence encoding amino acid sequence. 35 (1812 bases) 36 (604 aa)
“BbrGlu5”, Bifidobacterium breve ACS-071-VSch8b alpha-glucosidase (wild type). 37 (1821 bases) 38 (606 aa)
“BbrGlu5”, Bifidobacterium breve ACS-071-VSch8b alpha-glucosidase, codon-optimized sequence. 39 (1818 bases)
“Gtf-S”, Streptococcus sp. C150 glucosyltransferase, GENBANK Gl No. 321278321. 40 (1570 aa)
“GTF0459”, Streptococcus sp. C150 glucosyltransferase, N-terminal-truncated version of GENBANK Gl No. 321278321. 41 (4179 bases) 42 (1392 aa)
“Gtf-C”, Streptococcus mutans MT-4239 glucosyltransferase, GENBANK Gl No. 3130088. 43 (1455 aa)
“GTF0088BsT1”, Streptococcus mutans MT4239 glucosyltransferase, N- and C-terminaltruncated version of GENBANK Gl No. 3130088. 44 (2715 bases) 45 (904 aa)
“MUT3325”, Penicillium marneffei ATCC 18224 mutanase, GENBANK Gl No. 212533325. 46 (1308 bases) 47 (435 aa)
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DETAILED DESCRIPTION OF THE INVENTION
The disclosures of ail cited patent and non-patent literature are incorporated herein by reference in their entirety.
As used herein, the term “invention” or “disclosed invention” is not meant to be limiting, but applies generally to any of the inventions defined in the claims or described herein. These terms are used interchangeably herein.
The terms “saccharide”, “saccharide molecule” and “carbohydrate” are used interchangeably herein and refer to a disaccharide or oligosaccharide, unless otherwise noted. A “disaccharide” herein refers to a carbohydrate having two monosaccharides joined by a glycosidic linkage. An “oligosaccharide” herein refers to a carbohydrate that consists of 2 to 9 monosaccharides, for example, joined by glycosidic linkages. An oligosaccharide can also be referred to herein as an “oligomer. Monosaccharides that are comprised within a disaccharide or oligosaccharide can be referred to as “monosaccharide units” or “monomeric units”, for example. Preferred monosaccharides herein are fructose and glucose.
The terms “glycosidic linkage” and “glycosidic bond” are used interchangeably herein and refer to the type of covalent bond that joins a carbohydrate molecule to another carbohydrate molecule.
The terms “alpha-1,3 glucosyl-glucose linkage”, “alpha-1,3 glucoseglucose linkage” and “glucose-alpha 1,3-glucose” herein refers to an alpha-1,3glycosidic linkage between two alpha-D-glucose molecules. The terms “alpha1,8 glucosyl-glucose linkage, “alpha-1,6 glucose-glucose linkage and “glucosealpha 1,8-glucose” herein refers to an alpha-1,8-glycosidic linkage between two alpha-D-glucose molecules. Alpha-1,3 glucosyl-glucose linkage(s) and/or alpha1,6 glucosyl-glucose linkage(s) herein are comprised within a disaccharide or oligosaccharide in certain embodiments.
The terms “alpha-1,5 glucosyl-fructose linkage”, “alpha-1,5 glucosefructose linkage” and “glucose-alpha-1,5-fructose” herein refers to an alpha-1,5glycosidic linkage between an alpha-D-glucose molecule and a fructose molecule. An alpha-1,5 glucosyl-fructose linkage herein is comprised within a disaccharide or oligosaccharide in certain embodiments.
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PCT/US2015/017644 “Alpha-D-glucose” herein can also be referred to as “glucose”.
A disaccharide containing an alpha-1,5 glucosyl-fructose linkage is referred to herein as leucrose. The terms “leucrose” and “D-glucopyranosylaipha(1-5)~D-fructopyranose” are used interchangeably herein. Leucrose has the following structure:
Figure AU2015223025B2_D0001
The terms “alpha-glucosidase”, “alpha-1,4-glucosidase”, and “alpha-D~ glucoside glucohydrolase” are used interchangeably herein. Alpha-glucosidases (EC 3.2.1.20) (“EC” refers to Enzyme Commission number) have previously been recognized as enzymes that catalyze hydrolytic release of terminal, non-reducing (1,4)-linked aipha-D-glucose residues from oligosaccharide (e.g., disaccharide) and polysaccharide substrates. Alpha-glucosidases are now disclosed herein to also have hydrolytic activity toward alpha-1,5 glucosyl-fructose linkages, and hydrolytic activity toward alpha-1,3 and alpha-1,6 glucosyl-glucose linkages. Transglucosidase and glucoamylase enzymes are examples of alphaglucosidases with such activity.
The terms Transglucosidase” (TG), “transglucosidase enzyme”, and “1,4alpha-giucan 6-alpha-glucosyltransferase” are used interchangeably herein. Transglucosidases (EC 2.4.1.24) have previously been recognized as Dglucosyltransferase enzymes that catalyze both hydrolytic and transfer reactions on incubation with certain alpha-D-gluco-oligosaccharides. Transglucosidases are now disclosed herein to also have hydrolytic activity toward alpha-1,5 glucosyl-fructose linkages, and hydrolytic activity toward alpha-1,3 and alpha-1,6 glucosyl-glucose linkages.
The terms “glucoamylase” (GA), “glucoamylase enzyme”, and “alpha-1,4glucan glucohydrolase” are used interchangeably herein. Glucoamylases (EC 3.2.1.3) have previously been recognized as exo-acting enzymes that catalyze
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PCT/US2015/017644 hydrolysis of both alpha-1,4 and alpha-1,8 glycosldic linkages from non-reducing ends of glucose-containing di-, oligo- and poly-saccharides. Glucoamylases are now disclosed herein to also have hydrolytic activity toward alpha-1,5 glucosylfructose linkages.
Enzymatic hydrolysis is a process in which an enzyme facilitates the cleavage of bonds in molecules with the addition of the elements of water. “Hydrolyzing”, “hydrolysis of’, or “hydrolytic activity toward” an alpha-1,3 or alpha 1,6 glucosyl-glucose linkage herein refers to enzymatic hydrolysis of the alpha1,3 or alpha-1,8 glycosidic linkage between two glucose molecules by an alphaglucosidase such as a transglucosidase. Such hydrolysis occurs when contacting a disaccharide or oligosaccharide containing an alpha-1,3 and/or alpha-1,8 glucosyl-glucose linkage with an alpha-glucosidase herein under suitable conditions. Thus, a “hydrolysis reaction herein comprises at least (i) a disaccharide or oligosaccharide containing an alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkage(s), and (ii) an alpha-glucosidase.
The term “saccharification” herein refers to a process of breaking a saccharide (disaccharide or oligosaccharide) info its monosaccharide components. A saccharide can be saccharified in a hydrolysis reaction herein.
“Suitable conditions” for contacting a saccharide (disaccharide or oligosaccharide) comprising at least one alpha-1,3 and/or alpha-1,8 glucosylglucose linkage with an alpha-glucosidase herein refer to those conditions (e.g., temperature, pH, time) that support the hydrolysis of one or more alpha-1,3 and/or alpha-1,8 glucosyl-glucose linkages in the saccharide by the alphaglucosidase. Suitable conditions can comprise “aqueous conditions”, for example, comprising at least 20 wt% wafer. Aqueous conditions may characterize a solution or mixture. The solution or mixture in which a saccharide comprising at least one alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkage is contacted with an alpha-glucosidase can be referred to as an alpha-glucosidase reaction, for example (e.g., a transglucosidase or glucoamylase reaction).
An “immobilized” enzyme herein refers to an enzyme that is attached to an inert, insoluble material. Methods for preparing immobilized enzymes are
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PCT/US2015/017644 disclosed, for example, in U.S. Patent No. 5541097, which is incorporated herein by reference.
The terms “glucan” and “glucan polymer” are used interchangeably herein and refer to a polysaccharide of glucose monomers linked by glycosidic bonds. An “aipha-glucan” herein refers to a glucan polymer comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% alpha-glycosidic linkages.
An “insoluble glucan” herein refers to a glucan polymer that is not soluble in aqueous conditions. An example of insoluble glucan herein is poly alpha-1,3glucan with a DP of at least 8 or 9. A glucosyltransferase reaction in certain embodiments as presently disclosed produces at least one insoluble glucan product.
The terms “soluble glucan”, “soluble aipha-glucan”, “soluble fiber”, “soluble glucan fiber, “soluble dietary fiber” and the like are used interchangeably herein to refer to a glucan polymer that is soluble in aqueous conditions. Examples of soluble glucan herein are certain oligosaccharides, such as poly alpha-1,3glucan with a DP less than 8, and certain oligosaccharides disclosed in the Examples provided below. A glucosyltransferase reaction in certain embodiments as presently disclosed produces at least one soluble glucan product. Another set of features that characterizes soluble aipha-glucan compounds in certain embodiments herein is that they are (I) water-soluble glucose oligomers having a degree of polymerization of 3 or more, (ii) digestionresistant (i.e., exhibit very slow or no digestibility) with little or no absorption in the human small intestine, and (ill) at least partially fermentable in the lower gastrointestinal tract. Digestibility of a soluble glucan fiber composition can be measured using AOAC method 2009.01, for example.
The terms “poly alpha-1,3-glucan” and “alpha-1,3-glucan polymer are used interchangeably herein. Poly alpha-1,3-glucan is a polymer comprising glucose monomeric units linked together by glycosidic linkages, wherein at least about 50% of the glycosidic linkages are alpha-1,3-glycosidic linkages. The term “alpha-1,3-glycosidic linkage” as used herein refers to the type of covalent bond
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PCT/US2015/017644 that joins alpha-D-glucose molecules to each other through carbons 1 and 3 on adjacent alpha-D-glucose rings.
The “molecular weight” of a glucan herein can be represented as numberaverage molecular weight (Mn) or as weight-average molecular weight (Mw). Alternatively, molecular weight can be represented as Daltons, grams/mole, DPW (weight average degree of polymerization), or DPn (number average degree of polymerization). Various means are known in the art for calculating these molecular weight measurements such as with high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).
The terms “glucosyltransferase enzyme”, “gtf enzyme”, “gtf enzyme catalyst”, “gtf, “giucansucrase” and the like are used interchangeably herein.
The activity of a gtf enzyme herein catalyzes the reaction of sucrose substrate to make the products glucan and fructose. Other products (byproducts) of a gtf reaction can include glucose (results from when glucose is hydrolyzed from the glucosyl-gtf enzyme intermediate complex), various soluble oligosaccharides (e.g., DP2-DP7), and leucrose (results from when glucose of the glucosyl-gtf enzyme intermediate complex is linked to fructose). Wild type forms of glucosyltransferase enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide, a variable domain, a catalytic domain, and a glucanbinding domain. A glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarol et al., Nucleic Acids Res. 37:0233-238, 2009).
The term “sucrose” herein refers to a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar.
The terms “glucan synthesis reaction”, “glucan reaction” “gtf reaction” and the like are used interchangeably herein and refer to a reaction that is performed by a glucosyltransferase enzyme. A glucan synthesis reaction as used herein generally refers to a solution comprising at least one active glucosyltransferase enzyme in a solution comprising sucrose and water, and optionally other
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PCT/US2015/017644 components. Other components that can be in a glucan synthesis reaction herein include fructose, glucose, leucrose, soluble oligosaccharides (e.g., DP2DP7), and soluble glucan product(s), for example. Also, one or more alphaglucanohydrolase enzymes can be comprised in a glucan synthesis reaction in some aspects. It would be understood that certain glucan products, such as poly alpha-1,3~glucan with a degree of polymerization (DP) of at least 8 or 9, are water-insoluble and thus are not dissolved in a glucan synthesis reaction, but rather may be present out of solution.
The terms “alpha-glucanohydrolase” and “glucanohydrolase” are used interchangeably herein and refer to an enzyme capable of hydrolyzing an alphagSucan oligomer. An alpha-glucanohydrolase can be defined by its endohydrolysis activity towards certain alpha-D-glycosidic linkages. Examples of alpha-glucanohydrolase enzymes herein include dextranases (EC 3.2.1.11; capable of endohydrolyzing alpha-1,6-linked glycosidic bonds), mutanases (EC 3.2.1.59; capable of endohydrolyzing alpha-1,3~linked glycosidic bonds), and alternanases (EC 3.2.1.-; capable of endohydrolytically cleaving alternan). Various factors including, but not limited to, level of branching, the type of branching, and the relative branch length within certain alpha-glucans may adversely impact the ability of an alpha-glucanohydrolase to endohydrolyze some glycosidic linkages.
The “percent dry solids” of a glucan synthesis reaction refers to the wt% of all the sugars in a glucan synthesis reaction. The percent dry solids of a gtf reaction can be calculated, for example, based on the amount of sucrose used to prepare the reaction.
A “fraction” of a glucan synthesis reaction herein refers to a liquid solution portion of a glucan synthesis reaction. A fraction can be a portion of, or all of, the liquid solution from a glucan synthesis reaction, and has been separated from a soluble or insoluble glucan product synthesized in the reaction. A fraction can optionally be referred to as a “mother liquor in embodiments in which the product is an insoluble (solid) glucan product. An example of a fraction is a filtrate of a glucan synthesis reaction. Since a fraction can contain dissolved sugars such as
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PCT/US2015/017644 sucrose, fructose, glucose, leucrose, soluble oligosaccharides (e.g., DP2-DP7), a fraction can also be referred to as a “mixed sugar solution derived from a glucan synthesis reaction. A “hydrolyzed fraction” herein refers to a fraction that has been treated with an alpha-glucosidase herein to hydrolyze leucrose and/or oligosaccharides present in the fraction.
The terms “filtrate”, “glucan reaction filtrate”, “glucan filtrate” and the like are used interchangeably herein and refer to a fraction that has been filtered away from a solid glucan product synthesized in a glucan synthesis reaction. A “hydrolyzed filtrate” herein refers to a filtrate that has been treated with an alphaglucosidase herein to hydrolyze leucrose and/or oligosaccharides present in the filtrate.
The terms “percent by volume”, “volume percent”, “vol %”, “v/v %” and the like are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute )/(volume of solution)] x 100%.
The terms “percent by weight”, “weight percentage (wt%)”, “weight-weight percentage (% w/w)” and the like are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution. All percentages herein are weight percentages, unless otherwise noted.
As used herein, “polydispersity index”, “PDI”, “heterogeneity index”, “dispersity” and the like refer to a measure of the distribution of molecular mass in a given polymer (e.g., a glucose oligomer such as a soluble aipha-glucan) sample and can be calculated by dividing the weight average molecular weight by the number average molecular weight (PDto Mw/Mn),
The terms “increased”, “enhanced” and “improved” are used interchangeably herein. These terms refer to a greater quantity or activity such as a quantity or activity slightly greater than the original quantity or activity, or a quantity or activity in large excess compared to the original quantity or activity, and including all quantities or activities in between. Alternatively, these terms may refer to, for example, a quantify or activity that is at least 1%, 2%, 3%, 4%,
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5%, 8%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the quantity or activity for which the increased quantity or activity is being compared.
The terms “sequence identity” or “identity” as used herein with respect to polynucleotide or polypeptide sequences refer to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, “percentage of sequence identity” or “percent identity” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.
The Basic Local Alignment Search Tool (BLAST) algorithm, which is available online at the National Center for Biotechnology Information (NCBI) website, may be used, for example, to measure percent identity between or among two or more of the polynucleotide sequences (BLASTN algorithm) or polypeptide sequences (BLASTP algorithm) disclosed herein. Alternatively, percent identity between sequences may be performed using a Clustal algorithm (e.g., CiustalW or CiustalV). For multiple alignments using a Clustal method of alignment, the default values may correspond to GAP PENALTY-10 and GAP LENGTH PENALTY-10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using a Clustal method may be KTUPLE-1, GAP PENALTY-3, WSNDOW=5 and DIAGONALS SAVED-5. For nucleic acids, these parameters may be KTUPLE-2, GAP PENALTY-5, WINDOW-4 and DIAGONALS SAVED-4. Alternatively still,
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PCT/US2015/017644 percent identity between sequences may be performed using an EMBOSS algorithm (e.g., needle) with parameters such as GAP ΟΡΕΝ=10, GAP EXTENDED.S, END GAP PENALTY-false, END GAP OPEN=10, END GAP EXTEND-0.5 using a BLOSUM matrix (e.g., BLOSUM62).
Various polypeptide amino acid sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used. Alternatively, a variant amino acid sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
1%.
5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. A variant amino acid sequence herein has the same function/activity of the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of a disclosed sequence.
The term “isolated” as used in certain embodiments refers to any cellular component that is completely separated from its native source (e.g., an isolated polynucleotide or polypeptide molecule), in some instances, an isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix. For example, the isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a heterologous manner. Another example is an isolated alpha-glucosidase (e.g., glucoamylase, transglucosidase), or glucosyltransferase enzyme. The enzyme reactions (e.g., alpha-glucosidase reaction, glucosyltransferase reaction) disclosed herein are synthetic, non-naturaily occurring processes.
Embodiments of the disclosed invention concern a method of hydrolyzing an alpha-1,3 or alpha-1,6 glucosyl-glucose linkage in a saccharide comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage. The saccharide is a disaccharide or oligosaccharide. This method comprises contacting the saccharide with an alpha-glucosidase enzyme under suitable conditions, in the
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PCT/US2015/017644 contacting step, the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the saccharide. Due to this hydrolysis, the amount of the saccharide is reduced compared to the amount of the saccharide that was present prior to the contacting step. Thus, this hydrolysis method can alternatively be referred to as a method of reducing the amount of a saccharide in a composition.
Significantly, it is believed to be previously unknown that alphaglucosidase enzymes can hydrolyze alpha-1,3 and alpha-1,8 glucosyl-glucose linkages. Alpha-glucosidase reactions following this hydrolysis method can thus be used to remove oligosaccharide byproducts containing these glucose-glucose linkages from a glucan synthesis reaction and/or a fraction obtained therefrom. Such removal represents an improvement over chemical processes of byproduct removal, such as acid hydrolysis, which can result in degradation of glucan product. Finally, a glucan reaction fraction that is treated according to the above hydrolysis method is better-suited for downstream applications such as fermentation, for example, since the level of glucose monosaccharides is increased in the fraction. Monosaccharides are generally more tractable for downstream processes compared to oligosaccharide byproducts.
An alpha-glucosidase (EC 3.2.1.20) is used in embodiments herein to hydrolyze alpha-1,3 and/or alpha-1,8 glucosyl-glucose linkages in a saccharide comprising at least one of these linkages. Alpha-glucosidase enzymes have previously been recognized to catalyze hydrolytic release of terminal, nonreducing (1,4)-linked alpha-D-glucose residues from oligosaccharide (e.g., disaccharide) and polysaccharide substrates. These enzymes are now disclosed herein to also have hydrolytic activity toward alpha-1,3 and alpha-1,8 glucosylglucose linkages, for example.
An alpha-glucosidase can be from any source (e.g., plant, animal, microbe such as a bacteria or fungus/yeast), for example, such as those sources disclosed below from which a transglucosidase and/or glucoamylase can be derived. For example, an alpha-glucosidase can be a fungal alpha-glucosidase.
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Other examples of suitable alpha-glucosidases herein include those disclosed in U.S. Patent Nos. 6355467, 5922580, 5795766, 5763252, and 8633006, which are all incorporated herein by reference.
An alpha-glucosidase enzyme in certain embodiments herein may comprise the amino acid sequence of SEQ ID NQ:5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or that of DIAZYME RDF ULTRA (DuPont Industrial Biosciences), Alternatively, an alpha-glucosidase enzyme may comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NQ:5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or to the amino acid sequence of DIAZYME RDF ULTRA, and have hydrolytic activity toward alpha1,3 and/or alpha-1,6 glucosyl-glucose linkages in saccharides. Several of the foregoing sequences, for example, are mature alpha-glucosidases that lack an N-terminal signal peptide. For such sequences, it would be understood that an N-terminal start-methionine would typically be added (if necessary) (directly or via an intervening heterologous amino acid sequence such as an epitope) if expressing it without using a signal peptide (such as with an expression system where the enzyme is expressed intracelluiarly and obtained from a cell lysate).
A transglucosidase (EC 2.4.1.24; 1,4-alpha-glucan 6-alphaglucosyltransferase) can be used in certain embodiments herein as an alphaglucosidase to hydrolyze alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkages in a saccharide comprising at least one of these linkages. This class of enzymes has previously been recognized as D-glucosyltransferase enzymes that catalyze hydrolytic and transfer reactions on incubation with certain alpha-D-glucooligosaccharides. Transglucosidases as now disclosed herein also have hydrolytic activity toward alpha-1,3 and alpha-1,6 glucosyl-glucose linkages.
A transglucosidase enzyme herein may be derived from any microbial source, such as a bacteria or fungus. Examples of fungal transglucosidases include, but are not limited to, those of Trichoderma species (e.g., T. reesei).. Aspergillus species and Neosartorya species (e.g., N. fischeri). Examples of Aspergillus species from which a transglucosidase may be derived include, but
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PCT/US2015/017644 are not limited to, A. niger, A. awamori, A. oryzae, A. terreus, A. clavatus, A. fumigatus and A. nidulans. Other examples of transglucosidase enzymes useful herein are described in Barker et al. (1953, J. Chem. Soc. 3588-3593); Pazur et al. (1986, Carbohydr. Res. 149:137-147), Nakamura et al. (1997, J. Biotechnol. 53:75-84), and U.S. Patent Appl. Publ. No. 2008/0229514, all of which are incorporated herein by reference. Still other examples of transglucosidase enzymes useful herein are those that are thermostable; U.S. Patent No.
4689296, which is incorporated herein by reference, discloses a process for producing thermostable transgiucosidase. Yet more examples of transglucosidase enzymes useful herein may be any of those in the GENBANK database (NCBI). such as accession numbers: D45356 (GID:2645159, A. niger}, BAD06006.1 (GID:4G31328, A. awamori), BAA08125.1 (GID:1054565, A. oryzae), XP...001210809.1 (610:115492363, A. terreus), XPJD01216899.1 (GID:115433524, A. terreus), XP__GQ 1271891.1 (GID:12170762G, A. clavatus), XP.. 751811.1 (GID:70993928, A. fumigatus), XP...659621.1 (610:67523121, A. nidulans), XP__001266999.1 (GID:119500484, N. fischen)an6 XP_QG 1258585.1 (GSD:119473371, N. fischen), which are all incorporated herein by reference. Alternatively, a transgiucosidase herein may have an amino acid sequence that is at least 90% or 95% identical with the amino acid sequence of any of the foregoing disclosed transgiucosidase sequences, and have hydrolytic activity toward alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkages in saccharides. All of the foregoing transglucosidases, when used in a hydrolysis reaction herein, are preferably in a mature form lacking an N-terminal signal peptide.
A transgiucosidase enzyme in certain embodiments herein may comprise the amino acid sequence of SEG ID NO:1 (TG L-2000), which is an A. niger transgiucosidase (U.S. Patent Appl. Publ. No. 2008/0229514). Alternatively, a transgiucosidase may comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1 and have hydrolytic activity toward alpha-1,3 and/or alpha-1,8 glucosylglucose linkages in saccharides. Any of SEQ ID NQ:1 or variants thereof can be produced following the disclosure of U.S. Patent Appl. Publ. No. 2008/0229514,
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PCT/US2015/017644 for example, which is incorporated herein by reference. SEQ ID NO:1 is a mature transglucosidase that lacks an N-terminal signal peptide. Since SEQ ID NO:1 does not begin with a methionine residue, it would be understood that an N-terminal start-methionine would typically be added to SEQ ID NO:1 (directly or via an intervening heterologous amino acid sequence such as an epitope) if expressing it without using a signal peptide (such as with an expression system where the enzyme is expressed intracellularly and obtained from a cell lysate).
A glucoamylase (EC 3.2.1.3; alpha-1,4-glucan glucohydrolase) can be used in certain embodiments herein as an alpha-glucosidase. For example, a glucoamylase can be included with a transglucosidase in each of the hydrolysis reaction settings/conditions disclosed herein. In this context, a glucoamylase can be used to hydrolyze (i) an alpha-1,5 glucosyi-fructose linkage, and/or (ii) an alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkage present in a saccharide containing any of these linkage types. This class of enzymes has previously been recognized as exo-acting enzymes that catalyze hydrolysis of both alpha1,4 and alpha-1,6 glycosidic linkages from non-reducing ends of glucosecontaining di-, oligo- and poly-saccharides. Glucoamylases as now disclosed herein also have hydrolytic activity toward alpha-1,5 glucosyi-fructose linkages.
In certain embodiments, an alpha-glucosidase is not a glucoamylase.
A glucoamylase enzyme herein may be derived from any microbial source, such as a bacteria or fungus. Examples of bacterial glucoamylases include, but are not limited to, those of Bacillus species (e.g., B. alkalophHus, B. amyloliquefaciens, B. lentus, B. licheniformis, B. stearothermophilus, B. subtilis,
B. thuringiensis) and Strepiomyces species (e.g., S. lividans). Examples of fungal glucoamylases include, but are not limited to, those of Trichoderma species (e.g., T. reesei, T. longibrachiatum, T. strictipilis, T. asperellum, T. koniiangbra, T. hazianum), Aspergillus species (e.g., A. niger, A. oryzae, A. terreus, A. clavatus, A. nidulans, A. kawachi, A. awamori), Rhizopus species (e.g., R. oryzae, R. niveus), Talaromyces species (e.g., T. emersonii, T. thermophilus, T. duponti), Mucor species, Hypocrea species (e.g., H. gelatinosa, H. orientalis, H. vinosa, H. citrina), Fusarium species (e.g., F. oxysporum, F.
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PCT/US2015/017644 roseum, F. venenatum), Neurospora species (e.g., N. crassa), Humicola species (e.g., H. gnsea, H. insolens, H. lanuginosa), Peniciliium species (e.g., P. notatum, P. chrysogenum) and Saccharomycopsis species (e.g., S. fibuiigera). Examples of these bacterial and fungal glucoamylases for use herein are disclosed in U.S. Pat. Appl. Publ. No. 2013/0102035, which is incorporated herein by reference. Other examples of glucoamylase enzymes useful herein are described in Svensson et al. (1983, Carlsborg Res. Commun. 48:529-544), Boel et al. (1984, EMBO J. 3:1097-1102); Hayashida et al. (1989, Agric. Biol. Chem, 53:923-929); U.S. Pat, No. 5024941, U.S. Pat, No. 4794175, U.S. Pat.
No. 4247637, U.S. Pat. No. 8255084, U.S. Pat. No. 8620924, Ashikari et al. (1986, Agric. Biol. Chem. 50:957-964), Ashikari et al. (1989, Appl. Microbiol, Biotechnol. 32:129-133), U.S. Pat. No. 4883864; U.S. Pat. No. 4818579, Houghton-Larsen et al. (2003, Appl. Microbiol. Biotechnol. 62:210-217) and U.S. Pat. No. 7413887, all of which are incorporated herein by reference.
Alternatively, a glucoamylase herein may have an amino acid sequence that is at least 90% or 95% identical with the amino acid sequence of any of the foregoing disclosed glucoamylase sequences, and have hydrolytic activity toward (i) alpha1,5 glucosyl-fructose linkages and/or (ii) alpha-1,3 and/or alpha-1,6 glucosylglucose linkages. All of the foregoing glucoamylases, when used in a hydrolysis reaction herein, are preferably in a mature form lacking an N-terminal signal peptide. Commercially available glucoamylases useful herein include OPTIDEX L-400, GC 147, GC 321, G ZYME G990 4X, OPTIMAX 7525, DEXTROZYME, DISTILLASE and GLUCZYME, for example.
A glucoamylase enzyme in certain embodiments herein may comprise the amino acid sequence of SEG ID NO:2 (GC 321), which is a T. reesei glucoamylase. Alternatively, a glucoamylase may comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEG ID NO:2 and have hydrolytic activity toward (i) alpha-1,5 glucosyl-fructose linkages and/or (ii) alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkages. Any of SEG ID NO:2 or variants thereof can be produced following the disclosures of U.S. Pat. No. 7413887 or U.S. Pat. Appl.
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Publ. No. 2013/0102035, for example, which are incorporated herein by reference. SEQ ID NQ:2 is a mature glucoamylase that lacks an N-terminal signal peptide. Since SEQ ID NO:2 does not begin with a methionine residue, it would be understood that an N-terminal start-methionine would typically be added to SEQ ID NQ:2 (directly or via an intervening heterologous amino acid sequence such as ars epitope) if expressing it without using a signal peptide (such as with an expression system where the enzyme is expressed intracellularly and obtained from a cell lysate).
An alpha-glucosidase enzyme herein such as a transglucosidase or glucoamylase may be from a commercial source (e.g., DuPont Industrial Biosciences / Genencor, USA: Megazyme international, Ireland; Amano Enzyme inc., Japan). Alternatively, such an enzyme may be produced by any means known in the art, such as described in U.S. Pat. Appl. Publ. No. 2008/0229514, U.S. Pat. No. 7413887 or U.S. Pat. Appl. Publ. No. 2013/0102035, which are incorporated herein by reference. For example, an alpha-glucosidase may be produced recombinantly in a heterologous expression system, such as a microbial or fungal heterologous expression system. Examples of heterologous expression systems include bacterial (e.g., E. coli, Bacillus sp.) and eukaryotic systems. Eukaryotic systems can employ yeast (e.g., Pichia sp.,
Saccharomyces sp.) or fungal (e.g., Trichoderma sp. such as T. reesei; Aspergillus species such as A. niger) expression systems, for example. The transglucosidase of SEQ ID NO:1 and glucoamylase of SEQ ID NO:2, and variants thereof, can be expressed in a T. reesei host, for example.
An alpha-glucosidase enzyme when used in a hydrolysis reaction herein is preferably in a mature form lacking an N-terminal signal peptide. An expression system for producing a mature alpha-glucosidase enzyme herein may employ an enzyme-encoding polynucleotide that further comprises sequence encoding an N-terminal signal peptide to direct extra-cellular secretion. The signal peptide in such embodiments is cleaved from the enzyme during the secretion process.
The signal peptide may either be native or heterologous to the transglucosidase or glucoamylase. Alternatively, an alpha-glucosidase enzyme in a mature form
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PCT/US2015/017644 cars be provided by expressing it without using a signal peptide, such as with an expression system where the enzyme is expressed intracellularly and obtained from a ceil lysate. In either scenario (secretion or intracellularly expressed), a heterologous amino acid sequence such as an epitope can optionally be included at the N-terminus of the alpha-glucosidase.
An alpha-glucosidase enzyme in certain embodiments may be provided in a hydrolysis reaction herein by direct use of a cell that expresses the enzyme(s). In other words, an alpha-glucosidase that is contacted with a saccharide can be present by virtue of its expression from a cell placed in the suitable conditions for hydrolysis. Such a cell could thus be used in place of adding an isolated alphaglucosidase preparation to the hydrolysis reaction. A cell for this purpose can be a bacterial, yeast, or fungal cell, for example. Examples of yeast include those from the genera Saccharomyces (e.g., S. cerews/ae), Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenuia, Kioeckera, and Schwanniomyces. Other expression systems useful herein are disclosed in U.S. Patent. Appl. Publ. No. 2013/0323822, which is incorporated herein by reference,
A saccharide herein comprises at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage. Thus, depending on the length of the saccharide, it may contain 1, 2, 3, 4, 5, 6, 7, or 8 alpha-1,5 glucosyl-glucose linkages, for example. A saccharide preferably contains 1, 2, or 3 linkages of this type. A saccharide in other preferred embodiments only has alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkages. In other embodiments, a saccharide can have one or more alpha-1,5 glucosyl-fructose linkages.
Since a saccharide herein comprises at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage, the saccharide comprises at least two glucose units. In certain embodiments, a saccharide herein comprises only glucose units, or both glucose and fructose units. Such a composition may characterize the disaccharide and oligosaccharide byproducts of a glucan synthesis reaction. Alternatively, a saccharide herein may contain other monosaccharides in addition to glucose and fructose, such as galactose, ribose and xylose.
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A saccharide hydrolyzed in certain embodiments of the disclosed invention can be an oligosaccharide. An oligosaccharide herein can have, for example, 2, 3, 4, 5, 6, 7, 8, or 9 monosaccharide units. As would be understood in the art, an oligosaccharide herein can be referenced with respect to its degree of polymerization (DP) number, which specifies the number of monomeric units in the oligosaccharide. A DPS oligosaccharide has 3 monomeric units, for example. Thus, the oligosaccharide can be a DPS, DP4, DP5, DP6, DP7, DP8, or DP9 oligosaccharide, for example. The DP of a saccharide in certain embodiments is 3 to 7 (i.e., DP 3-7).
An oligosaccharide herein with 3 or more monosaccharide units, for example, can comprise other linkages in addition to at least one alpha-1,3 or alpha-1,8 glucosyl-glucose linkage. For example, there may also be one or more alpha-1,5 glucosyl-fructose linkages in the oligosaccharide, which are also susceptible to hydrolysis by alpha-glucosidases as shown herein.
An oligosaccharide in certain embodiments comprises only glucose monomers linked by alpha-1,3 and/or alpha-1,8 glycosidic linkages. Thus, such oligosaccharides comprise only alpha-1,3 glucosyl-glucose and/or alpha-1,8 glucosyl-glucose linkages. Examples of such an oligosaccharide contain only alpha-1,3 linkages or alpha-1,8 linkages. An oligosaccharide can comprise at least 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% glucosyl-glucose linkages in certain embodiments, in other embodiments, there can be about 75-85% alpha-1,3 glucosyl-glucose linkages and about 15-25% alpha-1,6 glucosyl-glucose linkages in oligosaccharides herein. Alternatively, oligosaccharides herein can comprise any percentage (any integer value between 1% and 99%) of alpha-1,3 glucosyl-glucose linkages and any percentage (any integer value between 1% and 99%) of alpha-1,8 glucosylglucose linkages, just so long that the total of these percentages is not greater than 100%. Any of these oligosaccharides can be in a fraction from a glucan synthesis reaction that produces (I) an insoluble alpha-glucan (e.g., poly alpha1,3-glucan), or (ii) a soluble alpha-glucan product, for example. This linkage content can characterize (I) each oligosaccharide individually, or (ii) a group of
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PCT/US2015/017644 oligosaccharides (i.e., average linkage content). Oligosaccharides comprising only glucose monomers linked by alpha-1,3 and/or alpha-1,6 glycosidic linkages can be DP2-DP7, or DP3-DP7, for example. It should be understood that the exact distribution of linkages in oligosaccharides can vary depending on the conditions of the glucan synthesis reaction (e.g., gtf enzyme) producing oligosaccharide byproducts, it should further be understood that the exact linkage distribution is not critical to the presently disclosed methods.
The Examples herein demonstrate that alpha-glucosidases (e.g., transglucosidase and glucoamylase enzymes) can hydrolyze both (i) leucrose, which comprises an alpha-1,5 glucosyl-fructose linkage, and (ii) oligosaccharides comprising only alpha-1,3 glucosyl-glucose and/or alpha-1,6 glucosyl-glucose linkages. Therefore, an alpha-glucosidase can be used, for example, in a reaction for hydrolyzing alpha-1,5 glucosyl-fructose linkages, alpha-1,3 glucosylglucose linkages and/or alpha-1,6 glucosyl-glucose linkages.
At least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage in a saccharide herein can be hydrolyzed by an alpha-glucosidase herein. Alternatively, it is believed that 2, 3, 4, 5, or more of such linkages in a saccharide can be hydrolyzed by an alpha-glucosidase, for example. Hydrolysis of at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage can occur at the non-reducing-end of a saccharide in certain embodiments.
The amount of a saccharide is reduced in the disclosed hydrolysis method compared to the amount of the saccharide that was present prior to the contacting step. This reduction results from hydrolytic cleavage of at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage in the saccharide. The amount (e.g., concentration) of a saccharide after the contacting step in a hydrolysis method herein can be less than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any integer value between 1% and 90%) of the amount of the saccharide that was present prior to the contacting step (prior to contacting alpha-glucosidase with a saccharide under suitable conditions).
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The amount of a saccharide Is reduced in the disclosed hydrolysis method compared to the amount of the saccharide that was present prior to the contacting step. It would be understood that such a comparison can be made in any number of ways. For example, the saccharide concentration can be measured both before and after performing the hydrolysis method. Alternatively, the comparison can be made with respect to a control reaction having the same conditions, except that an alpha-glucosidase as presently disclosed is not added to the control reaction.
An alpha-glucosidase in certain embodiments herein may be immobilized. The enzyme may be immobilized using any method and/or means known in the art, such as those disclosed in U.S. Pat. Nos. 5541097 and 4713333, both of which are incorporated herein by reference. For example, one or more enzymes can be immobilized by contacting the enzyme(s) with a solution of an aminereactive material (e.g., glutaraldehyde) to form an adduct (e.g., enzymeglutaraldehyde adduct), after which the adduct is bonded to a solid carrier that has been treated with a polyamine (e.g., a poiyethylenimine such as EPOMIN P1050).
A solid carrier (solid support) to which an alpha-glucosidase enzyme can be immobilized in certain embodiments can be an inorganic or organic material. Such materials include, for example, gamma-alumina, titania, activated granular carbon, granular diatomaceous earth, glass beads, porous glass, pumice-stone, silica gel, metal oxide and aluminum oxide.
A polyamine can be used to treat a solid carrier such that subsequent exposure of the solid carrier to an adduct comprising an enzyme and aminereactive material leads to attachment of the enzyme to the solid carrier.
Examples of polyamines useful herein include polyethylenediamine, a poiyethylenimine (e.g., polydiethylenetriamine, polytriethylenetetramine, polypentaethylenehexamine, polyhexamethylenediamine), polymethylenedicyclohexylamine, polymethylenedianiline, polytetraethylenepentamine, polyphenylenediamine and blends of two or more of
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PCT/US2015/017644 these polyamine compounds. Preferred polyamines are water-soluble and/or have a molecular weight of about from 500 to 100,000 Daltons. A polyethySenimine such as EPOMIN P-1050 can be used in certain embodiments.
An amine-reactive material useful for preparing an adduct comprising an enzyme herein can be, for example, an aldehyde, organic halide, anhydride, azo compound, isothiocyanate, and/or isocyanate. Examples of these amine-reactive materials include glutaraldehyde, succindialdehyde, terephfhaldehyde, bisdiazobenzidine-2,2’-disulfonic acid, 4,4’-difluoro-3,3’-dinitrodiphenylsulfone, diphenyl-4,4’-dithiocyanate-2,2’-disulfonic acid, 3-methoxydiphenylmethane-4,4’diisocyanate, toluene-2-isocyanate-4-isothiocyanate, toluene-2,-4diisothiocyanate, diazobenzidine, diazobenzidine-3,S’-dianisidine, N,N’~ hexamethylene bisiodoacetamide, hexamethylene diisocyanate, cyanuric chloride, and/or 1,5-difluoro-2,4-dinitrobenzene. Preferably, the amine-reactive material is an aldehyde such as glutaraldehyde.
An alpha-glucosidase enzyme adducted with an amine-reactive compound can be contacted with a polyamine-treated solid carrier, thereby immobilizing the enzyme onto the solid carrier. An immobilized enzyme herein can be employed in various reactor systems, such as in a column (e.g., packed column) or stirred tank reactor, for performing hydrolysis reaction as disclosed herein.
Suitable conditions for contacting a saccharide herein with an alphaglucosidase (e.g., transglucosidase) are those conditions that support the hydrolysis of one or more alpha-1,3 or alpha-1,6 glucosyl-glucose linkages in the saccharide by the alpha-glucosidase. Examples of suitable conditions are disclosed in the below Examples. Conditions (e.g., temperature, pH, time) for contacting an alpha-glucosidase with a sugar substrate are also disclosed in U.S. Pat. Appl. Publ. No. 2008/0229514, U.S. Pat. No. 7413887 and U.S. Pat. Appl. Publ. No. 2013/0102035 (all of which are incorporated herein by reference), and may also be applicable to the disclosed hydrolysis method.
The disaccharides and oligosaccharides in the disclosed hydrolysis method are typically soluble in water or an aqueous solution. Thus, contacting a
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PCT/US2015/017644 saccharide herein with an alpha-glucosidase is preferably performed under suitable conditions that are aqueous, in which the saccharide is dissolved. Aqueous conditions can characterize a solution or mixture comprising at least about 20 wt% water. Alternatively, aqueous conditions herein are at least about 20, 30, 40, 50, 60, 70, 80, 85, 90, or 95 wt% water (or any integer value between 20 and 95 wt%). for example. Aqueous conditions can further comprise a buffer, for example, such as an acidic, neutral, or alkaline buffer, at a suitable concentration and selected based on the pH range provided by the buffer. Examples of buffers/buffering agents include citrate, acetate (e.g., sodium acetate), KH2PO4, MOPS, CHES, borate, sodium carbonate, and sodium bicarbonate.
The pH of a hydrolysis reaction herein can be about 3.0 to 9.0, for example. Hydrolysis reaction pH can be, for example, about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0. Alternatively, the pH can be about 4-5. Techniques for setting pH include the use of buffers, alkalis, and/or acids, for example, and are well known in the art.
The temperature of a hydrolysis reaction herein can be about 20 °C to about 80 °C, for example. Hydrolysis reaction temperature can be, for example, about 20, 30, 40, 50, 60, 70, or 80 °C (or any integer value between 20 and 80 °C). A hydrolysis temperature of about 60 °C, 65 °C, or 60-65 °C is preferred in certain embodiments.
A hydrolysis reaction herein can be performed for a period of at least about 10 minutes to about 90 hours, for example. The time of a hydrolysis reaction can be, for example, at least about 0.5, 1,2, 3, 4, 8, 12, 16, 20, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, or 90 hours (or any integer value between 0.5 and 72 hours). In certain embodiments, a hydrolysis reaction can be performed in less than 4 hours (e.g., 0.5-4 hours) for example. The time period required to achieve a desired level of hydrolysis will vary on the exact conditions used, and would be understood by one skilled in the art. For example, increasing the amount of enzyme added to a reaction or immobilized on a solid support used in a reaction will reduce the contact time.
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One or more of alpha-glucosidase enzymes herein may be used in a hydrolysis reaction in certain embodiments. Both a transglucosidase and giucoamylase can be used in a reaction, for example. The amount of an alphaglucosidase in a hydrolysis reaction herein can be plus/minus 10% to 20% (or 5% to 10%) from any of the amounts used in the Examples below (e.g., Example 2), for example. Alternatively, about 0.1-0.5 vol% or 0.1-1.0 vol% of alphaglucosidase can be used in a hydrolysis reaction. Alternatively still, an alphaglucosidase herein can be used at about, or at least about, 1,2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ppm in a hydrolysis reaction. A transglucosidase unit (TGU) can be defined as the amount of a transglucosidase enzyme that will produce one micromole of panose per minute under the conditions of the following assay, for example. Transglucosidase activity can be assayed as follows, for example: a transglucosidase is brought up in 100 mM sodium acetate buffer, pH 4.5, containing 4 mM para-nitrophenyl-alpha-glucoside and 1 mg/ml bovine serum albumin (BSA). After 30 min incubation at 30 °C, the reaction is terminated by the addition of an equal volume 1 M sodium carbonate and OD405 is recorded. A giucoamylase unit (GAU) can be defined, for example, as the amount of giucoamylase enzyme that will produce 1 g of reducing sugar calculated as glucose per hour from a soluble starch substrate (4% DS [degree of substitution]) at pH 4.2 and 60 °C.
The initial concentration of a saccharide in a hydrolysis reaction in certain embodiments of the disclosed invention can be about 1 wt% to 50 wt%, for example. For example, the concentration of leucrose can be about 5, 10, 15, 20, 25, 30, 35, or 40 wf% (or any integer value between 5 and 40 wt%). As another example, the concentration of one or more oligosaccharides (e.g., DP2, DPS, DP4, DP2-DP7, DP3-DP7) in a hydrolysis reaction herein can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 wt%. Those skilled in the art would recognize that the concentration of total sugars (which includes disaccharides and oligosaccharides) can have an impact on the activity of alpha-glucosidase enzymes; preferred concentrations of total sugars in a hydrolysis reaction to
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PCT/US2015/017644 maximize enzyme activity can be less than 50 wt% dry solids (DS), with a most preferred concentration of 20-35 wt% DS in some aspects.
Suitable conditions in certain embodiments for contacting a saccharide with an alpha-glucosidase herein can comprise (i) a glucan synthesis reaction, or (ii) a fraction obtained from a glucan synthesis reaction, where the saccharide is a byproduct of the glucan synthesis reaction, in other words, a hydrolysis reaction herein may be conducted in the context of a glucan synthesis reaction or a fraction of a glucan synthesis reaction, though it is typically conducted in the latter. A glucan synthesis reaction herein can produce one or more insoluble and/or soluble alpha-glucan products, for example. Thus, a glucan synthesis reaction can be characterized in some embodiments herein as an “alpha-glucan synthesis reaction.
A glucan synthesis reaction generally refers to a solution comprising at least sucrose, water and one active glucosyltransferase enzyme, and optionally other components. Other components that can be in a glucan synthesis reaction include fructose, glucose, leucrose, soluble oligosaccharides (e.g., DP2-DP7), and soluble glucan product(s). Also, one or more alpha-glucanohydrolase enzymes can be comprised in a glucan synthesis reaction in some aspects. It would be understood that certain glucan products, such as poly alpha-1,3-glucan with a DP of at least 8 or 9, may be water-insoluble and thus are not dissolved in a glucan synthesis reaction, but rather may be present out of solution. Thus, a glucan produced by glucan synthesis reaction herein can be insoluble. An alphaglucosidase enzyme herein can be added to a glucan synthesis reaction at any stage thereof, such as during initial preparation of the reaction or when the reaction is near (e.g., 80 to 90% complete) or at completion, where the latter two time points are preferred.
A glucan synthesis reaction herein may be one that, in addition to producing a glucan product, produces byproducts such as leucrose and/or soluble oligosaccharides. A glucan in some aspects is a poly alpha-glucan.
Thus, a glucan synthesis reaction herein can be for producing poly alpha-1,330
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PCT/US2015/017644 glucan or mutan, for example, which are typically co-produced with at least leucrose and/or oligosaccharide byproducts in a glucan synthesis reaction.
A glucan synthesis reaction in certain embodiments comprises a glucosyitransferase enzyme that produces a poly alpha-glucan such as poly alpha-1,3-glucan. Examples of such glucosyitransferase enzymes useful herein are disclosed in U.S. Pat. No. 7000000, and U.S. Pat. Appl. Publ. Nos. 2013/0244288, 2013/0244287 and 2014/0087431 (all of which are incorporated herein by reference.
A glucosyitransferase enzyme herein may be derived from any microbial source, such as a bacteria or fungus. Examples of bacterial glucosyitransferase enzymes are those derived from a Streptococcus species, Leuconostoc species or Lactobacillus species. Examples of Streptococcus species include S. saiivarius, S. sobrinus, S. dentirousettL S. downei, S. mutans, S. oralis, S. gailolyticus and S. sanguinis. Examples of Leuconostoc species include L. mesenteroides, L. amelibiosum, L. argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicum and L. fructosum. Examples of Lactobacillus species include L. acidophilus, L. delbrueckii, L. heiveticus, L. saiivarius, L. cases, L. curvatus, L. plantarum, L. sakes, L. brevis, L. buchneri, L. fermentum and L. reuteri.
A glucosyitransferase enzyme herein can be primer-independent or primer-dependent. Primer-independent glucosyitransferase enzymes do not require the presence of a primer to perform glucan synthesis. A primerdependent glucosyitransferase enzyme requires the presence of an initiating molecule in the reaction solution to act as a primer for the enzyme during glucan polymer synthesis. The term “primer as used herein refers to any molecule that can act as the initiator for a glucosyitransferase enzyme. Primers that can be used in certain embodiments include dextran and other carbohydrate-based primers, such as hydrolyzed glucan, for example. U.S. Appl. Publ. No. 2013/0244287, which is incorporated herein by reference, discloses preparation of hydrolyzed glucan using poly alpha-1,3-glucan as the starting material.
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Dextran for use as a primer can be dextran T10 (i.e., dextran having a molecular weight of 10 kD), for example.
A glucosyltransferase enzyme for a glucan synthesis reaction herein may be produced by any means known in the art. For example, a glucosyltransferase enzyme may be produced recombinantly in a heterologous expression system, such as a microbial heterologous expression system. Examples of heterologous expression systems include bacterial (e.g., E. coli such as TOP10 or MG1655; Bacillus sp.) and eukaryotic (e.g., yeasts such as Pichia sp. and Saccharomyces sp.) expression systems.
A glucosyltransferase enzyme described herein may be used in any purification state (e.g., pure or non-pure). For example, a glucosyltransferase enzyme may be purified and/or isolated prior to its use. Examples of glucosyltransferase enzymes that are non-pure include those in the form of a cell lysate. A cell lysate or extract may be prepared from a bacteria (e.g,, E. coli) used to heterologously express the enzyme. For example, the bacteria may be subjected to disruption using a French pressure cell, in alternative embodiments, bacteria may be homogenized with a homogenizer (e.g., APV, Rannie, Gaulin).
A glucosyltransferase enzyme is typically soluble in these types of preparations.
A bacterial cell lysate, extract, or homogenate herein may be used at about 0.150.3% (v/v), for example, in a reaction solution for producing a poly alpba-glucan such as poly alpha-1,3-glucan from sucrose.
The temperature of a glucan synthesis reaction herein can be controlled, if desired. In certain embodiments, the temperature of the reaction is between about 5 °C to about 50 °C. The temperature in certain other embodiments is between about 20 °C to about 40 °C.
The initial concentration of sucrose in a glucan synthesis reaction herein can be about 20 g/L to about 400 g/L, for example. Alternatively, the initial concentration of sucrose can be about 75 g/L to about 175 g/L, or from about 50 g/L to about 150 g/L. Alternatively still, the initial concentration of sucrose can be about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 g/L (or any integer value between 40 and 160 g/L), for example, “initial concentration of
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PCT/US2015/017644 sucrose” refers to the sucrose concentration in a gtf reaction solution just after all the reaction solution components have been added (at least water, sucrose, gtf enzyme).
Sucrose used in a glucan synthesis reaction herein can be highly pure (> 99.5%) or be of any other purity or grade. For example, sucrose can have a purity of at least 99.0%, or can be reagent grade sucrose. As another example, incompletely refined sucrose can be used. Incompletely refined sucrose herein refers to sucrose that has not been processed to white refined sucrose. Thus, incompletely refined sucrose can be completely unrefined or partially refined. Examples of unrefined sucrose are raw sucrose” (“raw sugar”) and solutions thereof. Examples of partially refined sucrose have not gone through one, two, three, or more crystallization steps. The ICUMSA (international Commission for Uniform Methods of Sugar Analysis) of incompletely refined sucrose herein can be greater than 150, for example. Sucrose herein may be derived from any renewable sugar source such as sugar cane, sugar beets, cassava, sweet sorghum, or corn. Suitable forms of sucrose useful herein are crystalline form or non-crystalline form (e.g., syrup, cane juice, beet juice), for example. Additional suitable forms of incompletely refined sucrose are disclosed in U.S. Appl. No. 61/969,958.
Methods of determining ICUMSA values for sucrose are well known in the art and disclosed by the International Commission for Uniform Methods of Sugar
Recommended by the International Commission for Uniform Methods of Sugar
Analysis (ICUMSA) (Ed. H.C.S. de Whalley, Elsevier Pub. Co., 1964), for example, which is incorporated herein by reference. ICUMSA can be measured, for example, by ICUMSA Method GS1/3-7 as described by R.J. McCowage, R.M. Urquhartand M.L. Burge (Determination of the Solution Colour of Raw Sugars, Brown Sugars and Coloured Syrups at pH 7.0 - Official, Verlag Dr Albert Bartens, 2011 revision), which is incorporated herein by reference.
The pH of a glucan synthesis reaction in certain embodiments can be between about 4.0 to about 8.0. Alternatively, the pH can be about 4.0, 4.5, 5.0,
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5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. The pH can be adjusted or controlled by the addition or incorporation of a suitable buffer, including but not limited to: phosphate, tris, citrate, or a combination thereof. Buffer concentration in a giucan synthesis reaction can be from 0 mM to about 100 mM, or about 10, 20, or 50 mM, for example.
Poly alpha-1,3-glucan produced In a giucan synthesis reaction herein may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any integer value between 50% and 100%) glycosidic linkages that are alpha-1,3. In such embodiments, accordingly, the poly alpha-1,3-glucan has less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or 0% (or any integer value between 0% and 50%) of glycosidic linkages that are not alpha-1,3.
Poly alpha-1,3-glucan herein preferably has a backbone that is linear/unbranched. In certain embodiments, the poly alpha-1,3-glucan has no branch points or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as a percent of the glycosidic linkages in the polymer. Examples of branch points include alpha-1,6 branch points.
The molecular weight of poly alpha-1,3-glucan produced in a giucan synthesis reaction herein can be measured as number-average molecular weight (Mn) or weight-average molecular weight (Mw). Alternatively, molecular weight can be measured in Daltons or grams/mole. It may also be useful to refer to the DPW (weight average degree of polymerization) or DPn (number average degree of polymerization) of the poly alpha-1,3-glucan polymer.
The Mr, or Mw of poly alpha-1,3-glucan herein may be at least about 1000. Alternatively, the Mn or Mw can be at least about 1000 to about 600000 (or any integer value between 1000 and 600000), for example. Alternatively still, poly alpha-1,3-glucan in can have a molecular weight in DRf, or DPW of at least about 100, or of at least about 100 to 1000 (or any integer value between 100 and 1000).
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A fraction of a glucan synthesis reaction may constitute suitable conditions for contacting a saccharide with an alpha-glucosidase as presently disclosed. A fraction can be a portion of, or all of, the liquid solution from a glucan synthesis reaction. Typically, a fraction has been separated from soluble or insoluble glucan product(s) synthesized in the reaction. For example, a fraction can be separated from one or more glucan products that are insoluble in water (e.g., poly alpha-1,3-glucan) which fall out of solution during their synthesis. A fraction in certain preferred embodiments of the present disclosure is from a poly alpha1,3-glucan synthesis reaction.
The volume of a fraction (before optionally diluting or concentrating the fraction, see below) in certain embodiments can be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any integer value between 10% and 90%) of the volume of the glucan synthesis reaction from which it is obtained. Typically, in glucan synthesis reactions producing an insoluble glucan (e.g., poly alpha-1,3-glucan), the fraction will be a portion of (not all of) the liquid solution component of the reaction. A fraction can be obtained at any stage of a glucan synthesis reaction, but is preferably obtained near (e.g., greater than 80 or 90% complete) or after completion of the reaction.
Examples of a fraction of a glucan synthesis reaction in certain embodiments include filtrates and supernatants. Thus, in those embodiments in which an insoluble glucan product is synthesized, a fraction herein can be obtained (separated) from a glucan synthesis reaction using a funnel, filter (e.g., press filter), centrifuge, or any other method or equipment known in the art that allows removal of some or all liquids from solids. Filtration can be by gravity, vacuum, or press filtration, for example. Filtration preferably removes all or most of an insoluble glucan; any filter material (e.g., filter paper) with an average pore size (e.g., -40-50 micron) sufficient to remove solids from liquids can be used. A fraction typically retains all or most of its dissolved components, such as byproducts of the glucan synthesis reaction.
A fraction herein can optionally be diluted or concentrated, if desired. Concentration of a fraction can be performed using any other method or
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PCT/US2015/017644 equipment known in the art suitable for concentrating a solution. For example, a fraction can be concentrated by evaporation, such as with a rotary evaporator (e.g., set at a temperature of about 40-50 °C). A fraction in some aspects herein can be concentrated down to a volume that is about 75%, 80%, 85%, 90%, or 95% of the original fraction volume. A concentrated fraction (e.g., concentrated filtrate) can optionally be referred to as a syrup.
A fraction in some aspects can comprise water that replaces the water that was present in the composition from which the fraction was obtained. For example, saccharide byproduct(s) from a glucan synthesis reaction can be separated in certain chromatographic methods in which the original solvent is replaced with another solvent (e.g., saccharide byproducts that are bound to a column [thus removed from the original solvent] can be eluted into a new solvent).
A fraction in some aspects may be treated in a manner to have any of the suitable conditions (e.g., temperature, pH and time) disclosed above for contacting a saccharide with an alpha-glucosidase. For example, a fraction can be modified to have a pH of about 4 to 5 before an alpha-glucosidase is added to the fraction. As another example, the temperature of a hydrolysis reaction with a fraction can be about 55-65 °C (e.g., about 60 °C). A fraction that has been concentrated down to a syrup can be used in a hydrolysis reaction in yet another example.
A fraction in certain preferred embodiments herein is from a poly alpha1,3-glucan synthesis reaction; such a fraction is preferably a filtrate. A fraction of a poly alpha-1,3-glucan synthesis reaction herein comprises at least water, fructose and one or more types of saccharide (leucrose and/or oligosaccharides such as DP2-DP7). Other components that may be in this type of fraction include sucrose (i.e., residual sucrose not consumed in the gtf reaction), one or more gtf enzymes, glucose, buffer, salts, FermaSure®, borates, sodium hydroxide, hydrochloric acid, cell lysate components, proteins and/or nucleic acids, for example. Minimally, the components of a fraction from a poly alpha1,3-glucan synthesis reaction include water, fructose, glucose, one or more types
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PCT/US2015/017644 of saccharide (ieucrose and/or oligosaccharides such as DP2-DP7), and optionally sucrose, for example. It would be understood that the composition of a fraction depends, in part, on the conditions of the glucan synthesis reaction from which the fraction is obtained. In those fractions containing one or more gtf enzymes, it is preferable that such one or more gtf enzymes are deactivated (e.g., heat-deactivated) before using the fraction in a hydrolysis reaction herein.
It should be understood that the exact distribution of sugar byproducts produced via polymerization of sucrose in a glucan synthesis reaction can vary based on the reaction conditions and gtf enzyme used, especially on temperature and sucrose concentration, it should also be understood that the exact composition of sugars in a fraction of a glucan synthesis reaction is not critical to the disclosed hydrolysis process. Generally, as the amount of sucrose is increased, the selectivity of the reaction towards both Ieucrose and oligosaccharides will increase. Conversely, as the temperature increases, the selectivity of the reaction towards Ieucrose tends to decrease, while the selectivity towards oligosaccharides is largely unaffected. If should also be understood that the ratio of sugars to water, i.e., wt% dry solids (DS), which is calculated by dividing the mass of sugar to total solution weight, can be adjusted either by evaporating water, preferably at temperatures below 50 °C under vacuum, or addition of water, without significant impact to the relative distribution of sugars in a fraction of a glucan synthesis reaction, it is also possible to increase the percentage of sucrose in a fraction by stopping the gtf reaction before complete conversion (to glucan) is achieved, either by reducing the pH below the active range for the gtf enzyme or by thermal deactivation of the gtf enzyme.
In certain embodiments, a glucan synthesis reaction herein can produce one or more soluble alpha-glucan products. A soluble alpha-glucan product (“soluble fiber”, alternatively) can be (I) a direct product of a glucosyltransferase, or (ii) a product of the concerted action of both a glucosyltransferase and an
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PCT/US2015/017644 alpha-glucanohydrolase capable of hydrolyzing glucan polymers having one or more alpha-1,3-glycosidic linkages or one or more alpha-1,6-glycosidic linkages.
A soluble alpha-glucan herein can comprise, for example:
a) at least 75% alpha-1,3-glycosidic linkages;
b) less than 25% alpha-1,6-glycosidic linkages;
c) less than 10% alpha-1,3,6-glycosidic linkages;
d) an Mw of less than 5000 Daltons;
e) a viscosity of less than 0.25 Pascal second (Pa*s) at 12 wt% in water at 20 °C;
f) a dextrose equivalence (DE) in the range of 4 to 40;
g) a digestibility of less than 10% as measured by the Association of Analytical Communities (AOAC) method 2009.01;
h) a solubility of at least 20% (w/w) in pH 7 water at 25 °C; and
i) a polydispersity index (PDI) of less than 5.
Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl. No. 62/004,290.
As an example, a soluble alpha-glucan fiber composition can comprise at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% alpha-(1,3) glycosidic linkages.
As another example, in addition to the alpha-(1,3) glycosidic linkage embodiments described above, a soluble alpha-glucan fiber composition can further comprise less than 25%, preferably less than 10%, more preferably 5% or less, and even more preferably less than 1% alpha-(1,6) glycosidic linkages.
As another example, in addition to the a!pba-(1,3) and alpha-(1,6) glycosidic linkage content embodiments described above, a soluble alpha-glucan fiber composition can further comprise less than 10%, preferably less than 5%, and most preferably less than 2.5% alpha-(1,3,6) glycosidic linkages.
As another example, a soluble alpha-glucan fiber composition can comprise 93 to 97% alpha-(1,3) glycosidic linkages and less than 3% a!pha-(1,6) glycosidic linkages and has a weight-average molecular weight corresponding to
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PCT/US2015/017644 a DP of 3 to 7 mixture. In a further embodiment, a soluble alpha-glucan fiber composition can comprise about 95% alpha-(1,3) glycosidic linkages and about 1% aSpha-(1,6) glycosidic linkages and has a weight-average molecular weight corresponding to a DP of 3 to 7 mixture. In a further aspect of the above embodiment, a soluble alpha-glucan fiber composition can further comprise 1 to 3% alpha~(1,3,8) linkages or preferably about 2 % alpba-(1,3,6) linkages.
As another example, in addition to the above-mentioned glycosidic linkage content embodiments, a soluble alpha-glucan fiber composition can further comprise less than 5%, preferably less than 1 %, and most preferably less than 0.5 % alpha-(1,4) glycosidic linkages.
As another example, in addition the above-mentioned glycosidic linkage content embodiments, a soluble alpha-glucan fiber composition can comprise a weight average molecular weight (Mw) of less than 5000 Daltons, preferably less than 2500 Daltons, more preferably between 500 and 2500 Daltons, and most preferably about 500 to about 2000 Daltons.
As another example, in addition to any of the above features, a soluble alpha-glucan fiber composition can comprise a viscosity of less than 250 centipoise (0.25 Pa-s), preferably less than 10 cP (0.01 Pa-s), preferably less than 7 cP (0.007 Pa-s), more preferably less than 5 cP (0.005 Pa-s), more preferably less than 4 cP (0.004 Pa-s), and most preferably less than 3 cP (0.003 Pa-s) at 12 wt% in water at 20 °C.
A soluble alpha-glucan fiber composition can have, in certain embodiments, a digestibility of less than 10%, or preferably less than 9%, 8%, 7%, 8%, 5%, 4%, 3%, 2%, or 1% digestibility as measured by the Association of Analytical Communities (AOAC) method 2009.01. In another aspect, the relative level of digestibilify may alternatively be determined using AOAC 2011.25 (Integrated Total Dietary Fiber Assay) (McCleary et al., 2012, J. AOAC Ini., 95 (3), 824-844).
In addition to any of the above embodiments, a soluble alpha-glucan fiber composition can have a solubility of at least 20% ( w/w), preferably at least 30%, 40%, 50%, 80%, or 70% in pH 7 water at 25 °C.
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In one embodiment, a soluble alpha-glucan fiber composition can comprise a reducing sugar content of less than 10 wt%, preferably less than 5 wt%, and most preferably 1 wt% or less.
In one embodiment, a soluble alpha-glucan fiber composition can comprise a caloric content of less than 4 kcal/g, preferably less than 3 kcal/g, more preferably less than 2.5 kcal/g, and most preferably about 2 kcal/g or less.
As another example, a soluble alpha-glucan herein can comprise:
a) 10% to 30% alpha-1,3-glycosidic linkages;
b) 65% to 87% alpha-1,6-glycosidic linkages;
c) less than 5% alpha-1,3,6-glycosidic linkages;
d) a weight average molecular weight (Mw) of less than 5000 Daltons;
e) a viscosity of less than 0.25 Pascal second (Pa»s) at 12 wt% in water at 20 °C;
f) a dextrose equivalence (DE) in the range of 4 to 40, preferably 10 to 40;
g) a digestibility of less than 10% as measured by the Association of
Analytical Communities (AOAC) method 2009.01;
h) a solubility of at least 20% (w/w) in pH 7 water at 25 °C; and
i) a polydispersity index (PDI) of less than 5.
Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl. No. 62/004,308.
As another example, a soluble alpha-glucan herein can comprise:
a) 25-35 alpha-1,3-glycosidic linkages;
b) 55-75% alpha-1,6-glycosidic linkages;
c) 5-15% alpha-1,3,6-glycosidic linkages;
d) a weight average molecular weight of less than 5000 Daltons ;
e) a viscosity of less than 0.25 Pascal second (Pa*s) at 12 wt% in water at 20 °C;
f) a dextrose equivalence (DE) in the range of 4 to 40;
g) a digestibility of less than 10% as measured by the Association of Analytical Communities (AOAC) method 2009.01;
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b) a solubility of at least 20% (w/w) in wafer at 25 °C; and i) a polydispersity index of less than 5.
Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl. No. 62/004,312.
As another example, a soluble alpha-glucan herein can comprise:
a) at least 95% alpha-1,6-glycosidic linkages;
b) 1 % or less alpba-1,3-glycosidic linkages;
c) less than 2% alpha-1,3,6-glycosidic linkages;
d) less than 1.5% alpha-1,4-glycosidic linkages;
e) a weight average molecular weight of less than 20000 Daltons;
f) a viscosity of less than 0.25 Pascal second (Pa*s) at 12 wt% in water at 20 °C;
g) a dextrose equivalence (DE) in the range of 1 to 30;
h) a digestibility of less than 10% as measured by the Association of Analytical Communities (AOAC) method 2009.01;
i) a solubility of at least 20% (w/w) in pH 7 water at 25 °C; and
j) a polydispersity index of less than 5.
Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl. No. 62/004,314.
As another example, a soluble alpha-glucan herein can comprise:
a) a range of:
i) 1 % to 50% of alpha-1,3-glycosidic linkages; or ii) greater than 10% but less than 40 % alpha-1,4-glycosidic linkages; or iii) any combination of i) and ii);
b) 1 to 50% alpha-1,2-glycosidic linkages;
c) 0-25% alpha-1,3,6-glycosidic linkages;
d) less than 98% alpha-1,6-glycosidic linkages;
e) a weight average molecular weight of less than 300 kDa;
f) a viscosity of less than 0.25 Pascal second (Pa»s) at 12 wt% in water at 20 °C;
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g) a digestibility of less than 20% as measured by the Association of Analytical Communities (AOAC) method 2009.01:
h) a solubility of at least 20% (w/w) in pH 7 water at 25 °C; and
i) a polydispersity index of less than 26, preferably less than 5.
Such a soluble alpha-giucan can be produced as disclosed in U.S. Appl.
No. 62/004,305.
In certain embodiments, a soluble alpha-giucan is a direct product of a glucosyltransferase. Such a glucosyltransferase, and conditions for use thereof in a suitable glucan synthesis reaction, can be as disclosed herein, or as disclosed in any of U.S. Patent Appl. Nos. 62/004,290, 62/004,308, 62/004,312, 62/004,314, and/or 62/004,305, for example.
A soluble alpha-giucan can alternatively be a product, for example, of the concerted action of both a glucosyltransferase and an alpha-glucanohydrolase that is capable of hydrolyzing glucan polymers having one or more alpha-1,3glycosidic linkages or one or more alpha-1,6-glycosidic linkages. In some aspects, a glucan synthesis reaction for producing a soluble alpha-giucan product can comprise both at least one glucosyltransferase and at least one alpha-glucanohydrolase. in other aspects, a glucan synthesis reaction can initially comprise one or more glucosyltransferases as the only enzyme component(s). Such a reaction produces a first alpha-giucan product that has not yet been subject to modification by an alpha-glucanohydrolase. Then, at least one alpha-glucanohydrolase is added to the reaction for a suitable period of time to allow modification of the first product to a soluble alpha-giucan product. Thus, there are different ways by which to synthesize a soluble alpha-giucan product through the concerted action of both a glucosyltransferase and an alphaglucanohydrolase. Conditions for performing a glucan synthesis reaction in which one or more alpha-glucanohydrolase enzymes are included during glucan synthesis reaction and/or after glucan synthesis can be as disclosed herein, or as disclosed in any of U.S. Patent Appl. Nos. 62/004,290, 62/004,308, 62/004,312, 62/004,314, and/or 62/004,305, for example.
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An alpha-glucanohydrolase herein can be, for example, a dextranase (capable of hydrolyzing alpha-1,8-linked glycosidic bonds; E.C. 3.2.1.11), a mutanase (capable of hydrolyzing alpha-1,3-linked glycosidic bonds; E.C. 3.2.1.59), a mycodextranase (capable of endohydrolysis of (1-4)-alpha-Dglucosidlc linkages in alpha-D-glucans containing both (1-3)- and (1-4)-bonds;
EC 3.2.1.81), a glucan 1,6-alpha-glucosidase (EC 3.2.1.70), and an alternanase (capable of endohydrolyticaliy cleaving alternan; E.C. 3.2.1.-; see U.S. Patent No. 5786198).
A mutanase comprising SEQ ID NG:47 can be used in certain aspects. Alternatively, a mutanase can comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:47 and have mutanase activity, for example.
A glucan synthesis reaction as presently disclosed for producing one or more soluble aipha-glucan products can serve directly as suitable conditions in which to perform a hydrolysis reaction herein in which an alpha-glucosidase is used to hydrolyze an alpha-1,5 glucosyl-fructose linkage. Such hydrolysis can be performed following any of the conditions disclosed above regarding hydrolytic treatment of a glucan synthesis reaction that produces poly alpha-1,3glucan, for example. Alternatively, a fraction (e.g., chromatographic fraction) of a glucan synthesis reaction for producing one or more soluble aipha-glucan products can be used as suitable conditions in which to perform alphaglucosidase-mediated hydrolysis of alpha-1,5 glucosyl-fructose linkages.
A fraction in certain embodiments herein can be a chromatographic fraction of a glucan synthesis reaction. For example, a fraction can be a chromatographic fraction of a glucan synthesis reaction that produces one or more soluble aipha-glucan products as disclosed herein. Such a reaction can optionally include one or more alpha-gSucanohydroSases during glucan synthesis, and/or after completion of glucan synthesis. A fraction in any of these types of embodiments typically has been obtained for the purpose of separating all of, or most of (e.g., at least about 80%, 70%, 80%, 90%, 95%), a soluble aipha-glucan product from a reaction composition from which it was produced. Once
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PCT/US2015/017644 separated from ah or most of a soluble alpha-glucan product, a fraction can be subjected to any of the alpha-1,5 gSucosyl-fructose hydrolysis processes disclosed herein using one or more alpha-glucanases.
A chromatographic fraction herein can typically be obtained using a suitable type of liquid chromatography. Liquid chromatography can be performed using size-exclusion chromatography (SEC), column chromatography, highperformance liquid chromatography (HPLC), ion-exchange chromatography, affinity chromatography, ultrafiltration, microfiltration, or dialysis, for example.
The disclosed invention also concerns a composition produced by contacting a saccharide with an alpha-glucosidase (e.g., transglucosidase), wherein (i) the saccharide is a disaccharide or oligosaccharide comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage, and (ii) the alphaglucosidase hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the saccharide. The composition produced in this manner comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to the contacting. Examples of the composition include any of those disclosed herein, such as a hydrolyzed filtrate from a glucan synthesis reaction, ora hydrolyzed fraction of a glucan synthesis reaction used to produce soluble alpha-glucan. Any of the features disclosed above and in the Examples regarding a hydrolysis method and products thereof can characterize the composition. The following features of the composition are examples.
An alpha-glucosidase enzyme in certain embodiments of the composition cars comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 28, 28, 30, 32, 34, 36, 38, or that of DIAZYME RDF ULTRA (DuPont Industrial Biosciences). A transglucosidase in certain embodiments of the composition can comprise an amino acid sequence that is at least 90% identical to SEQ ID NO:1. Alternatively, any of the alpha-glucosidases disclosed herein can be used to produce the disclosed composition.
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A saccharide in certain embodiments of the composition has a degree of polymerization before hydrolysis of 3 to 7.
A composition produced by a hydrolysis method herein can have, for example, a concentration of a saccharide that is less than 50% of the concentration of the saccharide that was present prior to contacting the saccharide with an alpha-glucosidase.
A composition produced by a hydrolysis method in certain embodiments herein can be a glucan synthesis reaction, or a fraction thereof, in which a saccharide byproduct of the glucan synthesis reaction is contacted with an alphaglucosidase. A fraction in this embodiment can be a filtrate of the glucan synthesis reaction, or a fraction of a glucan synthesis reaction used to produce soluble alpha-glucan, for example. The saccharide in this embodiment can have a degree of polymerization of 3 to 7 before hydrolysis, for example.
It would be understood by a skilled artisan that the presently disclosed embodiments are useful, in part, for saccharifying disaccharides and oligosaccharides that can otherwise be difficult to breakdown. This feature can be taken advantage of to perform enhanced methods of (i) fructose enrichment and (ii) fermentation, for example.
Example 6 below demonstrates that fructose enrichment by chromatography is enhanced when using a glucan filtrate hydrolyzed by an alpha-glucosidase (transglucosidase), as compared to using a filtrate that was not hydrolyzed.
Thus, the disclosed invention further concerns a method of enriching fructose that is present in a fraction of a glucan synthesis reaction. This method comprises (a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase (e.g., transglucosidase) under suitable conditions, wherein the enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosylglucose linkage of a disaccharide or oligosaccharide comprised within the fraction; and (b) separating fructose from the hydrolyzed fraction of step (a) to
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PCT/US2015/017644 obtain a composition having a higher concentration of fructose compared to the fructose concentration of the fraction of step (a).
The features of the disclosed fructose enrichment method regarding alpha-glucosidase (e.g., transglucosidase) enzymes, and fractions of a glucan synthesis reaction, for example, can be according to any of the disclosures provided herein concerning each of these features.
Step (b) of separating fructose can be performed by any means known in the art. For example, chromatography can be employed as disclosed in the below Examples, or by following the disclosure of European Patent Publ. No. EP2292803B1, which is incorporated herein by reference.
A composition (e.g., fructose solution or fructose syrup) having a higher concentration of fructose resulting from the disclosed enrichment method can have at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt% fructose.
A fructose enrichment method herein can perform better than one which utilizes a filtrate that has not been hydrolyzed with an alpha-glucosidase as presently disclosed. Such increased performance can be measured in terms of a percent fructose recovery of at least 40%, 45%, or 50%.
The present disclosure further concerns a fermentation method comprising (a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme (e.g., transglucosidase or glucoamylase) under suitable conditions, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,5 glucosyl-fructose linkage of a disaccharide or oligosaccharide comprised within the fraction; (b) fermenting the fraction of step (a) with a microbe to yield a product; and (c) optionally, isolating the product of (b). The fermenting step of (b) can be performed after step (a) or simultaneously with step (a). Significantly, this method can be used to produce ethanol, for example, by fermenting a hydrolyzed filtrate of a glucan synthesis reaction. The ethanol yield from such a process is higher than the ethanol yield obtained when fermenting a glucan filtrate that has not been hydrolyzed.
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The features of the disclosed fermentation method regarding alphaglucosidase (e.g., transglucosidase or glucoamylase) enzymes, disaccharides and oligosaccharides, fractions of a glucan synthesis reaction, and suitable contacting conditions, for example, can be according to any of the disclosures provided herein concerning each of these features.
A microbe for use in a fermentation method herein can be a bacteria, yeast, or fungus, for example. Examples of bacteria useful herein include Lactobacillus species, Streptococcus species, Bifidobacterium species, Leuconostoc species, Escherichia species (e.g,, E. cols) and Bacillus species. Examples of yeast useful herein include Saccharomyces species such as S. cerevisiae and S. hayanus,
A fermentation method herein can yield a product such as ethanol or an acid (e.g., lactic acid). It is believed, however, that other products can be produced if desired. It would be understood by one of skill in the art that production of certain products using a fermentation method as disclosed would depend on various conditions such as the microbe(s) used in the fermentation. Conditions for fermentation herein can be as disclosed in the below Examples, or as disclosed in El-Mansi et al. (2006, Fermentation Microbiology and Biotechnology, Second Edition, CRC Press) and Stanbury et al. (1999, Principles of Fermentation Technology, Second Edition, Butterworth-Heinemann), for example, which are both incorporated herein by reference.
The yield of a product in certain embodiments of a fermentation method herein is higher than the product yield obtained when fermenting a glucan filtrate that has not been hydrolyzed with an alpha-glucosidase herein. This comparison can be with respect to a control fermentation, for example, which used a nonhydrolyzed fraction of a glucan synthesis reaction. Product yield of a fermentation herein can be increased by at least about 10%, 20%, 40%, 60%, 80%, or 100% (or any integer value between 10% and 100%), for example. In addition, the rate of product formation by a fermentation herein can be increased.
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Example 7 below demonstrates that leucrose can be fermented to ethanol by yeast provided a feed comprising giucan filtrate that had not been hydrolyzed. Thus, further disclosed herein is a method of using a microbe to ferment leucrose to a product (e.g., ethanol). Such a method can comprise fermenting a giucan filtrate that (I) has, or (ii) has not been, hydrolyzed with an alpha-glucosidase as disclosed herein. Regardless of whether the leucrose is provided in a giucan filtrate or another form (e.g., semi-purified or enriched form), a method for fermenting leucrose can comprise adapting a microbe (e.g., yeast such as S. cerev/s/ae) for utilizing leucrose. Such adaptation can comprise growing a microbe in the presence of leucrose, and optionally other sugars, over at least 2 or 3 growth cycles, for example, afterwhich the microbe utilizes more leucrose for fermenting a product. In certain embodiments, a microbe can be (I) grown in a first feed comprising leucrose (1 cycle complete), (ii) removed from the first feed, (iii) grown in a second feed comprising leucrose (two cycles complete), (iv) optionally removed from the second feed, and (v) optionally grown in a third feed (three cycles complete). A microbe adapted in this manner can have an increased capacity to ferment leucrose in certain embodiments.
Example 9 below demonstrates that almost all (e.g., >98% or >99%) the leucrose present in a giucan filtrate can be used for fermentation by yeast when the giucan filtrate is hydrolyzed with a transglucosidase while at the same time fermented with yeast. Thus, an enhanced leucrose fermentation method herein can comprise hydrolysis of leucrose with an alpha-glucosidase (e.g., transglucosidase or glucoamylase) while simultaneously fermenting the leucrose with a microbe.
Non-limiting examples of compositions and methods disclosed herein include:
1. A method of hydrolyzing an alpha-1,3 or alpha-1,6 glucosyl-glucose linkage in a saccharide comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage, wherein the saccharide is a disaccharide or oligosaccharide, and wherein the method comprises:
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2. The method of embodiment 1, wherein the alpha-glucosidase enzyme is immobilized.
3. The method of embodiment 1 or 2, wherein the degree of polymerization of the saccharide before hydrolysis is 3 to 7.
4. The method of embodiment 1, 2, or 3, wherein the concentration of the saccharide after the contacting step is less than 50% of the concentration of the saccharide that was present prior to the contacting.
5. The method of embodiment 1,2,3, or 4, wherein the suitable conditions comprise (i) a glucan synthesis reaction, or (ii) a fraction obtained from the glucan synthesis reaction;
wherein the saccharide is a byproduct of the glucan synthesis reaction.
6. The method of embodiment 5, wherein the glucan synthesis reaction produces at least one insoluble alpha-glucan product.
7. The method of embodiment 6, wherein the fraction is a filtrate of the glucan synthesis reaction.
8. The method of embodiment 5, wherein the glucan synthesis reaction produces at least one soluble alpha-glucan product that is (i) a product of a glucosyltransferase, or (ii) a product of the concerted action of both a glucosyltransferase and an alpha-glucanohydrolase capable of hydrolyzing glucan polymers having one or more alpha-1,3-glycosidic linkages or one or more alpha-1,6glycosidic linkages.
9. The method of embodiment 8, wherein the fraction is a chromatographic fraction of the glucan synthesis reaction.
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10. The method of any one of embodiments 1-9, wherein the alphaglucosidase enzyme is a transglucosidase.
11. A composition produced by contacting a saccharide with an alphaglucosidase enzyme, wherein the saccharide is a disaccharide or oligosaccharide and comprises at least one alpha-1,3 or alpha-1,8 glucosyl-glucose linkage, wherein the enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the saccharide, and wherein the composition comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to the contacting.
12. The composition of embodiment 11, wherein the degree of polymerization of the saccharide before hydrolysis is 3 to 7.
13. The composition of embodiment 11 or 12, wherein the saccharide is in (i) a glucan synthesis reaction, or (ii) a fraction obtained from the glucan synthesis reaction;
wherein the saccharide is a byproduct of the glucan synthesis reaction.
14. A method of enriching fructose present in a fraction of a glucan synthesis reaction, comprising:
(a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of a disaccharide or oligosaccharide comprised within the fraction; and (b) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition having a higher concentration of fructose compared to the fructose concentration of the fraction of step (a).
15. A fermentation method comprising:
(a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or
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(b) fermenting the fraction of step (a) with a microbe to yield a product, wherein the fermenting is performed after step (a) or simultaneously with step (a); and (c) optionally, isolating the product of (b);
wherein the yield of the product of (b) is increased compared to the product yield of fermenting a fraction of the glucan synthesis reaction that has not been contacted with the alpha-glucosidase enzyme.
EXAMPLES
The disclosed invention is further defined in the following Examples, it should be understood that these Examples, while indicating certain preferred aspects of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt if to various uses and conditions.
Abbreviations
The meaning of some of the abbreviations used herein is as follows: “g” means gram(s), “h” means hour(s), “mL” means miliiliter(s), “psi” means pound(s) per square inch, “wt%” means weight percentage, “pm means micrometer(s), “%” means percent, “°C” means degrees Celsius, “mg” means milligram(s), “mm” means millimeter(s), “mL/min” means milliliters per minute, “m” means meter(s), “pL” means microliter(s), “mmol” means millimole(s), “min” means minute(s), “mol%” means mole percent, “M” means molar, “mg/g” means milligram per gram, “rpm” means revolutions per minute, “MPa” means megaPascals. GENERAL METHODS
All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Sucrose was obtained from VWR (Radnor, PA).
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Preparation of Crude Extracts of Glucosyltransferase (gtf) Enzymes
The Streptococcus salivarius gtfJ enzyme (SEG ID NOG) was expressed in E. co//strain DH10B using an isopropyl beta-D-1-thiogaSactopyranoside (IPTG)-induced expression system. SEQ ID NOG has an N-terminal 42-residue deletion compared to the S. salivarius gtfJ amino acid sequence in GENBANK identification No. 47527, but includes a start methionine. Briefly, E. coli DH10B cells were transformed to express SEG ID NO:3 from a DNA sequence codonoptimized to express the gtfJ enzyme in E. coli. This DNA sequence was contained in the expression vector, pJexpress404® (DNA 2.0, Menlo Park CA). The transformed cells were inoculated to an initial optical density (OD at 600nm) of 0.025 in LB medium (10 g/L Tryptone; 5 g/L yeast extract, 10 g/L NaCI) and allowed to grow at 37 °C in an incubator while shaking at 250 rpm. The cultures were induced by addition of 1 mM IPTG when they reached an ΟΟθοο of 0.8-1.0. Induced cultures were left on the shaker and harvested 3 hours post induction.
GtfJ enzyme (SEQ ID NOG) was harvested by centrifuging cultured cells (25 °C, 16000 rpm) in an Eppendorf® centrifuge, re-suspending the cells in 5.0 mM phosphate buffer (pH 7.0) and cooling to 4 °C on ice. The cells were broken using a bead beater with 0.1-mm silica beads, and then centrifuged at 16000 rpm at 4 °C to pellet the unbroken cells and cell debris. The crude extract (containing soluble GtfJ enzyme, SEQ ID NOG) was separated from the pellet and analyzed by Bradford protein assay to determine protein concentration (mg/mL).
The Streptococcus sp. C150 gtf-S enzyme (SEQ ID NO:40) was prepared as follows. SG1184 is a Bacillus subtilis expression strain that expresses a truncated version of the glycosyltransferase Gtf-S (“GTF0459”) from Streptococcus sp.C150 (GENBANK® GL321278321). The gene (SEQ ID NO:41) encoding an N-terminal truncated protein GTF0459 (SEQ ID NO:42)from E. coli expression plasmid pMP79 was cloned into the Nhel and Hindlll sites of the Bacillus subtilis integrative expression plasmid p4JH under the aprE promoter and fused with the B. subtilis AprE signal peptide on the vector. The construct was first transformed into E. coli DH10B and selected on LB with ampicillin (100 pg/mL) plates. The confirmed construct pDCQ984 expressing GTF0459 was
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PCT/US2015/017644 then transformed into B. subtilis BG6006 containing nine protease deletions (amyE::xylRPxylAcomK-ermC, degUHy32, oppA, AspollE3501, AaprE, AnprE, Aepr, AispA, Abpr, Avpr, AwprA, Ampr-ybfJ, AnprB} and selected on LB plates with chloramphenicol (5 ug/mL). The colonies grown on LB plates with 5 pg/mL chloramphenicol were streaked several times onto LB plates with 25 pg/mL chloramphenicol. The resulting B. subtilis expression strain, SG1184, was first grown in LB medium with 25 pg/mL chloramphenicol and then subcultured into Grantsli medium containing 25 pg/mL chloramphenicol grown at 30 °C for 2-3 days. The cultures were spun at 15,000 g for 30 min at 4 °C and the supernatant was filtered through 0.22-pm filters. The filtered supernatant was aliquoted and frozen at -80 °C.
S. subtilis SG1184 strain, expressing GTF0459 (SEG ID NO:42), was grown under an aerobic submerged condition by conventional fed-batch fermentation. A nutrient medium was used containing 0-0.25% corn steep solids (Roquette), 5-25 g/L sodium and potassium phosphate, a solution of 0.3-0.6 M ferrous sulfate, manganese chloride and calcium chloride, 0.5-4 g/L magnesium sulfate, and a solution of 0.01-3.7 g/L zinc sulfate, cuprous sulfate, boric acid and citric acid. An antifoam agent, FOAMBLAST 882, at 2-4 mL/L was added to control foaming. A 10-L fermentation was fed with 50% (w/w) glucose feed when initial glucose in batch was non-detectable. The glucose feed rate was ramped over several hours. The fermentation was controlled at 30 °C and 20% DO, and at initial agitation of 750 rpm. The pH was controlled at 7.2 using 50% (v/v) ammonium hydroxide. Fermentation parameters such as pH, temperature, airflow, and DO% were monitored throughout the entire 2-day fermentation run. The culture broth was harvested at the end of the run and centrifuged to obtain supernatant. The supernatant containing GTF0459 (SEG ID NO:42) was then stored frozen at -80 °C.
The S. mutans MT-4239 gtf-C enzyme (SEG ID NO:43) was prepared as follows. A gene encoding a truncated version of a glucosyitransferase (gtf) enzyme identified in GENBANK® as Gl:3130088 (SEG ID NO:43; gtf-C from S. mutans MT-4239) was synthesized using codons optimized for expression in
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Bacillus subtilis and synthesized by GenScript. The gene (SEQ ID NO:44) encoding GTF0088BsT1 with an N-terminal truncation and a C-terminai T1 truncation (SEQ ID NO:45) was amplified from the GENSCRIPT plasmid and cloned into the Nhel and Hindlll sites of the Bacillus subtilis integrative expression plasmid p4JH under the aprE promoter and fused with the B. subtilis AprE signal peptide on the vector. The construct was first transformed into E. coil DH10B and selected on LB with ampicillin (100 pg/ml) plates. The confirmed construct pDCQ1021 expressing GTF0088BsT1 was then transformed into B. subtilis BG6006 containing nine protease deletions (amyE::xylRPxylAcomK-ermG, degUHy32, oppA, Aspol!E3501, AaprE, AnprE, Aepr, AispA, Abpr, Avpr, AwprA, Ampr-ybfJ, AnprB} and selected on the LB plates with chloramphenicol (5 pg/mL). The colonies grown on LB plates with 5 pg/mL chloramphenicol were streaked several times onto LB plates with 25 pg/mL chloramphenicol. The resulting B. subtilis expression strain SG1221 was first grown in LB medium with 25 pg/mL chloramphenicol and then subcultured into Grantsll medium containing 25 pg/mL chloramphenicol grown at 30 °C for 2-3 days. The cultures were spun at 15,000 g for 30 min at 4 °C and the supernatant was filtered through 0.22-pm filters. The filtered supernatant was aliquoted and frozen at -80 °C.
S. subtilis SG1221 strain, expressing GTF0088BsT1 (SEQ ID NQ:45), was grown under an aerobic submerged condition by conventional fed-batch fermentation. A nutrient medium was used containing 0-0.25% corn steep solids (Roquette), 5-25 g/L sodium and potassium phosphate, a solution of 0.3-0.6 M ferrous sulfate, manganese chloride and calcium chloride, 0.5-4 g/L magnesium sulfate, and a solution of 0.01-3.7 g/L zinc sulfate, cuprous sulfate, boric acid and citric acid. An antifoam agent, FQAMBLAST 882, at 2-4 mL/L was added to control foaming. A 2-L fermentation was fed with 50% (w/w) glucose feed when initial glucose in batch was non-detectable. The glucose feed rate was ramped over several hours. The fermentation was controlled at 30 °C and 20% DO, and at an initial agitation of 400 rpm. The pH was controlled at 7.2 using 50% (v/v) ammonium hydroxide. Fermentation parameters such as pH, temperature,
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PCT/US2015/017644 airflow, and DO% were monitored throughout the entire 2-day fermentation run. The culture broth was harvested at the end of run and centrifuged to obtain supernatant. The supernatant containing GTF088BsT1 (SEQ ID NO:45) was then stored frozen at -80 °C.
Determination of the Glucosyltransferase GTF0459 and GTF0088BsT1 Activity
Glucosyltransferase activity assay was performed by incubating 1-10% (v/v) crude protein extract containing GTF enzyme with 200 g/L sucrose in 25 mM or 50 mM sodium acetate buffer at pH 5.5 in the presence or absence of 25 g/L dextran (MW -1500, Sigma-Aidrich, Cat.#31394) at 37 °C and 125 rpm orbital shaking. One aliquot of reaction mixture was withdrawn at 1 h, 2 h and 3 h and heated at 90 °C for 5 min to inactivate the GTF. The insoluble material was removed by centrifugation at 13,000xg for 5 min, followed by filtration through 0.2-pm RC (regenerated cellulose) membrane. The resulting filtrate was analyzed by HPLC using two AMINEX HPX-87C columns series at 85 °C (BioRad, Hercules, CA) to quantify sucrose concentration. The sucrose concentration at each time point was plotted against the reaction time and the initial reaction rate was determined from the slope of the linear plot. One unit of GTF activity was defined as the amount of enzyme needed to consume one micromole of sucrose in one minute under the assay conditions.
Preparation of a Crude Extract of Alpha-(1,3)-GSucanohydrolase (mutanase)
A gene encoding the Penicillium marneffei ATGC®' 18224™ mutanase identified in GENBANKitt> as GL212533325 was synthesized by GenScript (Piscataway, NJ). The nucleotide sequence (SEQ ID NO:46) encoding protein sequence (MUT3325; SEQ ID NG:47) was subcloned into plasmid pTrex3 at Sacll and Ascl restriction sites, a vector designed to express the gene of interest in Trichoderma reesei, under control of CBH/ promoter and terminator, with Aspergillus niger acetamidase for selection. The resulting plasmid was transformed into T. reesei by biolistic injection. The detailed method of biolistic transformation is described in International PCT Patent Application Publication W02009/126773 A1, which is incorporated herein by reference. A 1-cm2 agar plug with spores from a stable clone, TRM05-3, was used to inoculate the
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PCT/US2015/017644 production media (described below). The culture was grown in shake flasks for 4-5 days at 28 °C and 220 rpm. To harvest the secreted proteins, the cell mass was first removed by centrifugation at 4000gfor 10 min and the supernatant was filtered through 0.2-pm sterile filters. The expression of mutanase MUT3325 (SEQ ID NG:47) was confirmed by SDS-PAGE.
The production media component is listed below. NREL-Trich Lactose Defined
Formula Amount Units
ammonium sulfate 5 g
DiDDQ ΓίΓΓΟ 33 g
BD BACTO casamino acid 9 g
KH2PO4 4.5 g
CaCI2.2H2Q 1.32 g
MgSO4.7H2Q 1 g
T. reesei trace elements 2.5 mL
NaOH pellet 4.25 g
Adjust pH to 5.5 with 50% NaOH
Bring volume to 920 mL
Add to each aliquot: FOAM BLAST 5 drops
Autoclave, then add 20 % lactose filter sterilized 80 mL
T. reesei trace elements
Formula Amount Units
citric acid.H2G 191.41 g
FeSQ4.7H2O 200 g
ZnSO4.7H2O 18 g
CuSO4.5H2O 3.2 g
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MnSO4.H2O 1.4 g
H3BO3 (boric acid) 0.8 g
Bring volume to 1 L
Fermentation seed culture was prepared by inoculating 0.5 L of minimal medium in a 2-L baffled flask with 1.0 mL frozen spore suspension of the MUT3325 expression strain TRM05-3 (The minimal medium was composed of 5 g/L ammonium sulfate, 4.5 g/L potassium phosphate monobasic, 1.0 g/L magnesium sulfate heptahydrate, 14.4 g/L citric acid anhydrous, 1 g/L calcium chloride dihydrate, 25 g/L glucose and trace elements Including 0.4375 g/L citric acid, 0.5 g/L ferrous sulfate heptahydrate, 0.04 g/L zinc sulfate heptahydrate, 0.003 g/L cupric sulfate pentahydrate, 0.0035 g/L manganese sulfate monohydrate and 0.002 g/L boric acid. The pH was 5.5.). The culture was grown at 32 ,;'C and 170 rpm for 48 hours before being transferred to 8 L of the production medium In a 14-L fermenter. The production medium was composed of 75 g/L glucose, 4.5 g/L potassium phosphate monobasic, 0.6 g/L calcium chloride dehydrate, 1.0 g/L magnesium sulfate heptahydrate, 7.0 g/L ammonium sulfate, 0.5 g/L citric acid anhydrous, 0.5 g/L ferrous sulfate heptahydrate, 0.04 g/L zinc sulfate heptahydrate, 0.00175 g/L cupric sulfate pentahydrate, 0.0035g/L manganese sulfate monohydrate, 0.002 g/L boric acid and 0.3 mL/L FOAM BLAST 882,
The fermentation was first run with batch growth on glucose at 34 °C, 500 rpm for 24 h. At the end of 24 h, the temperature was lowered to 28 °C and the agitation speed was increased tolOOO rpm. The fermenter was then fed with a mixture of glucose and sophorose (62% w/w) at a specific feed rate of 0.030 g glucose-sophorose solids / g biomass / hr. At the end of run, the biomass was removed by centrifugation and the supernatant containing the MUT3325 mutanase (SEG ID NO:47) was concentrated about 10-fold by ultrafiltration using 10-kD Molecular Weight Cut-Off ultrafiltration cartridge (UFP-10-E-35; GE Healthcare, Little Chalfont, Buckinghamshire, UK). The concentrated protein was stored at -80 °C.
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Determination of Alpha-Glucanohydrolase (Mutanase) Activity insoluble mutan polymers required for determining mutanase activity were prepared using secreted enzymes produced by Streptococcus sobrinus ATCC® 33478™. Specifically, one loop of glycerol stock of S. sobrinus ATCC® 33478™ was streaked on a BHi agar plate (Brain Heart Infusion agar, Teknova, Hollister, CA), and the plate was incubated at 37 °C for 2 days. A few colonies were picked using a loop to inoculate 2X 100 mL BHI liquid medium in the original medium bottle from Teknova, and the culture was incubated at 37 °C, held static for 24 h. The resulting cells were removed by centrifugation and the resulting supernatant was filtered through a 0.2-pm sterile filter; 2X 101 mL of filtrate was collected. To the filtrate was added 2X 11.2 mL of 200 g/L sucrose (final sucrose 20 g/L). The reaction was incubated at 37 °C with no agitation for 67 h. The resulting polysaccharide polymers were collected by centrifugation at 5000xg for 10 min. The supernatant was carefully decanted. The insoluble polymers were washed 4 times with 40 mL of sterile water. The resulting mutan polymers were lyophilized for 48 h, Mutan polymer (390 mg) was suspended in 39 mL of sterile water to make a 10 mg/mL suspension. The mutan suspension was homogenized by sonication (40% amplitude until large lumps disappear, -TO min in total). The homogenized suspension was aliquoted and stored at 4 °C.
A mutanase assay was initiated by incubating an appropriate amount of enzyme with 0.5 mg/mL mutan polymer (prepared as described above) in 25 mM KOAc buffer at pH 5.5 and 37 °C. At various time points, an aliquot of reaction mixture was withdrawn and quenched with equal volume of 100 mM glycine buffer (pH 10). The insoluble material in each quenched sample was removed by centrifugation at 14,000xg for 5 min. The reducing ends of oligosaccharide and polysaccharide polymer produced at each time point were quantified by the ρ-hydroxybenzoic acid hydrazide solution (PAHBAH) assay (Lever M.. Anal. Biochem., (1972) 47:273-279) and the initial rate was determined from the slope of the linear plot of the first three or four time points of the time course. The PAHBAH assay was performed by adding 10 pL of reaction sample supernatant to 100 μL of PAHBAH working solution and heated at 95 °C for 5 min. The
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PCT/US2015/017644 working solution was prepared by mixing one part of reagent A (0.05 g/mL phydroxy benzoic acid hydrazide and 5% by volume of concentrated hydrochloric acid) and four parts of reagent B (0.05 g/mL NaOH, 0.2 g/mL sodium potassium tartrate). The absorption at 410 nm was recorded and the concentration of the reducing ends was calculated by subtracting appropriate background absorption and using a standard curve generated with various concentrations of glucose as standards.
Analysis of Reaction Profiles by HPLC
Periodic samples from reactions were taken and analyzed using an Agilent® 1260 HPLC equipped with a refractive index detector. An Aminex® HP87C column (BioRad, Hercules, CA) having deionized water at a flow rate of 0.6 mL/min and 85 °C was used to quantitate the level of sucrose, glucose, leucrose and fructose in gtf reactions. An Aminex®' HP-42A column (BioRad) having deionized water at a flow rate of 0.6 mL/min and 85 °C was used to quantitate soluble oligosaccharide byproducts (DP2-DP7) in gtf reactions.
A Dionex® UltiMate1M 3000 HPLC (Thermo Scientific) equipped with a refractive index detector was used for samples involving immobilized enzymes (Example 4). A Phenomenex® Rezex'M calcium monosaccharide column having deionized water at a flow rate of 0.3 mL/min and 85 °C was used to analyze the sugars.
Analysis of oligosaccharide linkage by NMR
NMR data were acquired on an Agilent DD2 spectrometer operating at 500 MHz for 1H using a 5-mm cryogenic triple-resonance pulsed-field gradient (PPG) probe. Water suppression was obtained by carefully placing the observe transmitter frequency on resonance for the residual water signal in a “presat” experiment, and then using the first slice of a NOESY experiment with a full phase cycle (multiple of 32) and a mix time of 10 ms. One-dimensional !H spectra were acquired with a spectral width of 6410 Hz, acquisition time of 5.1 s, 65536 data points, 4 s presaturation and a 90-degree pulse of 5.85 ps. Sample temperature was maintained at 25 °C. Samples were prepared by adding 50 pL to a 5-mm NMR tube along with 450 pL of D2O and 60 pL of D2O containing 12.4
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PCT/US2015/017644 mM DSS (4,4-dimethyi-4-siiapentane-1 -sulfonic acid sodium salt) internal reference with the methyl resonance set to 0 ppm. Chemical shift assignments for different anomeric linkages were taken from: Goffin et al. (2009, Bull Korean Chem. Soc. 30:2535-2541. Peak assignments were 5.35 ppm for alpha(1,3) linkages, 5.1 ppm for leucrose, and 4.95 for alpha(1,6) linkages. Reducing ends (RE) were assigned to be 5.2 for alpha RE and 4.65 for beta RE.
EXAMPLE 1
Production of Sugar Syrup by Polymerization of Sucrose
This example discloses the general manner in which a mixture of soluble sugars was produced by polymerization of sucrose with a gtf enzyme in a glucan synthesis reaction. Specifically, a filtrate of a glucan synthesis reaction was prepared, which was then concentrated to a syrup.
Sucrose (3000 g) was added to a clean 5-gallon polyethylene bucket. Water (18.1 L) and PermasureiM (10 mL) were added to the bucket, and the pH was adjusted to 7.0 by addition of 5 vol% NaOH and 5 vol% H2SO4. The final volume was ~20 L and the initial concentration of sucrose as measured by HPLC was 152.5 g/L. The glucan polymerization reaction was initiated by adding 0.3 vol% of crude gtf enzyme (SEQ ID NO:3) extract prepared as described in the General Methods section. This extract contained about 2.9 mg/mL of protein. Agitation to the reaction solution was provided using an overhead mechanical motor equipped with a glass shaft and PTFE blade.
After 48 hours, HPLC analysis revealed that 96% of the sucrose had been consumed and the reaction was deemed to be complete. The insoluble polyalpha-1,3~glucan product of the reaction was removed by filtration with a Buchner filter funnel using 325-mesh steel screen and 40-micron filter paper. The mother liquor (filtrate) was then concentrated using a rotary evaporator (bath temp of 4050 °C) to a total sugar concentration of about 320 g/L sugars. The composition of the concentrated filtrate is provided in Table 2.
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Table 2
Composition of a Concentrated Filtrate of a Glucan Synthesis Reaction
Sucrose Leucrose Glucose Fructose DP2 DP3+ Total
g/L 13.5 130.6 25.5 103.8 18.3 28.3 320.1
wt% 4.2 40.8 8 32.4 5.7 8.9 100
Table 2 indicates that the concentrated filtrate of the glucan synthesis 5 reaction contains sucrose, fructose, glucose, leucrose and oligosaccharides of
DP2-DP7.
EXAMPLE 2
Effect of Enzymes on Hydrolysis of Sugars in a Filtrate of a Glucan Synthesis
Reaction
This example measures the activity of various glucoamylase (EC 3.2.1.3), transglucosidase (EC 2.4.1.24), beta-glucosidase (EC 3.2.1.21), alpha-amylase (EC 3.2.1.1) and glucosidase (EC 3.2.1) enzymes for the purpose of reducing the concentration of leucrose and/or oligosaccharide byproducts in a concentrated filtrate of a glucan synthesis reaction. Certain enzymes such as DIAZYME RDF
ULTRA, transglucosidase (EC 2,4.1.24) and glucoamylase (EC 3,2.1.3), which are all alpha-glucosidase, were found to be particularly effective at reducing the amount of these byproducts, resulting in a corresponding increase in monosaccharides (glucose and fructose) in the treated filtrate.
A filtrate of a glucan synthesis reaction was first prepared and concentrated to a syrup according to the procedure outline in Example 1. The composition of this concentrated filtrate is provided in Table 3. NMR analysis revealed that the ratio of alpha(1,3) to alpha (1,6) linkages present in the syrup was 78:22.
Table 3
Composition of a Concentrated Filtrate of a Glucan Synthesis Reaction
Sucrose Leucrose Glucose Fructose DP2 DP3-i- Total
g/L 161 210 93 302 33 61 860
wt% 18.7 24.4 10.8 35.1 3.8 7.1 100.0
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The syrup of Table 3 was used to test the hydrolytic activity of various enzymes against leucrose and oligosaccharide byproducts of the glucan synthesis reaction. It was not obvious at the outset of these experiments what enzyme could be used to hydrolyze both these byproducts, given that leucrose contains an unusual linkage [alpha(1,5)-glucosyl fructose] and that the oligosaccharides comprise primarily alpha(1.3) and alpha(1,6) glucosyl-glucose linkages. Enzymes with various activities were selected for this analysis (Table 4).
Table 4
Enzymes Evaluated for Leucrose and Oligosaccharide Hydrolysis
Enzyme Source Function Activity or protein concentration
DIAZYME RDF ULTRA DuPont IB3 1,4-alpha-glucosidase 710 U/g
Oligo-l,6-glucosidase Megazyme 1,6-alpha-giucosidase 320 U/mg
SPEZYME FRED DuPont IB Alpha-amylase 1-5%
SPEZYME RSL DuPont IB Alpha-amylase 1-5%
OPTIMAX L-1000 DuPont IB Puiluianase 1-5%
TRANSGLUCOSIDASE L-2000 DuPont IB Transgiucosidase >1700 TGU/g
purified TRANSGLUCOSIDASE DuPont IB Transgiucosidase 22.7 mg/mL
L-2000 !
ACCELLERASE BG DuPont IB Beta-glucosidase 3000 U/g
NOVO 188 Sigma-Aidrich Beta-glucosidase > 250 U/g
SUMiZYME BFS-L Shin Nihon Chemical Beta-glucosidase 100 U/g
SUMIZYME BGA Shin Nihon Chemical Beta-glucosidase 2000 U/g
ACCELERASE TRIO DuPont IB Celluiase 5-10%
ACCELERASE 1500 DuPont IB Cellulase/Beta- glucosidase 5-10%/0.5-4%
OPTIDEX L-4Q0 DuPont IB Glucoamylase > 350 GAU/g
GC 147 DuPont IB Glucoamylase 400 GAU/g
GC321 DuPont IB Glucoamylase > 350 GAU/g
a DuPont Industrial Biosciences
Conditions for treating the syrup of Table 3 with each of the above enzymes are provided in Table 5 (enzyme loading, time, temperature, pH, sugar concentration). The syrup was diluted with water to reach the sugar
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PCT/US2015/017644 concentration used in each hydrolysis reaction. Table 5 further provides the percent hydrolysis of the leucrose and DP3+ (at least DP3-DP7) oligosaccharides by each enzyme. Percent DP3+ hydrolysis was calculated as (1 --- (wt% DP3+ oligosaccharides in the final syrup) / (wt% DP3+ oligosaccharides in the initial syrup)). Similarly, percent leucrose hydrolysis was calculated as (1 - (wt% leucrose in the final syrup) / (wt% leucrose in the initial syrup)).
Table 5
Hydrolysis of Leucrose and Oligosaccharides in a Concentrated Filtrate by
Various Enzymes
Example Enzyme Enzyme loading (vol %) Temp CO Time (hr) pH Sis gar concentration (g/L)a DP3-P hydrolysis (%) Leucrose hydrolysis (%)
2.1 Di ΑΖΥΜΕ RDF ULTRA 0.5 60 88 4.0 300 36 13
2.2 Oligo-1,6- giucosidase 5 40 72 5.5 400 43 <2
2.3 SPEZYME FRED 0.5 60 66 4.0 280 <2 <2
2.4 SPEZYME RSL 0.5 60 48 4 290 <2 <2
2.5 OPTIMAX L-1000 0.5 60 48 4 290 <2 <2
2.6 TG L-2000 0.25 60 70 4.0 260 54 >98
2.7 TG L-2000 2 60 48 4.5 300 96 >98
2.8 PURIFIED TG L-2000 0.5 60 48 4.5 260 56 >98
2.9 ACCELERASE BG 0.5 60 70 4 300 11 <2
2.10 NOVO 188 0.25 60 70 4 300 49 36
2,11 NOVO 188 5 60 40 5.5 340 93 29
2.12 SUMIZYME BFS-L 0.5 60 48 4.5 2.60 55 46
2.13 SUMIZYME BGA 0.1 wt% 60 48 4.5 2.60 2.6 77
2.14 ACCELERASE TRIO 0.5 60 48 4 290 28 6.6
2.15 ACCELERASE 1500 0.5 60 66 4 2.80 26 4
2,16 GC 147 0.5 60 40 4 300 55 12
2,17 GC 321 5 60 72. 5.5 400 74 64
2.18 OPTIDEX L-400 0.5 60 70 4 300 27 25
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PCT/US2015/017644 3 Sugar concentration (total concentration of sucrose, glucose, fructose, leucrose and oligosaccharides) measured by HPLC; reported values are rounded to nearest 10 g/L increment.
b DP3+ contains DP3-DP7, but may also contain larger soluble oligosaccharides that have a high ratio of alpha-1,6 linkages to alpha-1,3 linkages, when produced using certain gtf enzymes.
Table 5 indicates that 1,4-alpha-glucosidase and 1,6-alpha-glucosidase showed some (Example 2.1) or very little (Example 2.2) hydrolysis of leucrose, but did release some glucose from the oligosaccharides. Use of alpha-amylase (Example 2.3 and Example 2.4) showed very little activity against the compounds of interest. Similarly, use of a pullulanase (Example 2.5) showed very little activity.
Cellulases (Examples 2.14 and 2.15) were largely ineffective at hydrolyzing leucrose, but did hydrolyze some of the oligosaccharides.
Although the oligosaccharides did not contain beta linkages, surprisingly, beta-glucosidase enzymes also showed a range of hydrolytic conversion from very low (ACCELERASE BG, Example 2.9) to very high (NOVO 188, Examples 2.10 and 2.11). The relative efficacy of these enzymes varied quite dramatically. In some cases, the amount of oligosaccharide that was hydrolyzed greatly exceeded (Example 2.11), or was close to (Example 2.12), the percentage of leucrose that was hydrolyzed. In other cases, leucrose was highly hydrolyzed by beta-glucosidase while the oligosaccharides were moderately hydrolyzed (Example 2.13). The high disparity amongst the results observed with betaglucosidase suggests that the presence of other enzymes in the tested betaglucosidase formulations, such as glucoamylase or another type of alphaglucosidase, could be responsible for the observed activity.
Conversely, the results in Table 5 indicate that transglucosidase (TG L2000, Example 2.6) showed very high activity at hydrolyzing both the oligosaccharides and leucrose. Leucrose hydrolysis by transglucosidase appeared quantitative under certain circumstances, and greater than 95% of the DP3+ material was hydrolyzed to glucose and DP2 at high enzyme loadings (Example 2.7). Use of a purified version of transglucosidase revealed similar
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PCT/US2015/017644 activity (Example 2.8), indicating that the observed hydrolysis is due to the transglucosidase enzyme and not background activity.
Giucoamyiases (Examples 2.16-2.18) showed a range of activity against leucrose and the oligosaccharides. Only one tested glucoamylase (Example 2.18) gave less than 30% hydrolysis of both the leucrose and oligosaccharides.
The results in Table 5 indicate that alpha-glucosidases such as DIAZYME RDF ULTRA, glucoamylase and transglucosidase can hydrolyze leucrose byproduct present in a glucan reaction filtrate. The ability of alpha-glucosidases to hydrolyze leucrose indicates that these enzymes can hydrolyze alpha-1,5 glucosyl-fructose linkages. While this activity was shown above using leucrose as a substrate, it is believed that this activity can also be extended to oligosaccharides comprising alpha-1,5 glucosyl-fructose linkages.
The results in Table 5 further indicate that alpha-glucosidases such as glucoamylase and transglucosidase can hydrolyze oligosaccharide byproducts present in a glucan reaction filtrate. Since these oligosaccharides are mostly comprised of glucose monomer units linked by alpha-1,3 and/or alpha-1,6 linkages (Example 3), the data in Table 5 indicate that alpha-glucosidase enzymes can hydrolyze alpha-1,3 glucosyl-glucose and/or alpha-1,6 glucosylglucose linkages.
Since alpha-glucosidase enzymes were generally effective at hydrolyzing the leucrose and/or oligosaccharide byproducts of a glucan synthesis reaction, these enzymes can be used alone or in combination to reduce the processing time necessary to generate a high purity syrup from a glucan reaction filtrate containing an increased amount of monosaccharides and reduced amount of sugar byproducts. An example of an effective enzyme combination could be a transglucosidase such as TG L-2000, for leucrose hydrolysis, and a glucoamylase (e.g., GC 321) enzyme that efficiently hydrolyzes oligosaccharide byproducts.
Thus, alpha-glucosidase enzymes can individually hydrolyze (i) alpba-1,5 glucosyl-fructose linkages and (ii) alpha-1,3 and alpha-1,6 glucosyl-glucose linkages in certain saccharides.
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EXAMPLE 3
Comparison of Linkage Distributions of Giucan Reaction Filtrate Components before and after Enzyme Hydrolysis
This example measures the hydrolytic activity of transglucosidase (EC 2.4.1.24) and beta-glucosidase (EC 3.2.1.21) enzymes against leucrose and oligosaccharide byproducts present in a concentrated filtrate of a giucan synthesis reaction. Transglucosidase was found to reduce the amount of these byproducts, resulting in a corresponding increase in monosaccharides (glucose and fructose) in the treated filtrate.
The oligosaccharide byproducts present in the filtrate of the above giucan synthesis reaction comprise >90% glucose-glucose linkages, as determined by NMR (General Methods). Of the glucose-glucose linkages, -78% represent alpha-1,3 linkages and -22% represent alpha-1,6 linkages.
NMR was used to determine the linkage profile of material generated in Example 2.11 above after hydrolysis. As shown in Figure 1, the peak corresponding to alpha-1,3 linkages was reduced by 86%, the peak corresponding to alpha-1,6 linkages was reduced by only 2.3%, and the peak corresponding to leucrose peaks was reduced by 21%. While sucrose was very nearly quantitatively hydrolyzed by this enzyme, Novo 188 does not appear to be capable of hydrolyzing alpha-1,6 linkages.
NMR was similarly used to determine the linkage profile of material generated using TG L-2000 (SEQ ID NGN) transglucosidase (Figure 2). 210 pL of concentrated filtrate from the material in Table 3, 300 pL of DsO, and 90 pL of D2O containing 12.4 mM DSS as internal reference were mixed in an NMR tube to give a total sugar concentration of 300 g/L and heated to 80 °C. A time zero spectrum (starting material in Figure 2) was acquired after thermal equilibration at 60 °C, and then 0.5 vol% of enzyme was added. The sample was reequilibrated in the probe at 60 °C and shimmed, and measurements were taken within a few minutes of analysis. After 10 hours of treatment with TG L-2000 enzyme (treated material in Figure 2), the peaks corresponding to alpha-1,3 linkages were reduced by 41%, the peaks corresponding to alpha-1,6 linkages
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PCT/US2015/017644 were reduced by 36%, and the peak corresponding to leucrose was reduced by >95% (Figure 2). An increase in both the alpha-reducing end and beta-reducing end peaks was observed, which corresponds to an increase in fructose and glucose (Figure 2).
These results demonstrate that a transglucosidase can convert oligosaccharides containing alpha-1,3 and alpha-1,8 linkages info glucose and can convert leucrose info fructose and glucose. Thus, transglucosidase can hydrolyze (I) alpha-1,5 glucosyl-fructose linkages and (ii) alpha-1,3 and alpha-1,6 glucosyl-glucose linkages in certain saccharides.
EXAMPLE 4
Hydrolysis of Leucrose and Oligosaccharides in Glucan Reaction Filtrate Using
Immobilized Enzymes
This Example describes using immobilized glucoamylase (EC 3.2.1.3) and transglucosidase (EC 2.4.1.24) to hydrolyze leucrose and other oligosaccharides present in filtrate obtained from a glucan synthesis reaction. Specifically, the effect of immobilized transglucosidase TG L-2000 (SEQ ID NO:1, obtained from Genencor/ DuPont Industrial Biosciences) and immobilized glucoamylase GC147 (obtained from Genencor / DuPont Industrial Biosciences) on the hydrolysis of leucrose and oligosaccharides DP2, DPS and HS (higher sugars, DP4+) in a filtrate of a glucan synthesis reaction was studied.
Immobilization of the glucoamylase and transglucosidase enzymes was carried out according to the method described in U.S. Patent No. 5541097, which is incorporated herein by reference.
In a typical process for immobilizing the glucoamylase or transglucosidase, two batches of about 8.0 g/batch of porous granular diatomaceous earth (EP Minerals, Reno, NV) were hydrated with distilled water and then transferred to a glass column reactor of 1,5-cm diameter and 30-cm height. Water was pumped upflow at about 8-7 mL/min to remove fines from all three columns. Generally, within an hour the water effluent was free of fines. Water was drained from the column to the top of the granular diatomaceous earth beds and replaced with 0.1% w/v aqueous solution of poiyethylenimine
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PCT/US2015/017644 (PEI, EPOMIN P-1050). 3500 mL of the PEI solution was then pumped upflow and effluent was recycled through the beds for 2 hours. The granular diatomaceous earth beds were then washed upflow with distilled water for 2 hours to remove free PEI at room temperature. In this manner, granular diatomaceous earth-PEI carriers were obtained.
In the meantime, 3.5 mL of glucoamylase GC-147 having activity defined in Table 4 was added to 315 ml of 0.02 M acetate buffer (pH 4.5). 1.575 g of 50% w/w glutaraldehyde (Protectol® GA-50) was then slowly added to the aqueous solution of glucoamylase with gentle mixing, and the glutaraldehyde was allowed to react with the aqueous glucoamylase solution for 4 hours at a temperature of 20-25 °C with gentle agitation, which resulted in formation of a treated enzyme-glutaraldehyde adduct containing treated glucoamylase. Separately, these steps were repeated using the transglucosidase TG L-2000 having activity defined in Table 4 instead of the glucoamylase, thereby resulting in the formation of a treated enzyme-glutaraldehyde adduct containing treated transglucosidase.
Each of the treated enzyme-glutaraldehyde adducts was then circulated for 4 hours (20-25 °C) in its own column prepared as above containing granular diatomaceous earth-PEI carrier. Excess treated adduct was then washed out of the carriers with water. Columns with immobilized glucoamylase or transglucosidase were thus prepared.
A glucan filtrate having the composition defined in Table 3 was diluted to 180 g/L, adjusted to pH 4.5, and passed through a column containing an immobilized enzyme. Column temperature was controlled to 60 °C. After 16 hours of column equilibration, samples were taken periodically at different flow rates. Sugar compositions of hydrolysis reaction products were determined by HPLC (Table 6). Every time the flow rate setting was changed, the column was allowed to re-equilibrate for at least 1-2 bed volumes before sampling. The degree of hydrolysis of leucrose and oligosaccharides was calculated using the manner described in Example 2. Three column configurations were tested: 1)
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Table 8
Application of Immobilized Glucoamylase and Transglucosidase Enzymes to
Hydrolyze Oligosaccharides and Leucrose
immobilized Enzyme Mean contact time (br) DP3+ hydrolysis (%) Leucrose hydrolysis (%)
GC 147 0.7 16 17
GC 147 1.0 20 22
GC 147 1.3 25 29
GC 147 3.0 39 47
TG L-2000 0.7 28 >95
TG L-2000 1.0 32 >95
TG L-2000 1.3 37 >95
TG L-2000 3.0 47 >95
GC 147 + TG L-2000 6.0 55 >95
Table 6 indicates that, as the mean contact time (defined as the nominal column volume divided by the mean flow rate) was increased, the degree of hydrolysis of leucrose and oligosaccharides generally increased. Use of the immobilized transglucosidase to hydrolyze leucrose was particularly effective, as no significant difference was observed even using the fastest flow rate that was tested. While each column individually showed reasonable conversion, the combination of the glucoamylase and transglucosidase gave the highest hydrolysis of oligosaccharides.
Thus, use of an immobilized glucoamylase or transglucosidase, or both types of immobilized enzymes, represents an effective technique to hydrolyze oligosaccharides containing alpha-1,3 and alpha-1,6 glucosyl-glucose linkages, as well as leucrose. These results are consistent with those of Example 2. Immobilization of other alpha-glucosidase enzymes should give similar results.
EXAMPLE 5
Enrichment of Fructose from a Glucan Reaction Filtrate by Chromatography
This example discloses how fructose in a glucan reaction filtrate can be further enriched through chromatography.
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Generally, when separating sugar molecules by chromatography, components elute inversely to molecular size so that the largest molecules elute first. Thus, with respect to a filtrate of a glucan synthesis reaction, oligosaccharides elute first, followed by disaccharides, and then 5 monosaccharides. Separations using a sodium cation resin did not separate fructose and glucose well, and all of leucrose, sucrose, and DP2 co-eluted. Use of ion exchange resins where the cation is calcium are preferred to separate glucose and fructose.
A filtrate of a glucan synthesis reaction was first prepared and 10 concentrated to a syrup according to the procedure outline in Example 1. The composition of this concentrated filtrate is provided in Table 7.
Table 7
Composition of a Concentrated Filtrate of a Glucan Synthesis Reaction
Sucrose Leucrose Glucose Fructose DP2 DP3+ Total
g/L 126 202 93 295 40 65 821
wt% 15.4 24.6 11.3 36.0 4.8 7.9 100
The syrup of Table 7 was filtered and diluted to 25 g dry solids/100 g solution with ion-exchanged water, and fed to a column containing a crosslinked strong acid ion exchange resin in the calcium form. The physical parameters of this column appear in Table 8. Diluted syrup (15.8 L) was fed to the column, which was maintained at 65 °C, afferwhich the column was eluted using water at a flow rate of 30 L/hr.
Table 8
Physical Parameters of the Column
Resin Type FINEX CS11GC
ion form Ca2+
Crosslinking, % divinyl benzene 5.5
Particle size (mm) 0.34
Bed length (m) 5.0
Column diameter (m) 0.225
In this separation, leucrose remained in the column longer than sucrose, 25 perhaps due to complexalson of leucrose with the calcium cation, and in fact, co71
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PCT/US2015/017644 eluted with glucose. Two fractions containing fructose were isolated. Fraction 5.1 eluted between 47 and 120 minutes, and fraction 5.2 eluted between 120 and 172 minutes. Of the fructose fed to the chromatographic separation, 95.7% of the fructose was isolated in >90% purity. The product distribution in each fraction (5.1 and 5.2) as measured by HPLC appears in Table 9.
Table 9
Product Distribution of Chromatographic Fractions Containing Significant
Amounts of Fructose
Fraction Sucrose Leucrose Glucose Fructose DPS-*- Others Total % Fructose recovered
5.1 31.9 34.8 20.8 3.9 5.4 4.8 too 3.9
5.2 0.0 1.0 0.8 97.7 0.0 0.6 too 95.7
As the feed composition for this separation comprised 36.0% fructose, a total of 34.5% of the total stream was recovered as a fructose syrup with >90 wt% DS fructose, if the sucrose in the feed is neglected, 40.7% of the sugars were recovered as a fructose syrup with >90 wt% DS fructose.
Thus, fructose in a glucan reaction filtrate can be further enriched through chromatography. Example 6 below demonstrates that this process can be enhanced using glucan filtrate hydrolyzed with a transglucosidase.
EXAMPLE 6
Enrichment of Fructose from a Hydrolyzed Glucan Reaction Filtrate by
This example demonstrates that fructose isolation from a glucan filtrate in which the oligosaccharides and Ieucrose have been hydrolyzed results in an increased yield of high purity fructose syrup compared to when isolating fructose from a non-hydrolyzed glucan filtrate.
A syrup was prepared by concentrating (vacuum at 50 °C) a glucan filtrate that had been treated with 1 voS% of transglucosidase TG L-2000 (SEQ ID NQ:1) for 24 hr at 60 °C and pH 4.5. Some oligosaccharide formation was observed during the concentration process, which was expected since transglucosidase enzymes are known to create oligosaccharides at high concentrations of
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PCT/US2015/017644 monosaccharides. The syrup had the final product distribution described in Table A.
Table A
Composition of a Concentrated Glucan Filtrate that Was Hydrolyzed before
Concentration
Sucrose Leucrose Glucose Fructose DP2 DP3+ Total
g/L 3 <10 294 409 73 81 -870
wt% 0.3 1.1 33.7 47.0 8.4 3.3 100
The syrup described in Table A was filtered and diluted to 25.4 g DS/100 g solution with ion-exchanged water and was fed to a column containing a crosslinked strong acid cation exchange resin in the calcium form. The physical parameters of the column appear in Table B. Diluted syrup (169 g) was then fed to the column, which was maintained at 65 °C, afterwhich the column was eluted using water at a flow rate of 50 mL/min.
Table B
Physical Parameters of the Column
Resin Type FINEX CS11GC
ion form Ca2+
Crosslinking, % divinyl benzene 5.5
Particle size (mm) 0.34
Bed length (m) 1.69
Column diameter (m) 0.093
Two fractions containing fructose were isolated. Fraction 6.1 eluted between 73 and 103 minutes, and fraction 6.2 eluted between 103 and 120 minutes. Of the fructose fed to the chromatographic separation, 93.0% of the fructose fed to the column was isolated in fraction 6.2 in >90% purity. The product distribution in each fraction (6.1 and 6.2) as measured by HPLC appears in Table C.
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Table C
Product Distribution of Chromatographic Fractions Containing Fructose from a
Hydrolyzed Glucan Filtrate
Fraction Sucrose Leucrose Glucose Fructose DF3+ Others Total % Fructose recovered
6.1 7.7 13.9 63.9 7.3 1.5 5.7 100 5.9
6.2 0.0 0.6 3.0 91.8 0.0 4.6 100 93.0
The reduced separation efficiency in this example compared to Example 5 can be attributed to differences in the scale of the column and the higher glucose fraction of the sample. Even so, chromatographic purification of this material resulted in an increased yield of high purity fructose syrup compared to that achieved in Example 5, in which syrup was chromatographically prepared from a glucan filtrate that had not been hydrolyzed by a transglucosidase. As the feed composition for this separation comprised 47% fructose (Table A), 43.7% of the total stream was recovered as a fructose syrup with >90 wt% DS fructose. This 43.7% recovery is significantly better than the 34.5% recovery in Example 5.
Thus, fructose isolation from a glucan filtrate that has been hydrolyzed with transglucosidase results in an increased yield of fructose compared to when isolating fructose from a non-hydrolyzed glucan filtrate.
EXAMPLE 7
Production of Ethanol by Fermenting a Filtrate of a Glucan Synthesis Reaction
This example discloses yeast fermentation of glucan filtrate to ethanol.
Yeast (S. cerevisiae) cream (Tonon mill, Brazil) was washed by suspending the cream in tap water (2.4 L, optical density of 65 at 600 nm) and then centrifuging the yeast cream for 5 minutes using a LEGEND XTR centrifuge (Thermo Scientific) at 4500 g. After decanting the supernatant, the yeast cells were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 2 by addition of 5 wt% sulfuric acid. The optical density was measured using a GENESYS 20 4001 spectrophotometer (Thermo Scientific) and adjusted to 100 at 600 nm by addition of tap water. The adjusted yeast cream (1.5 L) was added to a 7.5-L BIOFLO310 fermenter vessel
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PCT/US2015/017644 (New Brunswick). The fermenter was set to maintain temperature at 30 °C and agitation at 100 rpm. Although pH was measured during fermentation, it was not controlled by the addition of acid or base solutions.
A feed solution containing yeast extract (10 g/L), peptone (20 g/L), and 200 g/L of sugars from a glucan filtrate was prepared and sterilized using a PHOENIX AV-250 PLUS autoclave at 121 °C for 15 minutes. The feed solution was allowed to cool to 25 °C (room temperature) before the fermentation began. The sterilized feed solution (3.5 L) was added to the fermenter over approximately 5 hours at a rate of 684 mL/hr, and the fermentation was allowed to proceed for 22 hours.
Periodic samples were taken during the fermentation and analyzed for optical density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL3 refractometer (Atago), and sugar and ethanol concentrations by HPLC (General Methods). These results are summarized in Table 10.
Table 10
Feed and Time Course Fermentation Profiles for the First Ethanol Fermentation
Time (hr) Sucrose Leucrose Glucose Fructose DP2 DP3+ Total Sugar EtOH
Feed 9 70 19 76 7 19 200 0
0 0.2 0.0 0.0 0,1 - - - 7
1 <1 18 0.3 <1 - - - 15
2 <1 30 0.4 <1 - - - 21
3 <1 40 0.0 <1 - - - 25
4 <1 46 0.0 <1 - - - 28
5 <1 49 0.0 <1 - - - 29
6 <1 S3 0.0 <1 - - 32
8 <1 S3 0.0 <1 - 33
22 <1 S3 0.0 <1 5 18 76 33
Concentrations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-22 hours) are listed.
When the fermentation was over, the yeast cells were separated by centrifugation using a LEGEND XTR centrifuge at 4500 g for 5 minutes. After decanting the supernatant, the yeast were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to
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PCT/US2015/017644 by addition of 5 wt% sulfuric acid. The optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 at 600 nm by addition of tap water. Two additional fermentation cycles, each using fresh feed, were performed using recycled yeast cells from the previous fermentation following the same conditions described above. The fermentation results obtained using first-time and second-time recycled yeast are provided in Tables 11 and 12, respectively.
Table 11
Feed and Time Course Fermentation Profiles Using the First Recycle of Yeast
Cells
Time (hr) Sucrose Leucrose Glucose Fructose DPS DP3+ Total Sugar EtOH
Feed 13 69 21 77 7 18 206 0
0 0 4 0 0 - - - 5
1 <1 19 0 <1 - - - 18
4 <1 35 0 <1 - - 23
4 <1 40 0 <1 - 26
5 <1 45 0 <1 - - 29
6 <1 53 0 <1 - 32
7 <1 50 0 <1 - - - 32
7 <1 0 <1 - - - 33
21 <1 42 0 <1 6 18 65 37
Concentrations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-21 hours) are listed.
Table 12
Feed and Time Course Fermentation Profiles Using the Second Recycle of Yeast
Cells
Time (hr) j Sucrose Leucrose Glucose Fructose DPS DP3+ Total Sugar EtOH
Feed 10 70 19 76 6 19 201 0
0 <1 0 0 <1 - - - 11
1 <1 32 0 <1 - - - 24
4 <1 40 0 <1 - 29
4 <1 45 0 <1 - - - 31
5 <1 46 0 <1 - - - 33
6 45 0 <1 - - - 34
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θ <1 16 0 <1 - - - 48
7 <1 7 0 <1 - - - 52
21 <1 5 0 <1 5 16 27 54
Concentrations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-21 hours) are listed.
Very little ieucrose was consumed in the first fermentation, although the yeast cells started to acclimate and consume ieucrose by the second recycle. Ethanol fermentation titers increased from 33 g/L (Table 10, 22 hours) to 54 g/L (Table 12, 21 hours) after three fermentation cycles with recycled yeast, although significant amounts of Ieucrose were present in the medium, even after the last cycle.
Thus, glucan filtrate can be used in a fermentation process to produce ethanol.
EXAMPLE 8
Production of Ethanol by Fermenting Hydrolyzed Glucan Filtrate
This example demonstrates that fermenting a glucan filtrate in which the Ieucrose and oligosaccharide byproduct components have previously been saccharified results in increased ethanol yields.
Fermentations were performed following the procedure outlined in Example 7, but using a glucan filtrate that was previously treated with a transglucosidase (TG L-2000, SEQ ID NO:1). Hydrolyzed glucan filtrate was prepared as follows, Glucan filtrate was adjusted to 300 g sugars/L and then the pH was adjusted to 4.0 using 1.0 M sodium hydroxide and 5 wt% sulfuric acid. The final volume of this preparation was 6,75 L. The filtrate solution was then sterilized using a PHOENIX AV-250 PLUS autoclave at 121 °C for 15 minutes, and then the temperature was adjusted to 60 °C. TG L-2000 enzyme extract as described in Table 4 (135 mL) was mixed with the sterilized filtrate and the solution was incubated in an incubator-shaker (SKA KS4000) at 60 °C and 100 rpm for 72 hours. Hydrolyzed glucan filtrate was thus prepared.
Yeast (S. cerevisiae) cream (Bom Retiro mill, Brazil) was washed by suspending the cream in tap wafer (2.4 L, optical density of 65 at 600 nm) and then centrifuging the yeast cream for 5 minutes using a LEGEND XTR centrifuge
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PCT/US2015/017644 at 4500 g. After decanting the supernatant, the yeast were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 4.5 by addition of 5 wt% sulfuric acid and the optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 at
600 nm by addition of tap water. The adjusted yeast cream (1.5 L) was added to a 7.5-L BIOFLO310 fermenter vessel. The fermenter was set to maintain temperature at 30 °C, agitation at 100 rpm, and pH at 4.5 using 4 M aqueous ammonium hydroxide or 5 wt% aqueous sulfuric acid.
A feed solution containing yeast extract (10 g/L), peptone (20 g/L), and 10 200 g/L of sugars from the hydrolyzed filtrate was prepared and sterilized using a
PHOENIX AV-250 Plus autoclave at 121 °C for 15 minutes. The feed solution was allowed to cool to 25 °C (room temperature) before the fermentation began. The sterilized feed solution (3.5 L) was added to the fermenter over approximately 5 hours at a rate of 684 mL/hr, and the fermentation was allowed to proceed for 22 hours.
Periodic samples were taken during the fermentation and analyzed for optical density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL3 refractometer, and sugar and ethanol concentrations by HPLC (General Methods). These results are summarized in Table 13.
Table 13
Feed and Time Course Fermentation Profiles for the First Ethanol Fermentation
Using Hydrolyzed Glucan Filtrate
Time (hr) Sucrose Leucrose Glucose Fructose BP2 DP3+ Total Sugar EtOH
Feed 7 4 65 97 11 3 186 0
0 0 0 0 0 - - - 7
1 <1 1 <1 <1 - - - 20
2 <1 3 <1 <1 - - - 32
3 <1 4 <1 <1 - - - 40
4 <1 4 <1 <1 - - - 49
5 <1 4 <1 <1 - - - 53
6 <1 5 <1 <1 - - - 55
8 <1 5 <1 <1 - - - 57
22 <1 5 <1 <1 8 3 16 54
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Concentrations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-22 hours) are listed.
When the fermentation was over, the yeast cells were separated by centrifugation using a LEGEND XTR centrifuge at 4500 g for 5 minutes. After decanting the supernatant, the yeast cells were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 2 by addition of 5 wt% sulfuric acid. The optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 at 600 nm by addition of tap water. Two additional fermentation cycles, each using fresh feed, were performed using recycled yeast cells from the previous fermentation following the same conditions described above. The fermentation results obtained using first-time and second-time recycled yeast cells are provided in Tables 14 and 15, respectively.
Table 14
Feed and Time Course Fermentation Profiles Using the First Recycle of Yeast
Cells with Hydrolyzed Giucan Filtrate
Time (hr) Sucrose Leucrose Glucose Fructose DP2 DP3+ Total Sugar EtOH
Feed 7 4 69 104 7 4 194 0
0 <1 0 <1 <1 - - - 10
1 <1 7 <1 <1 - - - 25
4 <1 5 <1 <1 - - - 39
4 <1 4 <1 <1 - - - 45
5 <1 5 <1 <1 - - - 51
6 <1 5 <1 <1 - - - 57
6,2 <1 5 <1 <1 - - - 60
7 <1 5 <1 <1 - - - 59
21 <1 5 <1 <1 9 5 19 58
Concen rations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-21 hours) are listed.
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Table 15
Feed and Time Course Fermentation Profiles Using the Second Recycle of Yeast
Cells with Hydrolyzed Glucan Filtrate
Time (hr) Sucrose Leucrose Glucose Fructose DP2 DPS* Tote! Sugar EtOH
Feed 7 4 70 105 7 5 197 0
0 0 0 0 0 - - - 10
1 <1 7 <1 <1 - 25
3.5 <1 5 <1 <1 - 39
4 <1 4 <1 <1 - 45
5 <1 5 <1 <1 - - - 51
6 <1 5 <1 <1 - - - 57
6.2 <1 5 <1 <1 - 60
7 <1 5 <1 <1 - - - 59
21 <1 5 <1 <1 9 5 19 58
Concentrations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-21 hours) are listed.
All of the fermentations were essentially complete within about six hours of initiating fermentation, and resulted in ethanol titers of 57-60.0 g/L. Comparing these fermentations with those in Example 7 demonstrates that hydrolyzing a glucan filtrate before subjecting it to fermentation results in faster and greater ethanol yields than those obtained from fermentations using non-hydrolyzed glucan filtrate.
Thus, fermenting a glucan filtrate in which the leucrose and oligosaccharide byproduct components have been saccharified results in increased ethanol yields at faster rates. This saccharification can be done using a transglucosidase, for example.
EXAMPLE 9
Simultaneous Saccharification and Fermentation of a Glucan Filtrate Solution
This example discloses that simultaneous saccharification and fermentation of a feed containing glucan filtrate can result in enhanced fermentation properties.
Yeast (S. cerevistae) cream (Bom Retiro mill, Brazil) was washed by suspending the cream in tap water (2.4 L, optical density of 65 at 600 nm) and
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PCT/US2015/017644 then centrifuging the yeast cream for 5 minutes using a LEGEND XTR centrifuge at 4500 g. After decanting the supernatant, the yeast cells were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 4.5 by addition of 5 wt% sulfuric acid and the optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 at 600 nm by addition of tap water. The adjusted yeast cream (1.5 L) was added to a 7.5-L BIOFLO310 fermenter vessel. The fermenter was set to maintain temperature at 30 °C, agitation at 100 rpm, and pH at 4.5 using 4 M aqueous ammonium hydroxide or 5 wt% aqueous sulfuric acid.
A feed solution containing yeast extract (10 g/L), peptone (20 g/L), and 200 g/L of sugars from a glucan filtrate was prepared and sterilized using a PHOENIX AV-250 PLUS autoclave at 121 °C for 15 minutes. The feed solution was allowed to cool to 25 °C (room temperature) before the fermentation began. TG L-2000 transglucosidase enzyme extract as described in Table 4 (1% v/v) was added to the sterilized feed solution immediately before adding the solution to the fermenter. The feed solution (3.5 L) containing TG L-2000 enzyme was added to the fermenter over approximately 5 hours at 684 mL/hr, and the fermentation was allowed to proceed for 48 hours.
Periodic samples were taken during the fermentation and analyzed for optical density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL3 refractometer (Atago), and sugar and ethanol concentrations by HPLC (General Methods). These results are summarized in Table 16.
Table 16
Feed and Time Course Fermentation Profiles for Simultaneous Saccharification and Ethanol Fermentation of Glucan Filtrate
Time (hr) Sucrose Leucrose Glucose Fructose DP2 DP3+ Total Sugar EtOH
Feed 7 82 12 79 6 20 206 0
0 0 0 0 0 0 0 0 2
1 <1 13 <1 3 - - - 11
2 <1 21 <1 4 - - - 30
3 <1 21 <1 3 - - - 38
4 <1 20 <1 3 11 16 50 43
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5 <1 14 <1 2 - - - 45
5 <1 <1 <1 2 - - - 59
22 <1 <1 <1 1 - · 62
25 <1 <1 <1 <1 - - - 63
27 <1 <1 <1 <1 - - - 63
31 <1 <1 <1 <1 - - - 57
46 <1 <1 <1 <1 - · 57
48 <1 <1 <1 <1 1 11 12 62
Concentrations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-48 hours) are listed.
The fermentation was nominally complete in 6 hours, similar to the 5 fermentations where the filtrate was hydrolyzed prior to the fermentation step (Example 8), and gave a slightly superior titer of ethanol (62 g/L) compared to using unhydrolyzed filtrate (Example 7). In addition, almost all of the leucrose was consumed by 6 hours (compare Table 16 with Tables 13-15). In addition to adding a saccharifying enzyme, such as TG L-2000, to a feed containing glucan filtrate just prior to fermentation, similar results should be obtained if the saccharifying enzyme is added to the fermentation directly.
Thus, simultaneous saccharification and fermentation of a feed containing glucan filtrate can result in enhanced fermentation properties such as increased (i) consumption of glucan filtrate components (e.g., leucrose) and (ii) ethanol yield and rate of production.
EXAMPLE 10
This example discloses preparing various alpha-glucosidases in addition to those alpha-glucosidases (transglucosidase, glucoamylase, DIAZYME RDF
ULTRA) used in some of the foregoing Examples. These additional alphaglucosidases were tested for hydrolytic activity against oligosaccharides comprising alpha-1,5 glucosyl-fructose linkages or alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkages in Examples 11, 12, 15 and 16 provided below.
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Discovery of an Aspergillus clavatus alpha-glucosidase (Aclglul}
A strain of Aspergillus clavatus was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Aspergillus clavatus encodes an alpha-glucosidase and the sequence of this gene, called “Aclglul”, is provided in SEQ ID NO:4. The corresponding protein encoded by SEQ ID NQ:4 is provided in SEQ ID NO:5. Aclglul belongs to Glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO:5) has a signal peptide with a length of 19 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen etal., 2011, Nature Methods, 8:785-786). The presence of a signal sequence suggests that Aclglul is a secreted enzyme.__The amino acid sequence of the predicted mature form of Aclglul is set forth as SEQ ID NQ:6.
Expression of Aspergillus clavatus alpha-olucosidase Aclglu l
A synthetic Aclglul gene was cloned info pTrexSgM expression vector (described in U.S. Patent Appl. Pub!. No. 2011/0136197, incorporated herein by reference) and the resulting plasmid was designated as pJG294. The sequence of the Aclglul gene was confirmed by DNA sequencing.
Plasmid pJG294 was transformed into a quad deleted Trichoderma reesei strain (described in W005/001036) using a biolistic method (Te’o VS et al., J Microbiol Methods, 51:393-9, 2002). The protein, which was predicted to comprise SEQ ID NQ:6, was secreted into the extracellular medium and filtered culture medium was used to perform 8DS-PAGE and alpha-glucosidase activity assays to confirm enzyme expression.
Discovery of Neosartorya fischeri alpha-glucosidase Nfiglul
A strain of Neosartorya fischeri was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Neosartorya fischeri encodes an alpha-glucosidase and the sequence of this gene, called “Nfiglul, is provided in SEQ ID NO:7. The corresponding protein encoded by SEQ ID NO:7 is provided in SEQ ID NO:8.
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Nfigiu 1 belongs to Glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NQ:8) has a signal peptide with a length of 19 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et al., 2011, Nature Methods, 8:785-786). The presence of a signal sequence suggests that Nfigiu 1 is a secreted enzyme. The amino acid sequence of the predicted mature form of Nfigiul is set forth as SEQ ID NO: 9.
Expression of Neosartorya fischeri alpha-glucosidase Nfigiul
A synthetic Nfigiul gene was cloned info pTrex3gM expression vector (described in U.S. Patent Appl. Publ. No. 2011/0136197) and the resulting plasmid was designated as pJG295. The sequence of the Nfigiul gene was confirmed by DNA sequencing.
Plasmid pJG295 was transformed into a quad deleted Trichoderma reesei strain (described in W005/001036) using a biolisfic method (Te’o VS ef al., J Microbiol Methods, 51:393-9, 2002). The protein, which was predicted to comprise SEQ ID NO:9, was secreted into the extracellular medium and filtered culture medium was used to perform SDS-PAGE and alpha-glucosidase activity assays to confirm enzyme expression.
Discovery of Neurospora crassa alpha-glucosidase Ncrgiul
A strain of Neurospora crassa was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Neurospora crassa encodes an alpha-glucosidase and the sequence of this gene, called “Ncrgiul, is provided in SEQ ID NO:10. The corresponding protein encoded by SEQ ID NO: 10 is provided in SEQ ID NO:11. Ncrgiul belongs to Glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO:11) has a signal peptide with a length of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen etai., 2011, Nature Methods, 8:785-788). The presence of a signal sequence suggests that Ncrgiul is a secreted enzyme. The
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PCT/US2015/017644 amino acid sequence of the predicted mature form of Ncrglul is set forth as SEQ
ID NO:12.
Expression oi Neurospora crassa aloha-glucosidase Ncrglul
A synthetic Ncrglul gene was cloned into pTrexSgM expression vector (described in U.S. Patent Appl. Publ. No. 2011/0136197) and the resulting plasmid was designated as pJG296. The sequence of the Ncrglul gene was confirmed by DNA sequencing.
Plasmid pJG296 was transformed into a quad deleted Trlchoderma reesei strain (described in W005/001036) using a biolisfic method (Te’o VS ef al., J Microbiol Methods, 51:393-399, 2002). The protein, which was predicted to comprise SEQ ID NO: 12, was secreted into the extracellular medium and filtered culture medium was used to perform SDS-PAGE and alpha-glucosidase activity assays to confirm enzyme expression.
Discovery of Rasamsonia composticola alpha-glucosidase TauSecO98
A strain of Rasamsonia composticola was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Rasamsonia composticola encodes an alpha-glucosidase and the sequence of this gene, called “TauSecO98”, is provided in SEQ ID NO: 13. The corresponding protein encoded by SEQ ID NO: 13 is provided in SEQ ID NO:14. TauSecO98 belongs to Glycosyl hydrolase family 31 and contains an N-terminal CBM 20 domain based on a REAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO:14) has a signal peptide with a length of 22 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen etal., 2011, Nature Methods, 8:785-788). The presence of a signal sequence suggests that TauSecO98 is a secreted enzyme. The amino acid sequence of the predicted mature form of TauSecO98 is set forth as SEQ ID NQ:15.
Expression of Rasamsonia composticola alpha-glucosidase TauSecO98
A synthetic TauSecO98 gene was cloned into the Trlchoderma reesei expression vector pGXT (a pTTT-derived plasmid) by Generay Biotech Co.
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PCT/US2015/017644 (Shanghai, China) and the resulting plasmid was designated as pGX256TauSecO98. The sequence of the TauSecO98 gene was confirmed by DNA sequencing.
Plasmid pGX258-TauSecO98 was transformed into a quad-deleted Trichoderma reesei strain (described in W005/001036) using protoplast transformation (Te’o et al., J. Microbiol. Methods 51:393-399, 2002). Transformants were selected on a medium containing acetamide as a sole source of nitrogen (acetamide 0.6 g/L; cesium chloride 1.68 g/L; glucose 20 g/L; potassium dihydrogen phosphate 15 g/L; magnesium sulfate heptahydrate 0,6 g/L; calcium chloride dihydrate 0.6 g/L; iron (II) sulfate 5 mg/L; zinc sulfate 1.4 mg/L; cobalt (II) chloride 1 mg/L; manganese (II) sulfate 1.6 mg/L; agar 20 g/L; pH 4.25). Transformed colonies (about 50-100) appeared in about 1 week. After growth on acetamide plates, the spores of transformants were collected and transferred info new acetamide agar plates. After 5 days of growth on acetamide plates, 1 *108 spores were inoculated into 30 ml Glucose/Sophorose defined media in a 250-ml shake flask. The shake flask was shook at 28 °C for 5 days. Supernatants from these cultures were used to confirm expression (SDS PAGE) and activity of mature TauSecO98 enzyme (SEQ ID NO: 15).
Discovery of Rasamsonia composticoia alpha-glucosidase TauSecO99
A strain of Rasamsonia composticoia was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Rasamsonia composticoia encodes an alpha-glucosidase and the sequence of this gene, called TauSecO99”, is provided in SEQ ID NO: 16. The corresponding protein encoded by SEQ ID NO: 16 is provided in SEQ ID NO:17. TauSecO99 belongs to Glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO: 17) has a signal peptide with a length of 17 amino acids as predicted by SignalP version 4.0 (Nordahl Petersen et at., 2011, Nature Methods, 8:785-786). The presence of a signal sequence suggests that TauSecO99 is a secreted
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PCT/US2015/017644 enzyme. The amino acid sequence of the predicted mature form of TauSecO99 is set forth as SEQ ID NO: 18.
Expression of Rasamsonia composticola alpha-glucosidase TauSecO99
A synthetic TauSecO99 gene was cloned into the Trichoderma reesei expression vector pGXT (a pTTT-derived plasmid) by Generay Biotech Co. (Shanghai, China) and the resulting plasmid was designated as pGX256TauSecO99. The sequence of the TauSecO998 gene was confirmed by DNA sequencing.
Plasmid pGX256-TauSecO99 was transformed into a quad-deleted Trichoderma reesei strain (described in W005/001036) using protoplast transformation (Te’o et al., J. Microbiol. Methods 51:393-399, 2002). Transformants were selected on a medium containing acetamide as a sole source of nitrogen (acetamide 0.6 g/L; cesium chloride 1.68 g/L; glucose 20 g/L; potassium dihydrogen phosphate 15 g/L; magnesium sulfate heptahydrate 0.6 g/L; calcium chloride dihydrate 0.6 g/L; iron (II) sulfate 5 mg/L; zinc sulfate 1.4 mg/L; cobalt (II) chloride 1 mg/L; manganese (II) sulfate 1.6 mg/L; agar 20 g/L; pH 4.25). Transformed colonies (about 50-100) appeared in about 1 week. After growth on acetamide plates, the spores of transformants were collected and transferred into new acetamide agar plates. After 5 days of growth on acetamide plates, 1><10s spores were inoculated into 30 ml Glucose/Sophorose defined media in a 250-mL shake flask. The shake flask was shook at 28 °C for 5 days. Supernatants from these cultures were used to confirm expression (SDS PAGE) and activity of mature TauSecO99 enzyme (SEQ ID NO: 18),
Sequences of Bifidobacterium longum alpha-glucosidase BloGlul
An alpha-glucosidase gene, “BloGlul”, was identified from Bifidobacterium Songum subsp. longum JDM301. The nucleic acid sequence for the BloGlul gene (SEQ ID NO:19, GENBANKAcc. No. NC014169.1, complement sequence from positions 140600 to 142414) and the amino acid sequence of the hypothetical protein (SEQ ID NO:20) encoded by SEQ ID NO: 19 were found in GENBANK Acc. No. YP„003660432.1.
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Expression of Bifidobacterium longum alpha-glucosidase BioGiul
The DNA sequence encoding the entire BioGiul protein (SEG ID NO:20) was optimized for expression in B. subtiiis, then synthesized (yielding SEG ID NO:21) and inserted into the p3JM plasmid by Generay Biotech Co. (Shanghai, China), resulting in p3JM-BloGlu1. The p3JM-BloGlu1 plasmid contains an aprE promoter to drive expression of the optimized BioGiul sequence (SEG ID NO:21),
Plasmid p3JM-BloGlu1 was used to transform B. subtiiis cells (degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with MBD medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCb) to express BloGlul protein (SEQ ID NO:20).
Sequences of Bifidobacterium longum alpha-glucosidase BloGlu2
An alpha-glucosidase, BloGlu2, was identified from Bifidobacterium longum. The amino acid sequence (SEQ ID NO:22) of BloGiu2 was found in the NCBI database (GENBANK Acc. No. WP__007054865.1).
Expression of Bifidobacterium longum alpha-glucosidase BloGlu2
A DNA sequence encoding BloGlu2 protein was optimized for expression in B. subtiiis, then synthesized (yielding SEQ ID NO:23) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in p3JM-BloGlu2. SEQ ID NO:23 encodes the amino acid sequence of SEQ ID NO:24. The p3JM-BioGlu2 plasmid contains an aprE promoter to drive expression of the optimized BloGlu2 sequence (SEQ ID NO:23).
Plasmid p3JM-BloGlu2 was used to transform B. subtiiis cells (degUHy32, AnprB, Avpr, Aepr,,AscoC, AwprA, Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with MBD
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PCT/US2015/017644 medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCI?) to express BloGlu2 protein (SEG ID NO:24).
Sequences of Bifidobacterium longum alpha-glucosidase BloGluS
An alpha-glucosidase gene, “BloGluS”, was identified from Bifidobacterium longum subsp. longum F8. The nucleic acid sequence for the BloGluS gene (SEQ ID NO:25, GENBANK Acc. No. NC_021008.1, positions 2130627 to 2132441), and the amino acid sequence of the hypothetical protein (SEQ ID NQ:26) encoded by SEQ ID NO:25 were found in GENBANK Acc. No. γρ 007768249.1.
Expression of Bifidobacterium longum alpha-glucosidase BloGluS
The DNA sequence encoding the entire BloGluS protein (SEQ ID NQ:26) was optimized for expression in B. subtilis, then synthesized (yielding SEQ ID NQ:27) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in p3JM-BloGlu3. The p3JM-BloGlu3 plasmid contains an aprE promoter to drive expression of the optimized BloGluS sequence (SEQ ID NO:27).
Plasmid p3JM-BloGlu3 was used to transform B. subtilis cells (degUHy32, AnprB. Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with MBD medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCb) to express BloGluS protein (SEQ ID NO:26).
An alpha-glucosidase, BpsGlul, was identified from Bifidobacterium pseudolongum. The amino acid sequence (SEQ ID NO:28) of BpsGlul was found in the NCBI database (GENBANK Acc. No. WP 022858408.1).
Expression of Bifidobacterium pseudolonqum alpha-qlucosidase BpsGlul
A DNA sequence encoding BpsGlul protein was optimized for expression in B. subtilis, then synthesized (yielding SEQ ID NO:29) and inserted into the
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PCT/US2015/017644 p3JM plasmid by Generay Biotech Co., resulting in p3JM-BpsGlu1. SEQ ID NO:29 encodes the amino acid sequence of SEQ ID NO:30. The p3JM-BpsGlu1 plasmid contains an aprE promoter to drive expression of the optimized BpsGlul sequence (SEQ ID NO:29)
Plasmid p3JM-BpsGSu1 was used to transform B. subtilis cells (degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA. Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with MBD medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCfe) to express BpsGlul protein (SEQ ID NOGO).
An alpha-glucosidase gene, “BthGlul”, was identified from Bifidobacterium thermophilum RBL67. The nucleic acid sequence of the BthGlul gene (SEQ ID NO:31, GENBANK Acc. No. NC_020546.1, positions 150690 to 152495), and the amino acid sequence of the hypothetical protein (SEQ ID NO:32) encoded by SEQ ID NO:31 were found in GENBANK Acc. No. YP__007592840.1.
Expression of Bifidobacterium thermophilum alpha-glucosidase BthGlul
The DNA sequence encoding the entire BthGlul protein (SEQ ID NO:32) was optimized for expression in B. subtilis, then synthesized (yielding SEQ ID NOGS) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in p3JM-BthGlu1. The p3JM-BthGSu1 plasmid contains an aprE promoter to drive expression of the optimized BthGlul sequence (SEQ ID NOGS).
Plasmid p3JM-BlhGlu1 was used to transform B. subtilis cells (degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with MBD medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCb) to express BthGlul protein (SEQ ID NOG2).
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Sequences of Bifidobacterium breve alpha-glucosidase BbrGlu2
An alpha-glucosidase, BbrGlu2, was identified from Bifidobacterium breve.
The amino acid sequence (SEQ ID NO:34) of BbrGlu2 was found in the NCBI database (GENBANK Acc. No. WP„003827971.1).
A DNA sequence encoding BbrGlu2 protein was optimized for expression in B. subtilis, then synthesized (yielding SEQ ID NO:35) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in pSJM-BbrGlu2. SEQ ID NO:35 encodes the amino acid sequence of SEQ ID NQ:36. The p3JM-BbrGSu2 plasmid contains an aprE promoter to drive expression of the optimized BbrGiu2 sequence (SEQ ID NO:35)
Plasmid p3JM-BbrGlu2 was used to transform B. subtiiis cells (degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with MBD medium (a MGPS-based defined medium supplemented with an additional 5 mM CaCb) to express SEQ ID NO:38.
Sequences of Bifidobacterium breve alpha-glucosidase BbrGluS
An alpha-glucosidase gene, “BbrGiuS”, was identified from Bifidobacterium breve ACS-071-V-Sch8b, The nucleic acid sequence of the BbrGiuS gene (SEQ ID NO:37, GENBANK Acc. No. NC..017218.1, complement of sequence from positions 2241075 to 2242895), and the amino acid sequence of the hypothetical protein (SEQ ID NO:38) encoded by SEQ ID NO:37 were found in GENBANK Acc. No. YPJD05583701,1.
Expression of Bifidobacterium breve alpha-glucosidase BbrGiuS
The DNA sequence encoding the entire BbrGiuS protein (SEQ ID NG:38) was optimized for expression in B. subtiiis, then synthesized (yielding SEQ ID NO:39) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in
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PCT/US2015/017644 p3JM-BbrGSu5. The p3JM-BbrGlu5 plasmid contains an aprE promoter to drive expression of the optimized BbrGiu5 sequence (SEG ID NO:39).
Plasmid p3JM-BbrGlu5 was used to transform β. subfflis cells (degUHy32, AnprB, Avpr. Aepr, AscoC, AwprA, Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with MBD medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCI2) to express BbrGluS protein (SEG ID NO:38).
Purification of alpha-glucosidases from expression cultures
AcIGIul and NcrGlul
Both AcIGIul (SEQ ID NO:6) and NcrGlul (SEG ID NO:12) alphaglucosidases were purified using two chromatography steps. For each purification, the crude broth from the shake flask was concentrated, after which ammonium sulfate was added to a final concentration of 2 M. The solution was loaded onto a 50-mL phenyl HP column pre-equilibrated with 20 mM Tris pH 8.0, 2 M ammonium sulfate. The target protein (SEG ID NO:6 or SEQ ID NO: 12) was eluted from the column with 1 M ammonium sulfate, 20 mM Tris pH 8.0. Respective fractions were pooled, concentrated and buffer-exchanged into 20 mM Tris pH 8.0 (buffer A), using a VIVAFLOW 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to a 40-mL Q HP column preequilibrated with buffer A. The target protein was eluted from the column with 0,3 M NaCI in buffer A. The fractions containing target protein were then pooled and concentrated using 10K AMICON ULTRA-15 devices, and stored in 40% glycerol at -20 °C until usage.
NfiGlul
NfiGlul alpha-glucosidase (SEG ID NO:9) was purified using two hydrophobic interaction chromatography steps. The crude broth from the shake flask was concentrated, after which ammonium sulfate was added to a final concentration of 1 M. The solution was loaded onto a 50-mL phenyl HP column
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PCT/US2015/017644 pre-equilibrated with 20 mM Tris pH 8.0, 1 M ammonium sulfate. The target protein (SEQ ID NO:9) flowed through the column. Flow-through fractions were pooled, after which ammonium sulfate was added to a final concentration of 2 M. The solution was loaded onto the same phenyl HP column pre-equilibrated with 20 mM Tris pH 8.0, 2 M ammonium sulfate. The target protein was eluted from the column with 1 M ammonium sulfate, 20 mM Tris pH 8.0. The fractions containing target protein were then pooled and concentrated using 10K AM ICON ULTRA-15 devices, and stored in 40% glycerol at -20 °C until usage.
TauSecO98 and TauSecO99
Both TauSecO98 (SEQ ID NOUS) and TauSecO99 (SEQ ID NO:18) alphaglucosidases were purified via hydrophobic interaction chromatography. For each purification, ammonium sulphate was added to about 180 mL of concentrated crude broth from a 7-L fermenter to a final concentration of 1 M. This solution was then loaded onto a 50-mL HIPREP phenyl-FF Sepharose column (GE Healthcare) pre-equilibrated with 20 mM sodium acetate pH 5.0,1 M ammonium sulphate (buffer A). After washing with the same buffer with three column volumes (CVs), the column was eluted stepwise with 75%, 50% and 0% buffer A using three CVs each, followed by two CVs of MILLIQ HsO. All fractions were analyzed by SDS-PAGE. The target protein (SEQ ID NQ:15 or SEQ ID NO: 18) was mainly present in the flow-through fraction, which was concentrated and buffer-exchanged to remove excess ammonium sulfate using 10 KDa AMICQN ULTRA-15 devices. The final product, which was greater than 90% pure, was stored in 40% glycerol at -80 °C until usage.
BloGlul, BloGlu2 and BloGlu3
BloGlul (SEQ ID NQ:20), BloGlu2 (SEQ ID NO:24) and BloGluS (SEQ ID NO:26) alpha-glucosidases were all purified in three steps. For each purification, the crude broth from a 1-L DASGIR fermenter was concentrated, after which ammonium sulfate was added to 60% saturation. The solution was stirred at 4 °C for 1 hr, and then centrifuged at 8000 X g for 30 min. The resulting pellet was re-suspended in 20 mM Tris pH 8.0 (buffer A). Ammonium sulfate was added to the resulting solution to a final concentration of 1 M; this preparation was then
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PCT/US2015/017644 loaded onto a 40-mL HiPrepPhenyl FF column pre-equilibrated with 20 mM Tris pH 8.0, 1 M ammonium sulfate (buffer B). After washing, the column was eluted stepwise with 75%, 50%, and 0% buffer B and H2O in three column volumes each. All fractions were analyzed using SDS-PAGE and activity assays. The fractions containing target protein (SEG ID NO:20, SEG ID NO:24, or SEG ID NO:26) were pooled, concentrated and subsequently loaded onto a HiLoad™ 28/60 Superdex™75 column pre-equilibrated with 20 mM sodium phosphate pH 7.0, 0.15 M NaCI. Flow-through fractions containing the target protein were then pooled and concentrated using 10K AMICON ULTRA-15 devices, and stored in 40% glycerol at -20 °C until usage.
BpsGlul and BthGlul
Both BpsGlul (SEG ID NG:30) and BthGlul (SEQ ID NO:32) alphaglucosidases were purified in two steps. For each purification, the crude broth from a 1-L DASGIP fermenter was concentrated, afterwhich ammonium sulfate was added to 80% saturation. The solution was stirred at 4 °C for 1 hr, and then centrifuged at 8000 X g for 30 min. The resulting pellet was re-suspended in 20 mM Tris pH 8.0 (buffer A). Ammonium sulfate was added to the resulting solution to a final concentration of 1 M; this preparation was then loaded onto a 40-mL HiPrep™ Phenyl FF column pre-equilibrated with 20 mM Tris pH 8.0, 1 M ammonium sulfate (buffer B). After washing, the column was eluted stepwise with 75%, 50%, and 0% buffer B and H2O in three column volumes each. All fractions were analyzed using SDS-PAGE and activity assays. The target protein (SEG ID NO:30 or SEG ID NO:32) was present in the eluate from the 0% buffer B elution step; this eluate was pooled and concentrated using 10K AMICON ULTRA-15 devices. The final product, which was greater than 95% pure, was stored in 40% glycerol at -20 °C until usage.
BbrGlu2 and BbrGluS
Both BbrGlu2 (SEG ID NO:36) and BbrGluS (SEQ ID NO:38) alphaglucosidases were purified in four steps. For each purification, the crude broth from a 1-L DASGIP fermenter was concentrated, after which ammonium sulfate was added to 60% saturation. The solution was stirred at 4 °C for 1 hr, and then
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PCT/US2015/017644 centrifuged at 8000 X g for 30 min. The resulting pellet was re-suspended in 20 mM HEPES pH 7.0 (buffer A). Ammonium sulfate was added to the resulting solution to a final concentration of 1 M; this preparation was then loaded onto a HiPrep'M Phenyl FF column pre-equilibrated with 20 mM HEPES pH 7.0, 1 M ammonium sulfate. The target protein (SEQ ID NQ:36 or SEQ ID NQ:38) was eluted from the column with 0.5 M ammonium sulfate. Respective fractions were pooled, concentrated and buffer-exchanged into buffer A using a VSVAFLOW 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to a HIPrepiMQ FF 16/10 column pre-equilibrated with buffer A. Target protein was eluted from the column with a linear gradient of 0-0.5 M NaCI in buffer A. Fractions containing target protein were pooled, concentrated and subsequently loaded onto a HILoad™ 26/60 Superdex™ 75 column pre-equilibrated with 20 mM HEPES pH 7.0, 0.15 M NaCI. The fractions containing target protein were then pooled and concentrated using 10K AMICQN ULTRA-15 devices, and stored in 40% glycerol at -20 °C until usage.
Thus, various additional alpha-glucosidases were expressed and purified. These alpha-glucosidases were tested for hydrolytic activity against alpha-1,5 glucosyi-fructose linkages and alpha-1,3 and/or alpha-1,6 glucosyl-glucose linkages in Examples 11, 12, 15 and 16 provided below.
EXAMPLE 11
Testing Alpha-Glucosidases for Hydrolytic Activity Against Various Glycosidic
This example discloses testing whether alpha-glucosidases have hydrolytic activity beyond that previously associated with this class of enzymes (EC 3.2.1.20), Alpha-glucosidases from Example 10 were shown to have hydrolytic activity against alpha-1,5 glucosyi-fructose linkages and alpha-1,3 and alpha-1,6 glucosyl-glucose linkages.
Substrate Specificity of Alpha-Glucosidases
The substrate specificity of each alpha-glucosidase disclosed in Example 10 was assayed based on the release of glucose from a particular substrate (isomaltose, maltose, panose, Ieucrose, or nigerose) when the substrate was
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PCT/US2015/017644 incubated with alpha-glucosidase. The rate of glucose release was measured using a coupled glucose oxidase/peroxidase (GOX/HRP) method (1980, Anal. Biochem. 105:389-397). Glucose release was quantified as the rate of oxidation of 2,2'-azino-bis 3-ethylbenzotbiazoline-6-sulfonic acid (ABTS) by peroxide that was generated from coupled GOX/HRP enzymes reacted with glucose.
Individual substrate solutions were prepared by mixing a 9 mL solution of substrate (1% in water, w/v) with 1 mL of 0.5 M pH 5.0 sodium acetate buffer and 40 pL of 0.5 M calcium chloride in a 15-mL conical tube. Coupled enzyme (GOX/HRP) solution with ABTS was prepared in 50 mM sodium acetate buffer (pH 5.0), with the final concentrations of 2.74 mg/mL ABTS, 0.1 U/mL HRP, and 1 U/mL GOX. Serial dilutions of individual alpha-giucosidase samples and glucose standard were prepared in MILLIG water. For nigerose, alphagiucosidase samples were tested with only one dosage at 10 ppm due to a limited stock of substrate solutions. Each alpha-giucosidase sample (10 pL) was transferred into a new microtiter plate (Coming 3641) containing 90 pL of substrate solution pre-incubated at 50 °C for 5 min at 600 rpm. Reactions were carried out at 50 °C for 10 min (for isomaltose, maltose, panose, and nigerose substrates), or for 60 min (for leucrose substrate) with shaking (600 rpm) in a THERMOMIXER (Eppendorf). 10 pL of each reaction mix, as well as 10 pL of serial dilutions of glucose standard, were then quickly transferred to new microtiter plates (Corning 3641), respectively, to which 90 pL of ABTS/GOX/HRP solution was then added accordingly. The microtiter plates containing reaction mixes were immediately measured at 405 nm at 11 second intervals for 5 min using a SOFTMAX PRO plate reader (Molecular Devices). The output was the reaction rate, Vo, for each enzyme concentration. Linear regression was used to determine the slope of the plot Vo vs. enzyme dose. The specific activity of each alpha-giucosidase was calculated based on the glucose standard curve using Equation 1:
Specific Activity (Unit/mg) ~ Slope (enzyme) / slope (std) * 1000 (1), where 1 Unit = 1 pmol glucose / min.
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For nigerose, the value of the reaction rate with enzyme dosage at 10 ppm was directly used to indicate enzyme activity.
Using the foregoing method, the specificity of each alpha-glucosidase was determined against each substrate. The activities of an oligo-1,6-glucosidase (purchased from Megazyme, see Table 4) and a transglucosidase (TG L-2000, see Table 4) against each substrate were also measured. The results of this analysis are provided in Table 17.
Table 17
Activity of Various Alpha-Glucosidases Against Different Substrates
Enzyme SEQ ID NO. Enzyme Activity (U/mg) as Measured on:
isomaitose Maltose Panose Leucrose Nigerose3
Oligo-1,6glucosidase 118.2 0.0 54.3 1.3 19.6
TG L-2000 1 194.0 235.6 127,7 68.9 254.0
AcIGIul 6 255.7 401.9 180.9 113.7 315,1
NfiGlul 9 521.2 360.0 126.9 89,4 264.3
NcrGiul 12 282.7 34,9 15.9 61.6 200.4
TauSecO98 15 54.9 123.8 23.8 1,8 305.8
TauSecO99 18 244.0 97.7 50.8 70.6 184.8
BioGiul 20 71,1 66.9 23.1 2.5 165.0
BloGiu2 24 85.9 86,7 19.9 3.5 217.9
BloGlu3 26 120.1 175.5 31.4 9,0 272.6
BspGIul 30 64.2 247.9 60.8 27.3 254.6
BthGlul 32 108.3 93.3 21.1 68,4 128.5
BbrGlu2 36 106.6 187.5 26.9 6.1 258.8
BbrGluS 38 925.8 0.0 279.7 2,8 22,1
a Each enzyme was used at one dosage (10 ppm) against nigerose.
Interestingly, if was found that alpha-glucosidases, besides exhibiting hydrolytic activity against alpha-1,4 glucosyi-glucose linkage (maltose), also exhibit hydrolytic activity against alpha-1,6 glucosyi-glucose linkage (isomaltose),
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Thus, alpha-glucosidases have hydrolytic activity beyond that previously associated with EC 3.2.1.20 enzymes. Specifically, alpha-glucosidases have hydrolytic activity against alpha-1,5 glucosyl-fructose linkages and alpha-1,3 and alpha-1,8 glucosyl-glucose linkages.
EXAMPLE 12
Hydrolysis of Leucrose and Oligosaccharides in Glucan Reaction Filtrate Using
Alpha-Glucosidase
This Example describes using alpha-glucosidase to hydrolyze leucrose and other oligosaccharides present in filtrate obtained from a glucan synthesis reaction. Specifically, the effect of alpha-glucosidases disclosed in Example 10 on the hydrolysis of leucrose and oligosaccharides DP2, DPS and HS (higher sugars, DP4+) in a filtrate of an insoluble glucan (poly alpha-1,3-glucan) synthesis reaction was studied.
isolation and Analysis of Oligosaccharides for Testing Against AlphaGlucosidase Activity
First, a concentrated filtrate of a glucan synthesis reaction was prepared as per Example 1.
Briefly, oligosaccharides were isolated from the concentrated filtrate by chromatographic separation, and analyzed for glycosidic linkage profile. Chromatographic separation employing a strong acid cation-exchange resin was used to isolate the oligosaccharide fraction of the concentrated filtrate. The physical parameters of the column used for this separation were as follows: FINEX CS11GC, #227 resin; Na* ion form; 5% divinyl benzene (crosslinking); 0.34 mm particle size; 1.64 m bed length; 0.093 m column diameter.
In more detail, the concentrated sugar solution (i.e., concentrated filtrate) described in Table 3 was filtered and diluted to 25 g dry solids/100 g solution using tap water. Prior to addition of this sugar solution to the column resin, the resin was washed with six bed volumes (BV) of sodium chloride solution (three BV at 10 wt% sodium chloride followed by three BV at 5 wt% sodium chloride) to
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Table 18
Sucrose Leucrose Glucose Fructose DP2 DP3 DP4 DPS DP6 DP7 Total
g/L 0,0 2.5 0.0 0.7 31.5 75.9 101.8 82.1 26.9 15.3 316.7
wt% 0.0 0.8 0.0 0.2 9.9 23.9 32.1 19.6 8.5 4.8 100
Primary Screening of Aloha-Glucosidases on Glucan Oligomer Hydrolysis
The activities of eleven different alpha-glucosidases (Example 10), as well 20 as the activities of two benchmark enzymes, oligo-1,6-glucosidase (purchased from Megazyme) and transglucosidase (TG L-2000), were individually evaluated against the purified oligosaccharide fraction prepared above (Table 18). Each alpha-glucosidase (dosed at 1 mg/mL) was incubated in a solution containing oligosaccharide substrates (2.9% dry solids) and 2 mM calcium chloride at pH
5.0 at 60 °C. Each reaction was quenched after 24 hours of incubation by adding pL of 0.5 M NaOH.
The oligosaccharide/monosaccharide contents of the quenched reactions were determined as follows. A sample of each reaction was diluted 5-fold in water for HPLC analysis. HPLC separation was done using an Agilent 1200
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Table 19
Analysis Glucan Filtrate Oligosaccharides Following Treatment with Alpha10 Glucosidase
Enzyme ID NO DP7% DP8% DP5% DP4% DP3% DP2% DP1%
Oligo-1,6qlucosidase 0.0 !!!!!l!l!!!l! 8.7 iiiiiii iiiiiiiii® 12.2 28./
TG L-2000 1 0.0 lllllill!!!!! iiiiiii! llllill! llllill! iiiiiii!
Nfiglul 9 llllllll!! lllllill!!!! 1111111111 11111111111 111111111 11111!!!!!
Ncrgiul 12 1111111111 lllllill!!!!! lllllill! llllllll! 11111!!!!! 70 Sil
TauSecOSS 15 llllllll!! lllllill!!!!! llllllll!! 15.3 llllllll! llllllll! S0.2
TauSecO99 18 !!!!!l!l!!!l! lllllill!!!!! llllill! 15.7 111111111 llllllll! iiiiiiii
BioGlul 20 0.0 lllllill!!!!! 8.7 36.3 35.8 llllill! 21.3
BloGluZ 24 0.0 0.7 8.6 31.2 34.9 14.0 10.7
B!oGlu3 26 0.0 0.6 8.0 28.4 33.5 13.9 15.6
BspGIul 30 0.0 0.5 5.2 15.4 15.2 8.2 54.5
BthGiul 32 0.0 0.0 13.0 12.6 2.0 1.0 70.5
BbrGiuZ 36 0.0 0.0 21.5 30.7 24.6 0.0 23.3
BbrGluS 38 0.0 0.0 8.1 23.8 12.1 15.5 40.5
Blank 0.0 0.0 16.2 38.6 30.4 llllill! 14.8
As indicated with shading in Table 19, the oligosaccharide content of the reactions generally shifted toward smaller sized sugars, in comparison with the control reaction (“Blank) in which there was no enzyme. These results indicate that alpha-glucosidase can be used to hydrolyze oligosaccharides comprised within a glucan synthesis reaction and a fraction thereof. Also, given the linkage profile of the oligosaccharides (Examples 3 and 4), and the activity of alphaglucosidase against various glycosidic linkages in addition to alpha~1,4 linkages (Example 11), it is apparent that alpha-glucosidase can be used to break down
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Products of Glucan Synthesis Reactions
Reactions were prepared comprising one or two alpha-glucosidases and a concentrated filtrate obtained from a poly alpha-1,3-glucan synthesis reaction (Table 3). Alpha-glucosidase reactions were dosed with enzyme at 4 ppm, or for blends, each enzyme was used at a 1:1 ratio with a final dosage of 4 ppm. The concentrated filtrate was loaded in each reaction at 10% dry solids. Each reaction further comprised 2 mM calcium chloride at pH 5.0, and was carried out at 80 °C or 65 °C. The reactions were quenched by adding 50 pL of 0.5 M NaOH after a 23-hour incubation.
The oligosaccharide/monosaccharide contents of the quenched reactions were determined as follows. A sample of each reaction diluted 25-fold in water for HPLC analysis. HPLC separation was done using an Agilent 1200 series HPLC system with an AMINEX HPX-42A column (300 mm x 7.8 mm) at 85 °C. The sample (10 pL) was applied to the HPLC column and separated with an isocratic gradient of MSLLI-Q water as the mobile phase at a flow rate of 0.8 mL/min. Oligosaccharide products were detected using a refractive index detector. The numbers provided in Table 20 below reflect the average of peak area percentages (from duplication of each sample) of each DPn as a fraction of the total. The results provided in Table 20 generally confirm the activity of certain alpha-glucosidases as discussed above regarding the results provided in Table 19.
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Thus, alpha-glucosidase can be used to hydrolyze leucrose and other oligosaccharides present in a fraction (e.g., filtrate) obtained from a glucan synthesis reaction, such as a poly alpha-1,3-glucan synthesis reaction.
EXAMPLE 13
Isolation of Oiigomer/Leucrose Fraction from gtf-S/MUT3325 Reaction
Sucrose (4.50 kg) was dissolved in distilled deionized water to a final total volume of 9.5 L and the resulting solution was heated with stirring at 80 °C for 5 minutes and then cooled to 47 °C. With stirring, 500 grams of a crude extract containing 0,6 g/L of gtf-S enzyme (GTF0459, SEQ ID NO:42) and 15.0 mL of a crude extract containing 10 g/L of mutanase (MUT3325, SEQ ID NO:47) was added with stirring (see General Methods for enzyme preparations). The pH of the resulting mixture was immediately adjusted to between pH 5.5 to pH 6.0 by slowly adding a 1:10 (v/v) dilution of 37 wt% HCI with stirring. The reaction temperature and pH were maintained at 47 °C and pH 5,5-6.0, respectively, until sucrose conversion was >95% per HPLC analysis, after which the reaction mixture was immediately adjusted to pH 7.0 to 7.5 and heated to 90 °C for 20 min, then cooled to 25 °C for immediate filtration to remove particulates and precipitate. The resulting filtrate was held at 5 °C prior to IEX/SEC column chromatography using the following resin and conditions: FINEX CS 11 GC SAC in Caz+ form, column i.d = 9.3 cm, resin bed height 1.58 m, T~ 70 °C, flow rate = 51 mL/min, linear flow rate = 0.44 m/h, feed size = 0.6 L = 171 g, feed RI-DS = 25.1 g/100 g, sample interval = 3 min. The column fractions collected between 30 min and 67 min were combined, concentrated by evaporation to 66% dissolved solids and analyzed by HPLC as described in the General Methods. Table 21 indicates the oligosacharide and monosaccharide components of the isolated fraction thus prepared.
Table 21
Analysis of Oiigomer/Leucrose Fraction from gtf-S/MUT3325 Reaction
DP7+ (%DS) DP6 (%DS) DPS (%DS) DP4 (%DS) DP3 (%DS) DP2 (%DS) sucrose (%DS) leucrose (%DS) glucose (%DS) fructose (%DS)
0 llllllll llllllll 7.2 16.2 12.7 13,5 30,7 78
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In this Example, a glucan synthesis reaction was used to produce at least one soluble alpha-glucan product. This soluble product resulted from the concerted action of both a glucosyltransferase (GTF0459, SEG ID NO:42) and an alpha-glucanohydrolase (MUT3325, SEQ ID NO:47) that were both present In the glucosyltransferase reaction. This Example also demonstrated the preparation of a chromatographic fraction from the glucan synthesis reaction.
This fraction was used in Examples 15 and 16 below to test the activity of alphaglucosidases thereupon.
EXAMPLE 14
Isolation of Oligomer/Leucrose Fraction from Gtf-C Reaction
Sucrose (4.50 kg) was dissolved in distilled deionized water to a final total volume of 9.5 L and the resulting solution was heated with stirring at 80 °C for 5 minutes and then cooled to 47 °C . With stirring, 500 grams of a crude extract containing 0.41 g/L of gtf-C enzyme (GTF0088BsT1, SEG ID NO:45) was added with stirring (see General Methods for enzyme preparation). The pH of the resulting mixture was immediately adjusted to between pH 5.5 to pH 6.0 by slowly adding a 1:10 (v/v) dilution of 37 wt% HCI with stirring. The reaction temperature and pH were maintained at 47 °C and pH 5.5-6.0, respectively, until sucrose conversion was >95% per HPLC analysis, after which the reaction mixture was immediately adjusted to pH 7.0 to 7.5 and heated to 90 °C for 20 min, then cooled to 25 °C for immediate filtration to remove particulates and precipitate. The resulting filtrate held at 5 °C prior to IEX/SEC column chromatography using the following resin and conditions: FiNEX CS 11 GC SAC in Caz+ form, column i.d ··· 9.3 cm, resin bed height 1.58 m, T~ 70 °C, flow rate ··· 50 mL/min, linear flow rate = 0.44 m/h, feed size = 0.6 L = 171 g, feed RI-DS = 25.8 g/100 g, sample interval ··· 3 min. The column fractions collected between 34 min and 72 min were combined, concentrated by evaporation to 67% dissolved solids and analyzed by HPLC as described in the General Methods. Table 22 indicates the ollgosacharide and monosaccharide components of the isolated fraction thus prepared.
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Table 22
Analysis of Qligamer/Leucrose Fraction from Gtf-C Reaction
DP7+ (%DS) DP8 (%DS) DP5 (%DS) DP4 (%DS) DP3 (%DS) DP2 (%DS) sucrose (%DS) leucrose (%DS) glucose (%DS) fructose (%DS)
iiiiiiii iiiiii iiiiii 2.8 5.4 13.2 It 2,5 5.7
In this Example, a glucan synthesis reaction was used to produce at least one soluble aipha-glucan product. This Example also demonstrated the preparation of a chromatographic fraction from a glucan synthesis reaction that produced a soluble aipha-glucan product. This fraction was used in Examples 15 and 16 below to test the activity of alpha-glucosidases thereupon.
EXAMPLE 15
Primary Screening of Alpha-Glucosidases Using Qligomer/Leucrose Fractions from Gtf-S/MUT3325 and Gtf-C Reactions
This Example describes using alpha-glucosidase to hydrolyze leucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that produced soluble aipha-glucan product. Specifically, study was made on the effect of alpha-glucosidases disclosed in Example 10 on the hydrolysis of leucrose and oligosaccharides in the fractions prepared in Examples 13 and 14.
A total of twelve alpha-glucosidases and two benchmark enzymes (oligo1,6-glucosidase and TG L-2000 transglucosidase) were screened using oligomer/leucrose fractions from gtf-S/MUT3325 (Example 13) and gtf-C (Example 14) reactions as substrate material. All the enzymes (alphaglucosidases and benchmark enzymes) were dosed at equal protein concentrations. Each alpha-glucosidase (dosed at 100 ppm) was incubated in a solution containing oligomer/leucrose substrates (10% dry solids) and 2 mM calcium chloride at pH 5.5 at 47 °C. Each reaction was quenched after 21 hours of incubation by adding 50 pL of 0.5 M NaOH.
The oligosaccharide/monosaccharide contents of the quenched reactions were determined as follows. A sample from each reaction was centrifuged and supernatant therefrom was diluted 25-fold in water for HPLC analysis (General
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Methods). The percentages reported in Table 23 reflect the average of peak area percentages (from duplicate analyses of each sample) of each DPn as a fraction of the total. The results indicate that the fungal alpha-glucosidases had better hydrolytic activity towards glucan oligomers when compared to the bacterial alpha-glucosidases.
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Figure AU2015223025B2_D0003
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Figure AU2015223025B2_D0004
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As indicated with shading in Table 23, the oligosaccharide content of the reactions generally shifted toward smaller sized sugars, in comparison with the control reactions (“Blank”) in which there was no enzyme. These results indicate that alpha-glucosidase can be used to hydrolyze oligosaccharides comprised within a glucan synthesis reaction and a fraction thereof, particularly a chromatographic fraction of a glucan synthesis reaction that produced soluble alpha-glucan product. Also, given the linkage profile of the oligosaccharides (Examples 13 and 14), and the activity of alpha-glucosidase against various glycosidic linkages in addition to alpha-1,4 linkages (Example 11), it is apparent that alpha-glucosidase can be used to break down oligosaccharides with alpha1,5 glucosyl-fructose linkages and also likely alpha-1,3 and alpha-1,6 glucosylglucose linkages. The results provided in Table 23 also suggest that fungal alpha-glucosidases have better hydrolytic activity towards soluble oligosaccharides compared with the bacterial alpha-glucosidases.
Thus, alpha-glucosidase can be used to hydrolyze leucrose and other oligosaccharides present in a fraction (e.g., chromatographic fraction) obtained from a glucan synthesis reaction, such as one that synthesizes a soluble alphaglucan product.
EXAMPLE 16
Select Screening of Alpha-Glucosidases using Oliqomer/Leucrose Fractions from
Gtf-8/MUT3325 and Gtf-C Reactions
This Example is further to Example 15, describing the use of alphaglucosidase to hydrolyze leucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that produced soluble alpha-glucan product.
Evaluation of aloha-glucosidases that were most active for hydrolysis of oligomer/ieucrose fractions from qtf-S/MUT3325 and gtf-C reactions (Exampie 15) was performed by analyzing sugar compositions resulting in reactions containing enzymes dosed at equal protein concentrations. Incubations of alphaglucosidases (dosed at 4 ppm; for blends, the ratio of the two enzymes was 1:1 and total dosage was 4 ppm) and oligomer/ieucrose substrate (10% ds) were
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The oligosaccharide/monosaccharide contents of the quenched reactions were determined as follows. A sample from each reaction was centrifuged and supernatant therefrom was diluted 25-fold in wafer for HPLC analysis (General Methods). The percentages reported in Table 24 (below) reflect the average of peak area percentages (from duplicate analyses of each sample) of each DPn as a fraction of the total. The results indicate that TauSecO98 was efficacious for hydrolysis of DP2 to DP7 oligomers and TauSecO99 outperformed TG L-2000 for Ieucrose hydrolysis when the incubation was performed at 65 °C. The blends of TauSecO98 with TauSecO99 (or TG L-2000) were effective for hydrolysis of oligomers and Ieucrose for DP1 production.
Thus, alpha-glucosidase can be used to hydrolyze Ieucrose and other oligosaccharides present in a fraction (e.g., chromatographic fraction) obtained from a glucan synthesis reaction, such as one that synthesizes a soluble alphaglucan product.
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Claims (4)

  1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
    1. A method comprising: (a) providing a glucan synthesis reaction; (b) obtaining a fraction of the glucan synthesis reaction that comprises a
    5 saccharide byproduct of the glucan synthesis reaction, wherein the saccharide byproduct is a disaccharide or oligosaccharide and comprises at least one alpha-1,3 glucosyl-glucose linkage; and (c) contacting the saccharide byproduct in the fraction with an alpha-glucosidase enzyme, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3
    10 glucosyl-glucose linkage of the saccharide byproduct.
  2. 2. The method of claim 1, wherein the alpha-glucosidase enzyme is immobilized.
    15
  3. 3. The method of claim 1 or claim 2, wherein the degree of polymerization of the saccharide byproduct before hydrolysis is 3 to 7.
  4. 4. The method of any one of claims 1 to 3, wherein the alpha-glucosidase enzyme is a transglucosidase.
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    CL6220WOPCT_SequenceListing_ST25.txt SEQUENCE LISTING <110> E.I. du Pont de Nemours & Company, Inc.
    Nagy, Kevin <120> ENZYMATIC HYDROLYSIS OF DISACCHARIDES AND OLIGOSACCHARIDES USING ALPHA-GLUCOSIDASE ENZYMES <130> CL6220 <150> US 61/945,233 <151> 2014-02-27 <150> US 61/945,241 <151> 2014-02-27 <150> US 62/004,305 <151> 2014-05-29 <150> US 62/004,314 <151> 2014-05-29 <150> US 62/004,300 <151> 2014-05-29 <150> US 62/004,312 <151> 2014-05-29 <150> US 62/004,308 <151> 2014-05-29 <150> US 62/004,290 <151> 2014-05-29 <160> 47 <170> PatentIn version 3.5 <210> 1 <211> 965 <212> PRT <213> Aspergillus niger <400> 1
    Ser Gln Ser 1 Leu Leu 5 Ser Thr Thr Ala Pro Ser Gln 10 Pro Gln Phe 15 Thr Ile Pro Ala Ser Ala Asp Val Gly Ala Gln Leu Ile Ala Asn Ile Asp 20 25 30 Asp Pro Gln Ala Ala Asp Ala Gln Ser Val Cys Pro Gly Tyr Lys Ala 35 40 45 Ser Lys Val Gln His Asn Ser Arg Gly Phe Thr Ala Ser Leu Gln Leu 50 55 60 Ala Gly Arg Pro Cys Asn Val Tyr Gly Thr Asp Val Glu Ser Leu Thr 65 70 75 80 Leu Ser Val Glu Tyr Gln Asp Ser Asp Arg Leu Asn Ile Gln Ile Leu
    Page 1
    CL6220WOPCT_SequenceListing_ST25.txt
    Pro Thr His Val 100 Asp Ser Thr Asn Ala 105 Ser Trp Tyr Phe Leu 110 Ser Glu Asn Leu Val Pro Arg Pro Lys Ala Ser Leu Asn Ala Ser Val Ser Gln 115 120 125 Ser Asp Leu Phe Val Ser Trp Ser Asn Glu Pro Ser Phe Asn Phe Lys 130 135 140 Val Ile Arg Lys Ala Thr Gly Asp Ala Leu Phe Ser Thr Glu Gly Thr 145 150 155 160 Val Leu Val Tyr Glu Asn Gln Phe Ile Glu Phe Val Thr Ala Leu Pro 165 170 175 Glu Glu Tyr Asn Leu Tyr Gly Leu Gly Glu His Ile Thr Gln Phe Arg 180 185 190 Leu Gln Arg Asn Ala Asn Leu Thr Ile Tyr Pro Ser Asp Asp Gly Thr 195 200 205 Pro Ile Asp Gln Asn Leu Tyr Gly Gln His Pro Phe Tyr Leu Asp Thr 210 215 220 Arg Tyr Tyr Lys Gly Asp Arg Gln Asn Gly Ser Tyr Ile Pro Val Lys 225 230 235 240 Ser Ser Glu Ala Asp Ala Ser Gln Asp Tyr Ile Ser Leu Ser His Gly 245 250 255 Val Phe Leu Arg Asn Ser His Gly Leu Glu Ile Leu Leu Arg Ser Gln 260 265 270 Lys Leu Ile Trp Arg Thr Leu Gly Gly Gly Ile Asp Leu Thr Phe Tyr 275 280 285 Ser Gly Pro Ala Pro Ala Asp Val Thr Arg Gln Tyr Leu Thr Ser Thr 290 295 300 Val Gly Leu Pro Ala Met Gln Gln Tyr Asn Thr Leu Gly Phe His Gln 305 310 315 320 Cys Arg Trp Gly Tyr Asn Asn Trp Ser Asp Leu Ala Asp Val Val Ala 325 330 335 Asn Phe Glu Lys Phe Glu Ile Pro Leu Glu Tyr Ile Trp Thr Asp Ile 340 345 350 Asp Tyr Met His Gly Tyr Arg Asn Phe Asp Asn Asp Gln His Arg Phe 355 360 365
    Page 2
    CL6220WOPCT_SequenceListing_ST25.txt
    Ser Tyr Ser Glu Gly Asp Glu 375 Phe Leu Ser Lys Leu 380 His Glu Ser Gly 370 Arg Tyr Tyr Val Pro Ile Val Asp Ala Ala Leu Tyr Ile Pro Asn Pro 385 390 395 400 Glu Asn Ala Ser Asp Ala Tyr Ala Thr Tyr Asp Arg Gly Ala Ala Asp 405 410 415 Asp Val Phe Leu Lys Asn Pro Asp Gly Ser Leu Tyr Ile Gly Ala Val 420 425 430 Trp Pro Gly Tyr Thr Val Phe Pro Asp Trp His His Pro Lys Ala Val 435 440 445 Asp Phe Trp Ala Asn Glu Leu Val Ile Trp Ser Lys Lys Val Ala Phe 450 455 460 Asp Gly Val Trp Tyr Asp Met Ser Glu Val Ser Ser Phe Cys Val Gly 465 470 475 480 Ser Cys Gly Thr Gly Asn Leu Thr Leu Asn Pro Ala His Pro Ser Phe 485 490 495 Leu Leu Pro Gly Glu Pro Gly Asp Ile Ile Tyr Asp Tyr Pro Glu Ala 500 505 510 Phe Asn Ile Thr Asn Ala Thr Glu Ala Ala Ser Ala Ser Ala Gly Ala 515 520 525 Ser Ser Gln Ala Ala Ala Thr Ala Thr Thr Thr Ser Thr Ser Val Ser 530 535 540 Tyr Leu Arg Thr Thr Pro Thr Pro Gly Val Arg Asn Val Glu His Pro 545 550 555 560 Pro Tyr Val Ile Asn His Asp Gln Glu Gly His Asp Leu Ser Val His 565 570 575 Ala Val Ser Pro Asn Ala Thr His Val Asp Gly Val Glu Glu Tyr Asp 580 585 590 Val His Gly Leu Tyr Gly His Gln Gly Leu Asn Ala Thr Tyr Gln Gly 595 600 605 Leu Leu Glu Val Trp Ser His Lys Arg Arg Pro Phe Ile Ile Gly Arg 610 615 620 Ser Thr Phe Ala Gly Ser Gly Lys Trp Ala Gly His Trp Gly Gly Asp 625 630 635 640
    Page 3
    CL6220WOPCT_SequenceListing_ST25.txt
    Asn Tyr Ser Lys Trp Trp Ser Met Tyr Tyr Ser Ile Ser Gln Ala 655 Leu 645 650 Ser Phe Ser Leu Phe Asp Ile Pro Met Phe Gly Ala Asp Thr Cys Gly 660 665 670 Phe Asn Gly Asn Ser Asp Glu Glu Leu Cys Asn Arg Trp Met Gln Leu 675 680 685 Ser Ala Phe Phe Pro Phe Tyr Arg Asn His Asn Glu Leu Ser Thr Ile 690 695 700 Pro Gln Glu Pro Tyr Arg Trp Ala Ser Val Ile Glu Ala Thr Lys Ser 705 710 715 720 Ala Met Arg Ile Arg Tyr Ala Ile Leu Pro Tyr Phe Tyr Thr Leu Phe 725 730 735 Asp Leu Ala His Thr Thr Gly Ser Thr Val Met Arg Ala Leu Ser Trp 740 745 750 Glu Phe Pro Asn Asp Pro Thr Leu Ala Ala Val Glu Thr Gln Phe Met 755 760 765 Val Gly Pro Ala Ile Met Val Val Pro Val Leu Glu Pro Leu Val Asn 770 775 780 Thr Val Lys Gly Val Phe Pro Gly Val Gly His Gly Glu Val Trp Tyr 785 790 795 800 Asp Trp Tyr Thr Gln Ala Ala Val Asp Ala Lys Pro Gly Val Asn Thr 805 810 815 Thr Ile Ser Ala Pro Leu Gly His Ile Pro Val Tyr Val Arg Gly Gly 820 825 830 Asn Ile Leu Pro Met Gln Glu Pro Ala Leu Thr Thr Arg Glu Ala Arg 835 840 845 Gln Thr Pro Trp Ala Leu Leu Ala Ala Leu Gly Ser Asn Gly Thr Ala 850 855 860 Ser Gly Gln Leu Tyr Leu Asp Asp Gly Glu Ser Ile Tyr Pro Asn Ala 865 870 875 880 Thr Leu His Val Asp Phe Thr Ala Ser Arg Ser Ser Leu Arg Ser Ser 885 890 895 Ala Gln Gly Arg Trp Lys Glu Arg Asn Pro Leu Ala Asn Val Thr Val 900 905 910
    Page 4
    CL6220WOPCT_SequenceListing_ST25.txt
    Leu Gly Val 915 Asn Lys Glu Pro Ser Ala 920 Val Thr Leu Asn 925 Gly Gln Ala Val Phe Pro Gly Ser Val Thr Tyr Asn Ser Thr Ser Gln Val Leu Phe 930 935 940 Val Gly Gly Leu Gln Asn Leu Thr Lys Gly Gly Ala Trp Ala Glu Asn 945 950 955 960 Trp Val Leu Glu Trp 965 <210> ' 2 <211> 599 <212> PRT <213> Trichoderma i eesei <400> 2 Ser Val Asp Asp Phe Ile Ser Thr Glu Thr Pro Ile Ala Leu Asn Asn 1 5 10 15 Leu Leu Cys Asn Val Gly Pro Asp Gly Cys Arg Ala Phe Gly Thr Ser 20 25 30 Ala Gly Ala Val Ile Ala Ser Pro Ser Thr Ile Asp Pro Asp Tyr Tyr 35 40 45 Tyr Met Trp Thr Arg Asp Ser Ala Leu Val Phe Lys Asn Leu Ile Asp 50 55 60 Arg Phe Thr Glu Thr Tyr Asp Ala Gly Leu Gln Arg Arg Ile Glu Gln 65 70 75 80 Tyr Ile Thr Ala Gln Val Thr Leu Gln Gly Leu Ser Asn Pro Ser Gly 85 90 95 Ser Leu Ala Asp Gly Ser Gly Leu Gly Glu Pro Lys Phe Glu Leu Thr 100 105 110 Leu Lys Pro Phe Thr Gly Asn Trp Gly Arg Pro Gln Arg Asp Gly Pro 115 120 125 Ala Leu Arg Ala Ile Ala Leu Ile Gly Tyr Ser Lys Trp Leu Ile Asn 130 135 140 Asn Asn Tyr Gln Ser Thr Val Ser Asn Val Ile Trp Pro Ile Val Arg 145 150 155 160 Asn Asp Leu Asn Tyr Val Ala Gln Tyr Trp Asn Gln Thr Gly Phe Asp 165 170 175
    Page 5
    CL6220WOPCT_SequenceListing_ST25.txt
    Leu Trp Glu Glu Val 180 Asn Gly Ser Ser 185 Phe Phe Thr Val Ala 190 Asn Gln His Arg Ala Leu Val Glu Gly Ala Thr Leu Ala Ala Thr Leu Gly Gln 195 200 205 Ser Gly Ser Ala Tyr Ser Ser Val Ala Pro Gln Val Leu Cys Phe Leu 210 215 220 Gln Arg Phe Trp Val Ser Ser Gly Gly Tyr Val Asp Ser Asn Ile Asn 225 230 235 240 Thr Asn Glu Gly Arg Thr Gly Lys Asp Val Asn Ser Val Leu Thr Ser 245 250 255 Ile His Thr Phe Asp Pro Asn Leu Gly Cys Asp Ala Gly Thr Phe Gln 260 265 270 Pro Cys Ser Asp Lys Ala Leu Ser Asn Leu Lys Val Val Val Asp Ser 275 280 285 Phe Arg Ser Ile Tyr Gly Val Asn Lys Gly Ile Pro Ala Gly Ala Ala 290 295 300 Val Ala Ile Gly Arg Tyr Ala Glu Asp Val Tyr Tyr Asn Gly Asn Pro 305 310 315 320 Trp Tyr Leu Ala Thr Phe Ala Ala Ala Glu Gln Leu Tyr Asp Ala Ile 325 330 335 Tyr Val Trp Lys Lys Thr Gly Ser Ile Thr Val Thr Ala Thr Ser Leu 340 345 350 Ala Phe Phe Gln Glu Leu Val Pro Gly Val Thr Ala Gly Thr Tyr Ser 355 360 365 Ser Ser Ser Ser Thr Phe Thr Asn Ile Ile Asn Ala Val Ser Thr Tyr 370 375 380 Ala Asp Gly Phe Leu Ser Glu Ala Ala Lys Tyr Val Pro Ala Asp Gly 385 390 395 400 Ser Leu Ala Glu Gln Phe Asp Arg Asn Ser Gly Thr Pro Leu Ser Ala 405 410 415 Leu His Leu Thr Trp Ser Tyr Ala Ser Phe Leu Thr Ala Thr Ala Arg 420 425 430 Arg Ala Gly Ile Val Pro Pro Ser Trp Ala Asn Ser Ser Ala Ser Thr 435 440 445
    Page 6
    Ile Pro 450 Ser Thr Cys CL6220WOPCT_SequenceListi i ng_ST25.txt Ser Tyr Ser Arg Ser Gly Ala Ser Val 455 Val Gly 460 Pro Thr Ala Thr Ser Phe Pro Pro Ser Gln Thr Pro Lys Pro Gly Val 465 470 475 480 Pro Ser Gly Thr Pro Tyr Thr Pro Leu Pro Cys Ala Thr Pro Thr Ser 485 490 495 Val Ala Val Thr Phe His Glu Leu Val Ser Thr Gln Phe Gly Gln Thr 500 505 510 Val Lys Val Ala Gly Asn Ala Ala Ala Leu Gly Asn Trp Ser Thr Ser 515 520 525 Ala Ala Val Ala Leu Asp Ala Val Asn Tyr Ala Asp Asn His Pro Leu 530 535 540 Trp Ile Gly Thr Val Asn Leu Glu Ala Gly Asp Val Val Glu Tyr Lys 545 550 555 560 Tyr Ile Asn Val Gly Gln Asp Gly Ser Val Thr Trp Glu Ser Asp Pro 565 570 575 Asn His Thr Tyr Thr Val Pro Ala Val Ala Cys Val Thr Gln Val Val 580 585 590 Lys Glu Asp Thr Trp Gln Ser 595 <210> : 3 <211> : 1477 <212> PRT <213> : Streptococcus salivarius <400> 3 Met Asp Glu Thr Gln Asp Lys Thr Val Thr Gln Ser Asn Ser Gly Thr 1 5 10 15 Thr Ala Ser Leu Val Thr Ser Pro Glu Ala Thr Lys Glu Ala Asp Lys 20 25 30 Arg Thr Asn Thr Lys Glu Ala Asp Val Leu Thr Pro Ala Lys Glu Thr 35 40 45 Asn Ala Val Glu Thr Ala Thr Thr Thr Asn Thr Gln Ala Thr Ala Glu 50 55 60 Ala Ala Thr Thr Ala Thr Thr Ala Asp Val Ala Val Ala Ala Val Pro 65 70 75 80 Asn Lys Glu Ala Val Val Thr Thr Asp Ala Pro Ala Val Thr Thr Glu
    Page 7
    CL6220WOPCT_SequenceListing_ST25.txt 85 90 95 Lys Ala Glu Glu Gln Pro Ala Thr Val Lys Ala Glu Val Val Asn Thr 100 105 110 Glu Val Lys Ala Pro Glu Ala Ala Leu Lys Asp Ser Glu Val Glu Ala 115 120 125 Ala Leu Ser Leu Lys Asn Ile Lys Asn Ile Asp Gly Lys Tyr Tyr Tyr 130 135 140 Val Asn Glu Asp Gly Ser His Lys Glu Asn Phe Ala Ile Thr Val Asn 145 150 155 160 Gly Gln Leu Leu Tyr Phe Gly Lys Asp Gly Ala Leu Thr Ser Ser Ser 165 170 175 Thr Tyr Ser Phe Thr Pro Gly Thr Thr Asn Ile Val Asp Gly Phe Ser 180 185 190 Ile Asn Asn Arg Ala Tyr Asp Ser Ser Glu Ala Ser Phe Glu Leu Ile 195 200 205 Asp Gly Tyr Leu Thr Ala Asp Ser Trp Tyr Arg Pro Ala Ser Ile Ile 210 215 220 Lys Asp Gly Val Thr Trp Gln Ala Ser Thr Ala Glu Asp Phe Arg Pro 225 230 235 240 Leu Leu Met Ala Trp Trp Pro Asn Val Asp Thr Gln Val Asn Tyr Leu 245 250 255 Asn Tyr Met Ser Lys Val Phe Asn Leu Asp Ala Lys Tyr Ser Ser Thr 260 265 270 Asp Lys Gln Glu Thr Leu Lys Val Ala Ala Lys Asp Ile Gln Ile Lys 275 280 285 Ile Glu Gln Lys Ile Gln Ala Glu Lys Ser Thr Gln Trp Leu Arg Glu 290 295 300 Thr Ile Ser Ala Phe Val Lys Thr Gln Pro Gln Trp Asn Lys Glu Thr 305 310 315 320 Glu Asn Tyr Ser Lys Gly Gly Gly Glu Asp His Leu Gln Gly Gly Ala 325 330 335 Leu Leu Tyr Val Asn Asp Ser Arg Thr Pro Trp Ala Asn Ser Asp Tyr 340 345 350 Arg Arg Leu Asn Arg Thr Ala Thr Asn Gln Thr Gly Thr Ile Asp Lys Page 8
    355 CL6220WOPCT_SequenceListing_ST25.txt 360 365 Ser Ile Leu Asp Glu Gln Ser Asp Pro Asn His Met Gly Gly Phe Asp 370 375 380 Phe Leu Leu Ala Asn Asp Val Asp Leu Ser Asn Pro Val Val Gln Ala 385 390 395 400 Glu Gln Leu Asn Gln Ile His Tyr Leu Met Asn Trp Gly Ser Ile Val 405 410 415 Met Gly Asp Lys Asp Ala Asn Phe Asp Gly Ile Arg Val Asp Ala Val 420 425 430 Asp Asn Val Asp Ala Asp Met Leu Gln Leu Tyr Thr Asn Tyr Phe Arg 435 440 445 Glu Tyr Tyr Gly Val Asn Lys Ser Glu Ala Asn Ala Leu Ala His Ile 450 455 460 Ser Val Leu Glu Ala Trp Ser Leu Asn Asp Asn His Tyr Asn Asp Lys 465 470 475 480 Thr Asp Gly Ala Ala Leu Ala Met Glu Asn Lys Gln Arg Leu Ala Leu 485 490 495 Leu Phe Ser Leu Ala Lys Pro Ile Lys Glu Arg Thr Pro Ala Val Ser 500 505 510 Pro Leu Tyr Asn Asn Thr Phe Asn Thr Thr Gln Arg Asp Glu Lys Thr 515 520 525 Asp Trp Ile Asn Lys Asp Gly Ser Lys Ala Tyr Asn Glu Asp Gly Thr 530 535 540 Val Lys Gln Ser Thr Ile Gly Lys Tyr Asn Glu Lys Tyr Gly Asp Ala 545 550 555 560 Ser Gly Asn Tyr Val Phe Ile Arg Ala His Asp Asn Asn Val Gln Asp 565 570 575 Ile Ile Ala Glu Ile Ile Lys Lys Glu Ile Asn Pro Lys Ser Asp Gly 580 585 590 Phe Thr Ile Thr Asp Ala Glu Met Lys Gln Ala Phe Glu Ile Tyr Asn 595 600 605 Lys Asp Met Leu Ser Ser Asp Lys Lys Tyr Thr Leu Asn Asn Ile Pro 610 615 620 Ala Ala Tyr Ala Val Met Leu Gln Asn Met Glu Thr Ile Thr Arg Val Page 9
    C _6220WOPCT_Sequei nceListii ng_S T25. txt 625 630 635 640 Tyr Tyr Gly Asp Leu Tyr Thr Asp Asp Gly His Tyr Met Glu Thr Lys 645 650 655 Ser Pro Tyr Tyr Asp Thr Ile Val Asn Leu Met Lys Ser Arg Ile Lys 660 665 670 Tyr Val Ser Gly Gly Gln Ala Gln Arg Ser Tyr Trp Leu Pro Thr Asp 675 680 685 Gly Lys Met Asp Asn Ser Asp Val Glu Leu Tyr Arg Thr Asn Glu Val 690 695 700 Tyr Thr Ser Val Arg Tyr Gly Lys Asp Ile Met Thr Ala Asn Asp Thr 705 710 715 720 Glu Gly Ser Lys Tyr Ser Arg Thr Ser Gly Gln Val Thr Leu Val Ala 725 730 735 Asn Asn Pro Lys Leu Asn Leu Asp Gln Ser Ala Lys Leu Asn Val Glu 740 745 750 Met Gly Lys Ile His Ala Asn Gln Lys Tyr Arg Ala Leu Ile Val Gly 755 760 765 Thr Ala Asp Gly Ile Lys Asn Phe Thr Ser Asp Ala Asp Ala Ile Ala 770 775 780 Ala Gly Tyr Val Lys Glu Thr Asp Ser Asn Gly Val Leu Thr Phe Gly 785 790 795 800 Ala Asn Asp Ile Lys Gly Tyr Glu Thr Phe Asp Met Ser Gly Phe Val 805 810 815 Ala Val Trp Val Pro Val Gly Ala Ser Asp Asn Gln Asp Ile Arg Val 820 825 830 Ala Pro Ser Thr Glu Ala Lys Lys Glu Gly Glu Leu Thr Leu Lys Ala 835 840 845 Thr Glu Ala Tyr Asp Ser Gln Leu Ile Tyr Glu Gly Phe Ser Asn Phe 850 855 860 Gln Thr Ile Pro Asp Gly Ser Asp Pro Ser Val Tyr Thr Asn Arg Lys 865 870 875 880 Ile Ala Glu Asn Val Asp Leu Phe Lys Ser Trp Gly Val Thr Ser Phe 885 890 895 Glu Met Ala Pro Gln Phe Val Ser Ala Asp Asp Gly Thr Phe Leu Asp Pag e 10
    900 CL6220WOPCT_SequenceListing_ST25.txt 905 910 Ser Val Ile Gln Asn Gly Tyr Ala Phe Ala Asp Arg Tyr Asp Leu Ala 915 920 925 Met Ser Lys Asn Asn Lys Tyr Gly Ser Lys Glu Asp Leu Arg Asp Ala 930 935 940 Leu Lys Ala Leu His Lys Ala Gly Ile Gln Ala Ile Ala Asp Trp Val 945 950 955 960 Pro Asp Gln Ile Tyr Gln Leu Pro Gly Lys Glu Val Val Thr Ala Thr 965 970 975 Arg Thr Asp Gly Ala Gly Arg Lys Ile Ala Asp Ala Ile Ile Asp His 980 985 990
    Ser Leu Tyr Val Ala Asn Ser Lys Ser Ser Gly Lys Asp Tyr Gln Ala 995 1000 1005
    Lys Tyr 1010 Gly Gly Glu Phe Leu 1015 Ala Glu Leu Lys Ala 1020 Lys Tyr Pro Glu Met Phe Lys Val Asn Met Ile Ser Thr Gly Lys Pro Ile Asp 1025 1030 1035 Asp Ser Val Lys Leu Lys Gln Trp Lys Ala Glu Tyr Phe Asn Gly 1040 1045 1050 Thr Asn Val Leu Glu Arg Gly Val Gly Tyr Val Leu Ser Asp Glu 1055 1060 1065 Ala Thr Gly Lys Tyr Phe Thr Val Thr Lys Glu Gly Asn Phe Ile 1070 1075 1080 Pro Leu Gln Leu Thr Gly Lys Glu Lys Val Ile Thr Gly Phe Ser 1085 1090 1095 Ser Asp Gly Lys Gly Ile Thr Tyr Phe Gly Thr Ser Gly Thr Gln 1100 1105 1110 Ala Lys Ser Ala Phe Val Thr Phe Asn Gly Asn Thr Tyr Tyr Phe 1115 1120 1125 Asp Ala Arg Gly His Met Val Thr Asn Ser Glu Tyr Ser Pro Asn 1130 1135 1140 Gly Lys Asp Val Tyr Arg Phe Leu Pro Asn Gly Ile Met Leu Ser 1145 1150 1155 Asn Ala Phe Tyr Ile Asp Ala Asn Gly Asn Thr Tyr Leu Tyr Asn
    Page 11
    CL6220WOPCT_SequenceListing_ST25.txt
    1160 1165 1170 Ser Lys Gly Gln Met Tyr Lys Gly Gly Tyr Thr Lys Phe Asp Val 1175 1180 1185 Ser Glu Thr Asp Lys Asp Gly Lys Glu Ser Lys Val Val Lys Phe 1190 1195 1200 Arg Tyr Phe Thr Asn Glu Gly Val Met Ala Lys Gly Val Thr Val 1205 1210 1215 Ile Asp Gly Phe Thr Gln Tyr Phe Gly Glu Asp Gly Phe Gln Ala 1220 1225 1230 Lys Asp Lys Leu Val Thr Phe Lys Gly Lys Thr Tyr Tyr Phe Asp 1235 1240 1245 Ala His Thr Gly Asn Gly Ile Lys Asp Thr Trp Arg Asn Ile Asn 1250 1255 1260 Gly Lys Trp Tyr Tyr Phe Asp Ala Asn Gly Val Ala Ala Thr Gly 1265 1270 1275 Ala Gln Val Ile Asn Gly Gln Lys Leu Tyr Phe Asn Glu Asp Gly 1280 1285 1290 Ser Gln Val Lys Gly Gly Val Val Lys Asn Ala Asp Gly Thr Tyr 1295 1300 1305 Ser Lys Tyr Lys Glu Gly Phe Gly Glu Leu Val Thr Asn Glu Phe 1310 1315 1320 Phe Thr Thr Asp Gly Asn Val Trp Tyr Tyr Ala Gly Ala Asn Gly 1325 1330 1335 Lys Thr Val Thr Gly Ala Gln Val Ile Asn Gly Gln His Leu Tyr 1340 1345 1350 Phe Asn Ala Asp Gly Ser Gln Val Lys Gly Gly Val Val Lys Asn 1355 1360 1365 Ala Asp Gly Thr Tyr Ser Lys Tyr Asn Ala Ser Thr Gly Glu Arg 1370 1375 1380 Leu Thr Asn Glu Phe Phe Thr Thr Gly Asp Asn Asn Trp Tyr Tyr 1385 1390 1395 Ile Gly Ala Asn Gly Lys Ser Val Thr Gly Glu Val Lys Ile Gly 1400 1405 1410 Asp Asp Thr Tyr Phe Phe Ala Lys Asp Gly Lys Gln Val Lys Gly
    Page 12
    1415 CL6220WOPCT_SequenceListing_ST25.txt 1420 1425 Gln Thr Val Ser Ala Gly Asn Gly Arg Ile Ser Tyr Tyr Tyr Gly 1430 1435 1440 Asp Ser Gly Lys Arg Ala Val Ser Thr Trp Ile Glu Ile Gln Pro 1445 1450 1455 Gly Val Tyr Val Tyr Phe Asp Lys Asn Gly Leu Ala Tyr Pro Pro 1460 1465 1470
    Arg Val Leu Asn 1475 <210> 4 <211> 3147 <212> DNA <213> Aspergillus clavatus <400> 4
    atggccagtc tcgtaggcct tcttgccagt gcttggctcc tcccgtcgac ctacggagcg 60 gcggttcact cgtttgctcc tagtacgacg gcaaccgcag cttactcgca gcatactttg 120 ccttcgtcca ttgacaatgg ggctcagttg attgccaata tcgatgaccc cttggcagtg 180 aatgcgcagt ccgtttgtcc aggctataag gcctcggatg tgcaacagac atcacggggt 240 ttcaccgcca gcctgcagct tgcgggcgag ccgtgcaatg cctatgggat tgacgttgac 300 tcgctgagtc tctcggtgga agttctggcg aaggatcgct tgaatatcca gattgtccct 360 acccatgtgg atagctctaa tgcctcttgg tacattcttc cagaggaccg ggtacccaaa 420 gcgcaagcat ctgccgacgc ctccgtctct caaagtgact tcgagatcga gtggtccaat 480 gacccctcgt tcaatatcaa gatcatccgg aaggcaactg gggatgccct gtttgacaca 540 gccgattcgg tcttggtctt tcagaatcag ttcatcgagt ttgtgtctgc tttgcctgag 600 ggttataatc tttatggatt gggggagcgg atggcccagc tgcggcttct gagaaacgct 660 accctgacca cctacgcggc agatgtcgga gacccgattg atgagtatgt gttagaatct 720 atgaatcctt gatctcgttg gctcacaatg gaccaatagc aacatctatg gacagcatcc 780 tttctacctc gacacgagat actataccaa agatgccaac ggctcatatt ctcttgtgaa 840 caccgatgat gccgatgcgt ctggagatta tgagtccttc tcccacggcg tgttcctgag 900 aaatgcacat ggacaggagg ttattctgca gtctcgcaac attacatggc ggacaatcgg 960 tggaagcatc gatttgacct tctactcagg tcccactcag gcagatgtca ccaagagcta 1020 ccagctcacc acgattggct tgcctgcgat gcaacagtac agcgcccttg gtttccatca 1080 atgccgctgg ggttaccgca gctggtctga actcgaggaa gtcgtaaaca cctttgaaca 1140 gtttgagatt cccctcgaat atatctggtg agctcaattc ttcgtaagaa aatatgggct 1200 cttggtgtct aacgcatttt cccaggaccg acattgacta catgcgtggt taccgcgatt 1260 tcgataatga ccaagtccat ttcccctacg atgaaggcga ggagttcctc gacagacttc Page 13 1320
    CL6220WOPCT_SequenceListing_ST25.txt
    acaaatcagg gcgtcactgg gtccccatcg tcgactccgc catctacatc cctaaccctg 1380 acaatgcgtc tgacgcgtaa gtcattgtgt cttatttggc cagcttttca aacagatgct 1440 tacagacgtg acttcaggta cgacacctat gctagaggag ccaaggatga tgttttcatc 1500 aagaaccctg atggcagcct ttatatcgga gctgtatggc ccggattcac agtcttccct 1560 gattggcaca accccaaagc agcggaatgg tggagcaacg agcttgtcac ttggttcgag 1620 aaagtgcagt atgatggcat ttggattgac atgagcgaag tctcctcttt ctgcgttggt 1680 agctgcggga caggaaatct gcacctgaac cccgcccacc caccatttca acttcctggc 1740 gagccgggta acattgagta tacctatccc gaggccttca atgtgaccaa ctccactgaa 1800 gctgcttctg cctctgctgc ctccgccagc cagtcctctg cagctgctgc gacccaaacc 1860 gacgtcagct cgaccacgac ctcgtacttg cggacgacac ccactcctgg tgtccgtgat 1920 atcaactatc ccccttacgt gatcaaccat gttcaatctg gccatgatct cgctgtccat 1980 gccatctctc ccaacgccac ccacgtggat ggtgttcagg agtacgatgt gcacagtctg 2040 tggggtcatc agatcctcaa cgccacttat cagggattgc tcgaggtctt cactgagaag 2100 cgacctttca tcatcggccg atccaccttt gccggctcag ggaaatgggc cggtcactgg 2160 ggcggtgata acaactctag atggggctct atgttccact ccatttcgca ggctctgtcg 2220 ttctctctct ttggcattcc catgtttggt gttgatactt gtggcttcaa tggcaacacc 2280 gacgaggaac tctgcaaccg atggatgcag ctatcggcct tcttcccatt ctaccgaaac 2340 cacaacactc tcgcggctct ttcgcaggag ccctaccggt gggcctccgt cactgaagca 2400 gccaagactg ccatgagcat tcgatatgcc ctcctgccat acttctacac tctgttccac 2460 caggcgcaca ccaccggttc caccgtcatg cgcgccctcg catgggagtt cccgaacgat 2520 ccctcccttg ccgccgtcga cacccagttc atggtcggac catccattct ggtcacgccc 2580 gtcctcgagc ctcttgccaa aacagtcaag ggcgtcttcc ccggcgtcgg caagggtcag 2640 gtctggtatg actggtacac ccaggccgct gtcgacgcca agcccggcgt caacaccacc 2700 atcccggccc cgctgggcca catccccgtc tatgtccgcg gcggcagcat cctgcccatg 2760 caggagcccg ctcttaccac ccgcgacgcc cgcaagaccc cctggtcgct gcttgccgct 2820 ctggacggta accagaccgc ctccggccag ctgtacctcg acgacgggag cagcgtcaac 2880 ccgtcttcga ccctcaacgt ggagttcgcg gctacccact cgagcatcaa ggtctcggcc 2940 aagggtgatt ggcgcgagaa gaatagtctg gatagcgtga ctgttctcgg tgtcgccaag 3000 gagcctgccc gcgtcacctt caaccgccgc agggtccccc ccgagtcggt ggagtataat 3060 gcgacctccc aggttttgac cgtgagcgga ctgcagaagc tgacgccccg cggggcttgg 3120 gctgaggatt ggattctgaa gtggtaa 3147
    <210> 5 <211> 990 <212> PRT <213> Aspergillus clavatus
    Page 14
    CL6220WOPCT_SequenceListing_ST25.txt
    <400> 5 Met Ala Ser Leu Val Gly Leu Leu Ala Ser Ala Trp Leu Leu Pro Ser 1 5 10 15 Thr Tyr Gly Ala Ala Val His Ser Phe Ala Pro Ser Thr Thr Ala Thr 20 25 30 Ala Ala Tyr Ser Gln His Thr Leu Pro Ser Ser Ile Asp Asn Gly Ala 35 40 45 Gln Leu Ile Ala Asn Ile Asp Asp Pro Leu Ala Val Asn Ala Gln Ser 50 55 60 Val Cys Pro Gly Tyr Lys Ala Ser Asp Val Gln Gln Thr Ser Arg Gly 65 70 75 80 Phe Thr Ala Ser Leu Gln Leu Ala Gly Glu Pro Cys Asn Ala Tyr Gly 85 90 95 Ile Asp Val Asp Ser Leu Ser Leu Ser Val Glu Val Leu Ala Lys Asp 100 105 110 Arg Leu Asn Ile Gln Ile Val Pro Thr His Val Asp Ser Ser Asn Ala 115 120 125 Ser Trp Tyr Ile Leu Pro Glu Asp Arg Val Pro Lys Ala Gln Ala Ser 130 135 140 Ala Asp Ala Ser Val Ser Gln Ser Asp Phe Glu Ile Glu Trp Ser Asn 145 150 155 160 Asp Pro Ser Phe Asn Ile Lys Ile Ile Arg Lys Ala Thr Gly Asp Ala 165 170 175 Leu Phe Asp Thr Ala Asp Ser Val Leu Val Phe Gln Asn Gln Phe Ile 180 185 190 Glu Phe Val Ser Ala Leu Pro Glu Gly Tyr Asn Leu Tyr Gly Leu Gly 195 200 205 Glu Arg Met Ala Gln Leu Arg Leu Leu Arg Asn Ala Thr Leu Thr Thr 210 215 220 Tyr Ala Ala Asp Val Gly Asp Pro Ile Asp Asp Asn Ile Tyr Gly Gln 225 230 235 240 His Pro Phe Tyr Leu Asp Thr Arg Tyr Tyr Thr Lys Asp Ala Asn Gly 245 250 255 Ser Tyr Ser Leu Val Asn Thr Asp Asp Ala Asp Ala Ser Gly Asp Tyr Page 15
    CL6220WOPCT_SequenceListing_ST25.txt
    260 265 270 Glu Ser Phe Ser His Gly Val Phe Leu Arg Asn Ala His Gly Gln Glu 275 280 285 Val Ile Leu Gln Ser Arg Asn Ile Thr Trp Arg Thr Ile Gly Gly Ser 290 295 300 Ile Asp Leu Thr Phe Tyr Ser Gly Pro Thr Gln Ala Asp Val Thr Lys 305 310 315 320 Ser Tyr Gln Leu Thr Thr Ile Gly Leu Pro Ala Met Gln Gln Tyr Ser 325 330 335 Ala Leu Gly Phe His Gln Cys Arg Trp Gly Tyr Arg Ser Trp Ser Glu 340 345 350 Leu Glu Glu Val Val Asn Thr Phe Glu Gln Phe Glu Ile Pro Leu Glu 355 360 365 Tyr Ile Trp Thr Asp Ile Asp Tyr Met Arg Gly Tyr Arg Asp Phe Asp 370 375 380 Asn Asp Gln Val His Phe Pro Tyr Asp Glu Gly Glu Glu Phe Leu Asp 385 390 395 400 Arg Leu His Lys Ser Gly Arg His Trp Val Pro Ile Val Asp Ser Ala 405 410 415 Ile Tyr Ile Pro Asn Pro Asp Asn Ala Ser Asp Ala Tyr Asp Thr Tyr 420 425 430 Ala Arg Gly Ala Lys Asp Asp Val Phe Ile Lys Asn Pro Asp Gly Ser 435 440 445 Leu Tyr Ile Gly Ala Val Trp Pro Gly Phe Thr Val Phe Pro Asp Trp 450 455 460 His Asn Pro Lys Ala Ala Glu Trp Trp Ser Asn Glu Leu Val Thr Trp 465 470 475 480 Phe Glu Lys Val Gln Tyr Asp Gly Ile Trp Ile Asp Met Ser Glu Val 485 490 495 Ser Ser Phe Cys Val Gly Ser Cys Gly Thr Gly Asn Leu His Leu Asn 500 505 510 Pro Ala His Pro Pro Phe Gln Leu Pro Gly Glu Pro Gly Asn Ile Glu 515 520 525 Tyr Thr Tyr Pro Glu Ala Phe Asn Val Thr Asn Ser Thr Glu Ala Ala Pag e 16
    CL6220WOPCT_SequenceListing_ST25.txt 530 535 540
    Ser Ala Ser 545 Ala Ala Ser Ala Ser Gln Ser Ser Ala Ala Ala Ala Thr 550 555 560 Gln Thr Asp Val Ser Ser Thr Thr Thr Ser Tyr Leu Arg Thr Thr Pro 565 570 575 Thr Pro Gly Val Arg Asp Ile Asn Tyr Pro Pro Tyr Val Ile Asn His 580 585 590 Val Gln Ser Gly His Asp Leu Ala Val His Ala Ile Ser Pro Asn Ala 595 600 605 Thr His Val Asp Gly Val Gln Glu Tyr Asp Val His Ser Leu Trp Gly 610 615 620 His Gln Ile Leu Asn Ala Thr Tyr Gln Gly Leu Leu Glu Val Phe Thr 625 630 635 640 Glu Lys Arg Pro Phe Ile Ile Gly Arg Ser Thr Phe Ala Gly Ser Gly 645 650 655 Lys Trp Ala Gly His Trp Gly Gly Asp Asn Asn Ser Arg Trp Gly Ser 660 665 670 Met Phe His Ser Ile Ser Gln Ala Leu Ser Phe Ser Leu Phe Gly Ile 675 680 685 Pro Met Phe Gly Val Asp Thr Cys Gly Phe Asn Gly Asn Thr Asp Glu 690 695 700 Glu Leu Cys Asn Arg Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr 705 710 715 720 Arg Asn His Asn Thr Leu Ala Ala Leu Ser Gln Glu Pro Tyr Arg Trp 725 730 735 Ala Ser Val Thr Glu Ala Ala Lys Thr Ala Met Ser Ile Arg Tyr Ala 740 745 750 Leu Leu Pro Tyr Phe Tyr Thr Leu Phe His Gln Ala His Thr Thr Gly 755 760 765 Ser Thr Val Met Arg Ala Leu Ala Trp Glu Phe Pro Asn Asp Pro Ser 770 775 780 Leu Ala Ala Val Asp Thr Gln Phe Met Val Gly Pro Ser Ile Leu Val 785 790 795 800 Thr Pro Val Leu Glu Pro Leu Ala Lys Thr Val Lys Gly Val Phe Pro
    Page 17
    CL6220WOPCT_SequenceListing_ST25.txt
    805 810 815 Gly Val Gly Lys Gly Gln Val Trp Tyr Asp Trp Tyr Thr Gln Ala Ala 820 825 830 Val Asp Ala Lys Pro Gly Val Asn Thr Thr Ile Pro Ala Pro Leu Gly 835 840 845 His Ile Pro Val Tyr Val Arg Gly Gly Ser Ile Leu Pro Met Gln Glu 850 855 860 Pro Ala Leu Thr Thr Arg Asp Ala Arg Lys Thr Pro Trp Ser Leu Leu 865 870 875 880 Ala Ala Leu Asp Gly Asn Gln Thr Ala Ser Gly Gln Leu Tyr Leu Asp 885 890 895 Asp Gly Ser Ser Val Asn Pro Ser Ser Thr Leu Asn Val Glu Phe Ala 900 905 910 Ala Thr His Ser Ser Ile Lys Val Ser Ala Lys Gly Asp Trp Arg Glu 915 920 925 Lys Asn Ser Leu Asp Ser Val Thr Val Leu Gly Val Ala Lys Glu Pro 930 935 940 Ala Arg Val Thr Phe Asn Arg Arg Arg Val Pro Pro Glu Ser Val Glu 945 950 955 960 Tyr Asn Ala Thr Ser Gln Val Leu Thr Val Ser Gly Leu Gln Lys Leu 965 970 975 Thr Pro Arg Gly Ala Trp Ala Glu Asp Trp Ile Leu Lys Trp
    980 985 990 <210> 6 <211> 971 <212> PRT <213> Aspergillus clavatus
    <400> 6 Ala Ala Val 1 His Ser 5 Phe Ala Pro Ser Thr 10 Thr Ala Thr Ala Ala 15 Tyr Ser Gln His Thr 20 Leu Pro Ser Ser Ile 25 Asp Asn Gly Ala Gln 30 Leu Ile Ala Asn Ile 35 Asp Asp Pro Leu Ala 40 Val Asn Ala Gln Ser 45 Val Cys Pro Gly Tyr Lys 50 Ala Ser Asp Val 55 Gln Gln Thr Ser Arg 60 Gly Phe Thr Ala
    Page 18
    CL6220WOPCT_SequenceListing_ST25.txt
    Ser 65 Leu Gln Leu Ala Gly 70 Glu Pro Cys Asn Ala Tyr Gly Ile Asp 75 Val 80 Asp Ser Leu Ser Leu Ser Val Glu Val Leu Ala Lys Asp Arg Leu Asn 85 90 95 Ile Gln Ile Val Pro Thr His Val Asp Ser Ser Asn Ala Ser Trp Tyr 100 105 110 Ile Leu Pro Glu Asp Arg Val Pro Lys Ala Gln Ala Ser Ala Asp Ala 115 120 125 Ser Val Ser Gln Ser Asp Phe Glu Ile Glu Trp Ser Asn Asp Pro Ser 130 135 140 Phe Asn Ile Lys Ile Ile Arg Lys Ala Thr Gly Asp Ala Leu Phe Asp 145 150 155 160 Thr Ala Asp Ser Val Leu Val Phe Gln Asn Gln Phe Ile Glu Phe Val 165 170 175 Ser Ala Leu Pro Glu Gly Tyr Asn Leu Tyr Gly Leu Gly Glu Arg Met 180 185 190 Ala Gln Leu Arg Leu Leu Arg Asn Ala Thr Leu Thr Thr Tyr Ala Ala 195 200 205 Asp Val Gly Asp Pro Ile Asp Asp Asn Ile Tyr Gly Gln His Pro Phe 210 215 220 Tyr Leu Asp Thr Arg Tyr Tyr Thr Lys Asp Ala Asn Gly Ser Tyr Ser 225 230 235 240 Leu Val Asn Thr Asp Asp Ala Asp Ala Ser Gly Asp Tyr Glu Ser Phe 245 250 255 Ser His Gly Val Phe Leu Arg Asn Ala His Gly Gln Glu Val Ile Leu 260 265 270 Gln Ser Arg Asn Ile Thr Trp Arg Thr Ile Gly Gly Ser Ile Asp Leu 275 280 285 Thr Phe Tyr Ser Gly Pro Thr Gln Ala Asp Val Thr Lys Ser Tyr Gln 290 295 300 Leu Thr Thr Ile Gly Leu Pro Ala Met Gln Gln Tyr Ser Ala Leu Gly 305 310 315 320 Phe His Gln Cys Arg Trp Gly Tyr Arg Ser Trp Ser Glu Leu Glu Glu 325 330 335
    Page 19
    CL6220WOPCT_SequenceListing_ST25.txt
    Val Val Asn Thr 340 Phe Glu Gln Phe Glu 345 Ile Pro Leu Glu Tyr 350 Ile Trp Thr Asp Ile Asp Tyr Met Arg Gly Tyr Arg Asp Phe Asp Asn Asp Gln 355 360 365 Val His Phe Pro Tyr Asp Glu Gly Glu Glu Phe Leu Asp Arg Leu His 370 375 380 Lys Ser Gly Arg His Trp Val Pro Ile Val Asp Ser Ala Ile Tyr Ile 385 390 395 400 Pro Asn Pro Asp Asn Ala Ser Asp Ala Tyr Asp Thr Tyr Ala Arg Gly 405 410 415 Ala Lys Asp Asp Val Phe Ile Lys Asn Pro Asp Gly Ser Leu Tyr Ile 420 425 430 Gly Ala Val Trp Pro Gly Phe Thr Val Phe Pro Asp Trp His Asn Pro 435 440 445 Lys Ala Ala Glu Trp Trp Ser Asn Glu Leu Val Thr Trp Phe Glu Lys 450 455 460 Val Gln Tyr Asp Gly Ile Trp Ile Asp Met Ser Glu Val Ser Ser Phe 465 470 475 480 Cys Val Gly Ser Cys Gly Thr Gly Asn Leu His Leu Asn Pro Ala His 485 490 495 Pro Pro Phe Gln Leu Pro Gly Glu Pro Gly Asn Ile Glu Tyr Thr Tyr 500 505 510 Pro Glu Ala Phe Asn Val Thr Asn Ser Thr Glu Ala Ala Ser Ala Ser 515 520 525 Ala Ala Ser Ala Ser Gln Ser Ser Ala Ala Ala Ala Thr Gln Thr Asp 530 535 540 Val Ser Ser Thr Thr Thr Ser Tyr Leu Arg Thr Thr Pro Thr Pro Gly 545 550 555 560 Val Arg Asp Ile Asn Tyr Pro Pro Tyr Val Ile Asn His Val Gln Ser 565 570 575 Gly His Asp Leu Ala Val His Ala Ile Ser Pro Asn Ala Thr His Val 580 585 590 Asp Gly Val Gln Glu Tyr Asp Val His Ser Leu Trp Gly His Gln Ile 595 600 605
    Page 20
    CL6220WOPCT_SequenceListing_ST25.txt
    Leu Asn Ala 610 Thr Tyr Gln Gly 615 Leu Leu Glu Val Phe Thr 620 Glu Lys Arg Pro Phe Ile Ile Gly Arg Ser Thr Phe Ala Gly Ser Gly Lys Trp Ala 625 630 635 640 Gly His Trp Gly Gly Asp Asn Asn Ser Arg Trp Gly Ser Met Phe His 645 650 655 Ser Ile Ser Gln Ala Leu Ser Phe Ser Leu Phe Gly Ile Pro Met Phe 660 665 670 Gly Val Asp Thr Cys Gly Phe Asn Gly Asn Thr Asp Glu Glu Leu Cys 675 680 685 Asn Arg Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn His 690 695 700 Asn Thr Leu Ala Ala Leu Ser Gln Glu Pro Tyr Arg Trp Ala Ser Val 705 710 715 720 Thr Glu Ala Ala Lys Thr Ala Met Ser Ile Arg Tyr Ala Leu Leu Pro 725 730 735 Tyr Phe Tyr Thr Leu Phe His Gln Ala His Thr Thr Gly Ser Thr Val 740 745 750 Met Arg Ala Leu Ala Trp Glu Phe Pro Asn Asp Pro Ser Leu Ala Ala 755 760 765 Val Asp Thr Gln Phe Met Val Gly Pro Ser Ile Leu Val Thr Pro Val 770 775 780 Leu Glu Pro Leu Ala Lys Thr Val Lys Gly Val Phe Pro Gly Val Gly 785 790 795 800 Lys Gly Gln Val Trp Tyr Asp Trp Tyr Thr Gln Ala Ala Val Asp Ala 805 810 815 Lys Pro Gly Val Asn Thr Thr Ile Pro Ala Pro Leu Gly His Ile Pro 820 825 830 Val Tyr Val Arg Gly Gly Ser Ile Leu Pro Met Gln Glu Pro Ala Leu 835 840 845 Thr Thr Arg Asp Ala Arg Lys Thr Pro Trp Ser Leu Leu Ala Ala Leu 850 855 860 Asp Gly Asn Gln Thr Ala Ser Gly Gln Leu Tyr Leu Asp Asp Gly Ser
    865 870 875 880
    Page 21
    CL6220WOPCT_SequenceListing_ST25.txt
    Ser Val Asn Pro Ser Ser 885 Thr Leu Asn Val 890 Glu Phe Ala Ala Thr 895 His Ser Ser Ile Lys Val Ser Ala Lys Gly Asp Trp Arg Glu Lys Asn Ser 900 905 910 Leu Asp Ser Val Thr Val Leu Gly Val Ala Lys Glu Pro Ala Arg Val 915 920 925 Thr Phe Asn Arg Arg Arg Val Pro Pro Glu Ser Val Glu Tyr Asn Ala 930 935 940 Thr Ser Gln Val Leu Thr Val Ser Gly Leu Gln Lys Leu Thr Pro Arg 945 950 955 960 Gly Ala Trp Ala Glu Asp Trp Ile Leu Lys Trp 965 970
    <210> 7 <211> 3158 <212> DNA <213> Neosartorya fischeri <400> 7
    atggccagcc tcctgggcct cgtcgccagt gcttggctcc tccccacggc ctatggtgca 60 agccattcgc ttgcgcctag cacgtccgca acctcagcac acgcgcaata cactttacca 120 tcttctattg acgttggtgc tcaattggtc gccaacatcg acgatcccct tgccgtcgac 180 gcacagtctg tgtgtccggg ctacaaagcc tcaaatgtgc accagacatc ccaaggtttc 240 accgccagcc tacagctcgc gggcgaccca tgcaacgtgt acgggacaga cgttgattcg 300 ctgtctctga cagtggatta tctggccaag gaccgcctga acatccaaat tgttcctacc 360 tacgtggatg cctccaacgc ttcttggtac ctcctctcgg aagacttggt gccccgggct 420 caaggctctg gcgtgtccgc ctctcaaagc gactttgatg tgaagtggtc caatgagcct 480 tctttcaacc tcaaggtcat tcgcaaggct actggagacg tcctcttcga taccgagggc 540 tccgtcctgg tctttgagaa ccagtttatc gagtttgtct cttcgttgcc cgagggttac 600 aacctgtacg gtttgggaga gcgcatggcc cagctgcggc tcttgagaaa cgcgaccctg 660 accacctatg cagcggatgt gggagacccg attgataggt atgttgctga ccatggttga 720 aacctaatgt acgaagtcga caagcttaca atcggctctc cagcaacatc tatggacagc 780 atccgttcta tctcgacact agatactata ctaaaggcac gaatgggtct tactcgcttg 840 tcaacacgga cgaggcggac ttgtcggagg attatgaatc attctcccac ggtgtctttc 900 tgagaaactc tcatggtcag gaggttcttc tgcaaccccg caacatcacc tggcgcacaa 960 ttggtggtag catcgatttg actttctact ccggtcccac gcaagcggac gtcacaaaga 1020 gctaccagct ctccaccatt ggacttcctg caatgcagca gtacagcacc cttggattcc 1080
    Page 22
    CL6220WOPCT_SequenceListing_ST25.txt
    accaatgccg ctggggctac cagaattggt ctcagctcga ggaagtggtc aacaactttg 1140 agcgatttga gattcccctg gaatacatct ggtcagtctg atttctgagt ttatacatat 1200 ttcccagttc cttttattta cattccttcc aggagcgaca tcgattacat gcttggctac 1260 cgtgactttg agaatgatcc cgaacggttc tcctacgatg aaggcgagga atttctgaac 1320 aaacttcaca agtcgggacg acactacgtt cctatcgttg actcggcaat ctatattccc 1380 aaccccgaca atgcatcgga tgcgtaagtc cttgttatct tatcctcctt gttagatggt 1440 caagttctca cgaacgtttg aattccaggt acgagcctta tgctcgcggg gcaaaggatg 1500 acgttttcat caagaaccct gatggcaccc tctacatcgg tgcagtgtgg ccgggcttta 1560 ctgtcttccc agattggctc aaccccaagg catttgacta ctgggccaac gaactcgtca 1620 tctggtcaaa gaaggttgcg ttcgatggca tctggattga tatgagcgaa gtatcctctt 1680 tctgcgttgg cagctgtgga acaggaaagc tacatctgaa cccggttcac ccaccattcc 1740 agcttcccgg tgaacctgga aatatcggct acgactaccc cgaggccttc aacgtgacga 1800 actctaccga agcggcctct gcctccgccg cctctgccag tcaggcttcg gctgctgctg 1860 ctacccaaac cgccactacg tcaacatcta catcgtatct gcggacgacg cccacgccgg 1920 gcgtccgtga cgtcaactac cctccatatg tgattaatca tgttcaggag ggtcatgacc 1980 ttgccgttca cgccatttct cccaactcta ctcatgcgga cggcgtccag gaatacgatg 2040 ttcacagtct gtggggccac cagatcctca atgccaccta ctacggactg cgccaggtct 2100 tcactgagaa gcgacccttc atcattggtc ggtctacctt tgctggctcg ggcaagtggg 2160 ccggtcactg gggcggtgat aacaactcca aatgggggtc catgttcctg tccatctcgc 2220 agggtctgtc gttctcgctg ttcggtattc ccatgttcgg cgtggatacc tgcggtttca 2280 acggtaacac cgacgaggag ctctgcagcc ggtggatgca gctgtcggcc ttcttcccct 2340 tctaccgcaa ccacaatgtc cttggggcta tcccccagga gccctaccgt tgggcctctg 2400 tcacccaagc ctccaaggct gccatgaaga tccgctattc catcctacct tacttctaca 2460 ctcttttcca ccaggcccac accactggct ctaccgtcat gcgcgctctc gcctgggagt 2520 tccccacgga cccgtccctc gccgccgtcg acacccagtt catggtcggt ccttccatca 2580 tggtcgtccc cgtgctcgag cccctcgccg ataccgtcaa gggcgcgttc ccaggcgtcg 2640 gcaaaggcga agtctggtac gactggtaca cccagaccgc cgtagacgcc aaacccggcg 2700 tcaacaccac cattcccgct ccgctgggcc acattcccgt ctatgtccgt ggaggcagca 2760 tcctgcccat gcaggaaccc gccctcacga ccagagacgc ccgtaacact ccctggtcgc 2820 tactcgtcgc tctgagcggc aaccagactg cctcgggctc gctgtatctc gacgacggaa 2880 ccagcctcaa cccgtcccgc actctcgatg tcgacttcca ggctaccgcc tggagcatca 2940 aggtctcggt caagggtacc tgggaggaga agaaccgcct ggataaggtg actgtcctcg 3000 gcgtgggtga gaagccttcc gctgtgacgt tcaacggccg caacgtccac cctggctcgg 3060 tgcactacaa tgctacctcc aaggtgctgt ctgtgcaggg attgcacagc atgacgcccc 3120
    Page 23
    3158
    CL6220WOPCT_SequenceListing_ST25.txt atggcgcctg ggctggaaac tgggttctga aatggtag <210> 8 <211> 988 <212> PRT <213> Neosartorya fischeri <400> 8
    Met Ala Ser 1 Leu Leu Gly Leu Val 5 Ala Ser 10 Ala Trp Leu Leu Pro 15 Thr Ala Tyr Gly Ala Ser His Ser Leu Ala Pro Ser Thr Ser Ala Thr Ser 20 25 30 Ala His Ala Gln Tyr Thr Leu Pro Ser Ser Ile Asp Val Gly Ala Gln 35 40 45 Leu Val Ala Asn Ile Asp Asp Pro Leu Ala Val Asp Ala Gln Ser Val 50 55 60 Cys Pro Gly Tyr Lys Ala Ser Asn Val His Gln Thr Ser Gln Gly Phe 65 70 75 80 Thr Ala Ser Leu Gln Leu Ala Gly Asp Pro Cys Asn Val Tyr Gly Thr 85 90 95 Asp Val Asp Ser Leu Ser Leu Thr Val Asp Tyr Leu Ala Lys Asp Arg 100 105 110 Leu Asn Ile Gln Ile Val Pro Thr Tyr Val Asp Ala Ser Asn Ala Ser 115 120 125 Trp Tyr Leu Leu Ser Glu Asp Leu Val Pro Arg Ala Gln Gly Ser Gly 130 135 140 Val Ser Ala Ser Gln Ser Asp Phe Asp Val Lys Trp Ser Asn Glu Pro 145 150 155 160 Ser Phe Asn Leu Lys Val Ile Arg Lys Ala Thr Gly Asp Val Leu Phe 165 170 175 Asp Thr Glu Gly Ser Val Leu Val Phe Glu Asn Gln Phe Ile Glu Phe 180 185 190 Val Ser Ser Leu Pro Glu Gly Tyr Asn Leu Tyr Gly Leu Gly Glu Arg 195 200 205 Met Ala Gln Leu Arg Leu Leu Arg Asn Ala Thr Leu Thr Thr Tyr Ala 210 215 220 Ala Asp Val Gly Asp Pro Ile Asp Ser Asn Ile Tyr Gly Gln His Pro 225 230 235 240 Page 24
    CL6220WOPCT_SequenceListing_ST25.txt
    Phe Tyr Leu Asp Thr Arg Tyr Tyr Thr 245 Lys Gly Thr Asn Gly Ser Tyr 250 255 Ser Leu Val Asn Thr Asp Glu Ala Asp Leu Ser Glu Asp Tyr Glu Ser 260 265 270 Phe Ser His Gly Val Phe Leu Arg Asn Ser His Gly Gln Glu Val Leu 275 280 285 Leu Gln Pro Arg Asn Ile Thr Trp Arg Thr Ile Gly Gly Ser Ile Asp 290 295 300 Leu Thr Phe Tyr Ser Gly Pro Thr Gln Ala Asp Val Thr Lys Ser Tyr 305 310 315 320 Gln Leu Ser Thr Ile Gly Leu Pro Ala Met Gln Gln Tyr Ser Thr Leu 325 330 335 Gly Phe His Gln Cys Arg Trp Gly Tyr Gln Asn Trp Ser Gln Leu Glu 340 345 350 Glu Val Val Asn Asn Phe Glu Arg Phe Glu Ile Pro Leu Glu Tyr Ile 355 360 365 Trp Ser Asp Ile Asp Tyr Met Leu Gly Tyr Arg Asp Phe Glu Asn Asp 370 375 380 Pro Glu Arg Phe Ser Tyr Asp Glu Gly Glu Glu Phe Leu Asn Lys Leu 385 390 395 400 His Lys Ser Gly Arg His Tyr Val Pro Ile Val Asp Ser Ala Ile Tyr 405 410 415 Ile Pro Asn Pro Asp Asn Ala Ser Asp Ala Tyr Glu Pro Tyr Ala Arg 420 425 430 Gly Ala Lys Asp Asp Val Phe Ile Lys Asn Pro Asp Gly Thr Leu Tyr 435 440 445 Ile Gly Ala Val Trp Pro Gly Phe Thr Val Phe Pro Asp Trp Leu Asn 450 455 460 Pro Lys Ala Phe Asp Tyr Trp Ala Asn Glu Leu Val Ile Trp Ser Lys 465 470 475 480 Lys Val Ala Phe Asp Gly Ile Trp Ile Asp Met Ser Glu Val Ser Ser 485 490 495 Phe Cys Val Gly Ser Cys Gly Thr Gly Lys Leu His Leu Asn Pro Val 500 505 510
    Page 25
    CL6220WOPCT_SequenceListing_ST25.txt
    His Pro Pro 515 Phe Gln Leu Pro Gly 520 Glu Pro Gly Asn Ile Gly Tyr Asp 525 Tyr Pro Glu Ala Phe Asn Val Thr Asn Ser Thr Glu Ala Ala Ser Ala 530 535 540 Ser Ala Ala Ser Ala Ser Gln Ala Ser Ala Ala Ala Ala Thr Gln Thr 545 550 555 560 Ala Thr Thr Ser Thr Ser Thr Ser Tyr Leu Arg Thr Thr Pro Thr Pro 565 570 575 Gly Val Arg Asp Val Asn Tyr Pro Pro Tyr Val Ile Asn His Val Gln 580 585 590 Glu Gly His Asp Leu Ala Val His Ala Ile Ser Pro Asn Ser Thr His 595 600 605 Ala Asp Gly Val Gln Glu Tyr Asp Val His Ser Leu Trp Gly His Gln 610 615 620 Ile Leu Asn Ala Thr Tyr Tyr Gly Leu Arg Gln Val Phe Thr Glu Lys 625 630 635 640 Arg Pro Phe Ile Ile Gly Arg Ser Thr Phe Ala Gly Ser Gly Lys Trp 645 650 655 Ala Gly His Trp Gly Gly Asp Asn Asn Ser Lys Trp Gly Ser Met Phe 660 665 670 Leu Ser Ile Ser Gln Gly Leu Ser Phe Ser Leu Phe Gly Ile Pro Met 675 680 685 Phe Gly Val Asp Thr Cys Gly Phe Asn Gly Asn Thr Asp Glu Glu Leu 690 695 700 Cys Ser Arg Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn 705 710 715 720 His Asn Val Leu Gly Ala Ile Pro Gln Glu Pro Tyr Arg Trp Ala Ser 725 730 735 Val Thr Gln Ala Ser Lys Ala Ala Met Lys Ile Arg Tyr Ser Ile Leu 740 745 750 Pro Tyr Phe Tyr Thr Leu Phe His Gln Ala His Thr Thr Gly Ser Thr 755 760 765 Val Met Arg Ala Leu Ala Trp Glu Phe Pro Thr Asp Pro Ser Leu Ala 770 775 780
    Page 26
    CL6220WOPCT_SequenceListing_ST25.txt
    Ala 785 Val Asp Thr Gln Phe 790 Met Val Gly Pro Ser 795 Ile Met Val Val Pro 800 Val Leu Glu Pro Leu Ala Asp Thr Val Lys Gly Ala Phe Pro Gly Val 805 810 815 Gly Lys Gly Glu Val Trp Tyr Asp Trp Tyr Thr Gln Thr Ala Val Asp 820 825 830 Ala Lys Pro Gly Val Asn Thr Thr Ile Pro Ala Pro Leu Gly His Ile 835 840 845 Pro Val Tyr Val Arg Gly Gly Ser Ile Leu Pro Met Gln Glu Pro Ala 850 855 860 Leu Thr Thr Arg Asp Ala Arg Asn Thr Pro Trp Ser Leu Leu Val Ala 865 870 875 880 Leu Ser Gly Asn Gln Thr Ala Ser Gly Ser Leu Tyr Leu Asp Asp Gly 885 890 895 Thr Ser Leu Asn Pro Ser Arg Thr Leu Asp Val Asp Phe Gln Ala Thr 900 905 910 Ala Trp Ser Ile Lys Val Ser Val Lys Gly Thr Trp Glu Glu Lys Asn 915 920 925 Arg Leu Asp Lys Val Thr Val Leu Gly Val Gly Glu Lys Pro Ser Ala 930 935 940 Val Thr Phe Asn Gly Arg Asn Val His Pro Gly Ser Val His Tyr Asn 945 950 955 960 Ala Thr Ser Lys Val Leu Ser Val Gln Gly Leu His Ser Met Thr Pro 965 970 975 His Gly Ala Trp Ala Gly Asn Trp Val Leu Lys Trp 980 985 <210> ' 9 <211> 969 <212> PRT <213> Neosartorya fischeri <400> 9 Ala Ser His Ser Leu Ala Pro Ser Thr Ser Ala Thr Ser Ala His Ala 1 5 10 15 Gln Tyr Thr Leu Pro Ser Ser Ile Asp Val Gly Ala Gln Leu Val Ala 20 25 30
    Page 27
    CL6220WOPCT_SequenceListing_ST25.txt
    Asn Ile Asp 35 Asp Pro Leu Ala Val 40 Asp Ala Gln Ser Val 45 Cys Pro Gly Tyr Lys Ala Ser Asn Val His Gln Thr Ser Gln Gly Phe Thr Ala Ser 50 55 60 Leu Gln Leu Ala Gly Asp Pro Cys Asn Val Tyr Gly Thr Asp Val Asp 65 70 75 80 Ser Leu Ser Leu Thr Val Asp Tyr Leu Ala Lys Asp Arg Leu Asn Ile 85 90 95 Gln Ile Val Pro Thr Tyr Val Asp Ala Ser Asn Ala Ser Trp Tyr Leu 100 105 110 Leu Ser Glu Asp Leu Val Pro Arg Ala Gln Gly Ser Gly Val Ser Ala 115 120 125 Ser Gln Ser Asp Phe Asp Val Lys Trp Ser Asn Glu Pro Ser Phe Asn 130 135 140 Leu Lys Val Ile Arg Lys Ala Thr Gly Asp Val Leu Phe Asp Thr Glu 145 150 155 160 Gly Ser Val Leu Val Phe Glu Asn Gln Phe Ile Glu Phe Val Ser Ser 165 170 175 Leu Pro Glu Gly Tyr Asn Leu Tyr Gly Leu Gly Glu Arg Met Ala Gln 180 185 190 Leu Arg Leu Leu Arg Asn Ala Thr Leu Thr Thr Tyr Ala Ala Asp Val 195 200 205 Gly Asp Pro Ile Asp Ser Asn Ile Tyr Gly Gln His Pro Phe Tyr Leu 210 215 220 Asp Thr Arg Tyr Tyr Thr Lys Gly Thr Asn Gly Ser Tyr Ser Leu Val 225 230 235 240 Asn Thr Asp Glu Ala Asp Leu Ser Glu Asp Tyr Glu Ser Phe Ser His 245 250 255 Gly Val Phe Leu Arg Asn Ser His Gly Gln Glu Val Leu Leu Gln Pro 260 265 270 Arg Asn Ile Thr Trp Arg Thr Ile Gly Gly Ser Ile Asp Leu Thr Phe 275 280 285 Tyr Ser Gly Pro Thr Gln Ala Asp Val Thr Lys Ser Tyr Gln Leu Ser 290 295 300
    Page 28
    CL6220WOPCT_SequenceListing_ST25.txt
    Thr 305 Ile Gly Leu Pro Ala 310 Met Gln Gln Tyr Ser 315 Thr Leu Gly Phe His 320 Gln Cys Arg Trp Gly Tyr Gln Asn Trp Ser Gln Leu Glu Glu Val Val 325 330 335 Asn Asn Phe Glu Arg Phe Glu Ile Pro Leu Glu Tyr Ile Trp Ser Asp 340 345 350 Ile Asp Tyr Met Leu Gly Tyr Arg Asp Phe Glu Asn Asp Pro Glu Arg 355 360 365 Phe Ser Tyr Asp Glu Gly Glu Glu Phe Leu Asn Lys Leu His Lys Ser 370 375 380 Gly Arg His Tyr Val Pro Ile Val Asp Ser Ala Ile Tyr Ile Pro Asn 385 390 395 400 Pro Asp Asn Ala Ser Asp Ala Tyr Glu Pro Tyr Ala Arg Gly Ala Lys 405 410 415 Asp Asp Val Phe Ile Lys Asn Pro Asp Gly Thr Leu Tyr Ile Gly Ala 420 425 430 Val Trp Pro Gly Phe Thr Val Phe Pro Asp Trp Leu Asn Pro Lys Ala 435 440 445 Phe Asp Tyr Trp Ala Asn Glu Leu Val Ile Trp Ser Lys Lys Val Ala 450 455 460 Phe Asp Gly Ile Trp Ile Asp Met Ser Glu Val Ser Ser Phe Cys Val 465 470 475 480 Gly Ser Cys Gly Thr Gly Lys Leu His Leu Asn Pro Val His Pro Pro 485 490 495 Phe Gln Leu Pro Gly Glu Pro Gly Asn Ile Gly Tyr Asp Tyr Pro Glu 500 505 510 Ala Phe Asn Val Thr Asn Ser Thr Glu Ala Ala Ser Ala Ser Ala Ala 515 520 525 Ser Ala Ser Gln Ala Ser Ala Ala Ala Ala Thr Gln Thr Ala Thr Thr 530 535 540 Ser Thr Ser Thr Ser Tyr Leu Arg Thr Thr Pro Thr Pro Gly Val Arg 545 550 555 560 Asp Val Asn Tyr Pro Pro Tyr Val Ile Asn His Val Gln Glu Gly His 565 570 575
    Page 29
    CL6220WOPCT_SequenceListing_ST25.txt
    Asp Leu Ala Val 580 His Ala Ile Ser Pro Asn 585 Ser Thr His Ala 590 Asp Gly Val Gln Glu Tyr Asp Val His Ser Leu Trp Gly His Gln Ile Leu Asn 595 600 605 Ala Thr Tyr Tyr Gly Leu Arg Gln Val Phe Thr Glu Lys Arg Pro Phe 610 615 620 Ile Ile Gly Arg Ser Thr Phe Ala Gly Ser Gly Lys Trp Ala Gly His 625 630 635 640 Trp Gly Gly Asp Asn Asn Ser Lys Trp Gly Ser Met Phe Leu Ser Ile 645 650 655 Ser Gln Gly Leu Ser Phe Ser Leu Phe Gly Ile Pro Met Phe Gly Val 660 665 670 Asp Thr Cys Gly Phe Asn Gly Asn Thr Asp Glu Glu Leu Cys Ser Arg 675 680 685 Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn His Asn Val 690 695 700 Leu Gly Ala Ile Pro Gln Glu Pro Tyr Arg Trp Ala Ser Val Thr Gln 705 710 715 720 Ala Ser Lys Ala Ala Met Lys Ile Arg Tyr Ser Ile Leu Pro Tyr Phe 725 730 735 Tyr Thr Leu Phe His Gln Ala His Thr Thr Gly Ser Thr Val Met Arg 740 745 750 Ala Leu Ala Trp Glu Phe Pro Thr Asp Pro Ser Leu Ala Ala Val Asp 755 760 765 Thr Gln Phe Met Val Gly Pro Ser Ile Met Val Val Pro Val Leu Glu 770 775 780 Pro Leu Ala Asp Thr Val Lys Gly Ala Phe Pro Gly Val Gly Lys Gly 785 790 795 800 Glu Val Trp Tyr Asp Trp Tyr Thr Gln Thr Ala Val Asp Ala Lys Pro 805 810 815 Gly Val Asn Thr Thr Ile Pro Ala Pro Leu Gly His Ile Pro Val Tyr 820 825 830 Val Arg Gly Gly Ser Ile Leu Pro Met Gln Glu Pro Ala Leu Thr Thr 835 840 845
    Page 30
    CL6220WOPCT_SequenceListing_ST25.txt
    Arg Asp Ala Arg Asn Thr Pro Trp Ser Leu Leu Val Ala Leu Ser Gly 850 855 860 Asn Gln Thr Ala Ser Gly Ser Leu Tyr Leu Asp Asp Gly Thr Ser Leu 865 870 875 880 Asn Pro Ser Arg Thr Leu Asp Val Asp Phe Gln Ala Thr Ala Trp Ser 885 890 895 Ile Lys Val Ser Val Lys Gly Thr Trp Glu Glu Lys Asn Arg Leu Asp 900 905 910 Lys Val Thr Val Leu Gly Val Gly Glu Lys Pro Ser Ala Val Thr Phe 915 920 925 Asn Gly Arg Asn Val His Pro Gly Ser Val His Tyr Asn Ala Thr Ser 930 935 940 Lys Val Leu Ser Val Gln Gly Leu His Ser Met Thr Pro His Gly Ala 945 950 955 960 Trp Ala Gly Asn Trp Val Leu Lys Trp 965
    <210> 10 <211> 3385 <212> DNA <213> Neurospora crassa <400> 10
    atggccgtct tcacatcggc ttccttcttt cttcttgcgg ctcttgtccc tcaatggaca 60 tctgctcaac atgtatctgt ggttgcaacg tcatctggac ctggtgtgtt gagcgggacg 120 gtggcagggg attcccctat gttcactttc ccagcttcgg ctgacatcgg accaaatgtc 180 ttaccgaaca ttttcgaccc gcaagcagtt aatgttcaaa gcgtctgtcc aggatataca 240 gctgctaatg cacaaaagac ggagaaggga ctcacggctg acttgaccct tgctggccct 300 ccctgcaatg tctatggcaa cgacatcgag cacctgaagc ttaccattga gtttcaggcg 360 gacaatcgga tcaatgtcca gattcaacct cgctatactg gccccggtaa tgaaacctgg 420 ttcatacttc ctgaggtgct cgtgccacga ccagaggccg agcctgatgc gaatgccgct 480 agaagcaagc tggaaatctc gtggtcgaat gagcccacct tctccttcac agtgaagcgt 540 aaggagactg gagatgtctt gttcacgacc gagggccgtg tccttgttta tgaggatcag 600 ttcatcgagt tcggctcctc tttacccgag aattacaacc tgtatggtct cggcgaagtt 660 atgcatggct ttagactggg gaacaatctg acacgtaagt ctctttactt aacatgaatg 720 tgcatcgtgg attgcatctg actaacacca tctccaggca cgctgttcgc tgcggatgtg 780 ggcgacaacc tcgatgccaa catctacggc aaccacccga tctatctcga caccagatac 840 ttcaccaagg acgagtctgg aaaattaagc tacgtttccg acccagcaga caagaatgcc 900
    Page 31
    CL6220WOPCT_SequenceListing_ST25.txt
    aaatatgttt cgtatacaaa cggtgtcttc cttcgaaatg ctcatgcaca ggaagtgctc 960 cttcgacccg aaggtatcac ctggaggact ctcggcggaa gcatcgattt gtacttcttt 1020 gagggacctt ttgctcagga tatcatcaag tcttatcagc tcagtaccgt tggtcttcct 1080 gcaatgcagc agtattggac tcttggcttt caccaatgtc gctggggata ctcgaactgg 1140 actgttgtga aggatgtcgt tgacaatttc cgcaagtttg gcatcccgtt ggagacaatc 1200 tggagtgagt gaacccaagt ccttcagcat cagaatcttc cccatccatg cttttactga 1260 cttttttaag ccgacattga ttacatgaag ggctatcgtg actttgaaaa cgaccctgat 1320 cagttcagct acgaggaagg cgccaggttc ctcgaggagc ttcacaagaa tcaccagcac 1380 tacgtgccga ttgtcgactc ggccatctat gttccaaatc ctgacaagcc agaagatgat 1440 tacgaacctt accaccgtgg actcgaagct gatgctttca tcatgaaccc agacggttcg 1500 ctctatatcg gtgcagtgtg gcctggctac accgtgtttc cggactggat tggtgccgcc 1560 ctcaacggta caggtaccgt cggctggtgg acggacgagt ttgttaggta ctataagaag 1620 gtcgcttttg atggcatctg gattgacatg agcgaggttg cttctttctg catcggaagc 1680 tgtggcacgg gcaacctgac gctcaatccg gttcatccac catggggtct tcccggtgag 1740 ccaggcgctc ttgtgctcga ttaccccgaa ggttttgaaa aaaccaacgc gagtgaggca 1800 tcctctgcaa cgtcggttta caaaacgcag aacccagacc ccacgactac agccagcacc 1860 actagcacta cttcttacct caggacgaca ccgactcctg gagtgcgcaa tatcaactat 1920 ccaccatatg tcatcaacaa cttccacggc gacattggta ctcacgccct gagccccaat 1980 ggtacccacc atggcggcac agtcgactat gacttccaca acttgttcgg ccatcagatc 2040 ctccacgcaa cctaccaggc acttctcaaa gtctttgagg gcaagcgtcc ttttatcatc 2100 ggccgcagca cctttgcggg ttctggcaaa tgggccggtc actggggtgg tgataactat 2160 tccctatggg cgtttctgta ctttagcatt ccccaggccc tatccttctc catcttcggt 2220 ttccccatgt tcggcgtaga tacctgcggc ttcaatggca acacggacca cgagctgtgc 2280 tcacgatgga tgcagctcag cgcctttttc ccattctacc gcaaccacaa cgtccgtggc 2340 gccatcagcc aggagcccta cgtgtggagc tctgtgatcg atgcgtccaa gaaggcgatg 2400 aggattcgat acctcctgct cccgtacatg tacacactta tggcccaggc cagcctgtct 2460 ggagatacgg tcatgcgcgc actctcgtgg gagttcccgc aagagccgtg gttggcggat 2520 gcggatcgcc agttcatgtt gggcagcgcg gtgatggtga caccatgtct tgttcaaggg 2580 gccaatacag tggacggcgt gtttcctgga gttggcgatg ggaccatctg gtatgattgg 2640 tatacataca aggccgccag tgagggtgtt cagcctgggg agaatgtaac gatcgatgca 2700 cctctgggac acattccggt ttttctacgg ggtggccatg tcattccagt gcaagagccg 2760 ggcatgacta cgacggagag cagacaaaat gagtggagtg tcatcgttgc tcttgatggt 2820 gcgggtaagg cgaatggtac gttgtatctg gatgatggtg agagtttgga gccgggtgag 2880 aatgtgaagt gggttgatgt gagtcttctt ccgttctttc tatttttctt ctcttttccg Page 32 2940
    CL6220WOPCT_SequenceListing_ST25.txt
    tgagtgttca gtcggtcgat tgccactcgc tcctctcgcg acaaggtcga aagtgctaac 3000 cgggtttggc tgtttctagt tcacggttga gaagaactca tttcgagtga cacctcaggg 3060 caagtacctt gaccgaaact cactggccaa cgtcacgatc ctgggagtgg ccgaggcacc 3120 tctgggagtg gctattaata gtcatctgct cggatcagct tcttggtcct acgactccga 3180 ggggaagttc ctttcggtaa ccgagctgca ggacaacttc aaggaagggg cgtgggcatc 3240 caactggacg ctgtcgtgga actcggcctc aaactcgggc tcgtctcctg ttcagggagg 3300 cggcggcagg ctcgagttca gcacgcccaa tttgctccat gcagctgctt tcggcatcct 3360 ttttggccgc atgtttgtag tttag 3385
    <210> 11 <211> 1044 <212> PRT <213> Neurospora crassa <400> 11
    Met Ala 1 Val Phe Thr 5 Ser Ala Ser Phe Phe 10 Leu Leu Ala Ala Leu 15 Val Pro Gln Trp Thr Ser Ala Gln His Val Ser Val Val Ala Thr Ser Ser 20 25 30 Gly Pro Gly Val Leu Ser Gly Thr Val Ala Gly Asp Ser Pro Met Phe 35 40 45 Thr Phe Pro Ala Ser Ala Asp Ile Gly Pro Asn Val Leu Pro Asn Ile 50 55 60 Phe Asp Pro Gln Ala Val Asn Val Gln Ser Val Cys Pro Gly Tyr Thr 65 70 75 80 Ala Ala Asn Ala Gln Lys Thr Glu Lys Gly Leu Thr Ala Asp Leu Thr 85 90 95 Leu Ala Gly Pro Pro Cys Asn Val Tyr Gly Asn Asp Ile Glu His Leu 100 105 110 Lys Leu Thr Ile Glu Phe Gln Ala Asp Asn Arg Ile Asn Val Gln Ile 115 120 125 Gln Pro Arg Tyr Thr Gly Pro Gly Asn Glu Thr Trp Phe Ile Leu Pro 130 135 140 Glu Val Leu Val Pro Arg Pro Glu Ala Glu Pro Asp Ala Asn Ala Ala 145 150 155 160 Arg Ser Lys Leu Glu Ile Ser Trp Ser Asn Glu Pro Thr Phe Ser Phe
    165 170 175
    Page 33
    CL6220WOPCT_SequenceListing_ST25.txt
    Thr Val Lys Arg Lys Glu Thr Gly Asp Val Leu Phe Thr Thr 190 Glu Gly 180 185 Arg Val Leu Val Tyr Glu Asp Gln Phe Ile Glu Phe Gly Ser Ser Leu 195 200 205 Pro Glu Asn Tyr Asn Leu Tyr Gly Leu Gly Glu Val Met His Gly Phe 210 215 220 Arg Leu Gly Asn Asn Leu Thr Arg Thr Leu Phe Ala Ala Asp Val Gly 225 230 235 240 Asp Asn Leu Asp Ala Asn Ile Tyr Gly Asn His Pro Ile Tyr Leu Asp 245 250 255 Thr Arg Tyr Phe Thr Lys Asp Glu Ser Gly Lys Leu Ser Tyr Val Ser 260 265 270 Asp Pro Ala Asp Lys Asn Ala Lys Tyr Val Ser Tyr Thr Asn Gly Val 275 280 285 Phe Leu Arg Asn Ala His Ala Gln Glu Val Leu Leu Arg Pro Glu Gly 290 295 300 Ile Thr Trp Arg Thr Leu Gly Gly Ser Ile Asp Leu Tyr Phe Phe Glu 305 310 315 320 Gly Pro Phe Ala Gln Asp Ile Ile Lys Ser Tyr Gln Leu Ser Thr Val 325 330 335 Gly Leu Pro Ala Met Gln Gln Tyr Trp Thr Leu Gly Phe His Gln Cys 340 345 350 Arg Trp Gly Tyr Ser Asn Trp Thr Val Val Lys Asp Val Val Asp Asn 355 360 365 Phe Arg Lys Phe Gly Ile Pro Leu Glu Thr Ile Trp Thr Asp Ile Asp 370 375 380 Tyr Met Lys Gly Tyr Arg Asp Phe Glu Asn Asp Pro Asp Gln Phe Ser 385 390 395 400 Tyr Glu Glu Gly Ala Arg Phe Leu Glu Glu Leu His Lys Asn His Gln 405 410 415 His Tyr Val Pro Ile Val Asp Ser Ala Ile Tyr Val Pro Asn Pro Asp 420 425 430 Lys Pro Glu Asp Asp Tyr Glu Pro Tyr His Arg Gly Leu Glu Ala Asp 435 440 445
    Page 34
    CL6220WOPCT_SequenceListing_ST25.txt
    Ala Phe Ile 450 Met Asn Pro Asp Gly Ser 455 Leu Tyr Ile 460 Gly Ala Val Trp Pro Gly Tyr Thr Val Phe Pro Asp Trp Ile Gly Ala Ala Leu Asn Gly 465 470 475 480 Thr Gly Thr Val Gly Trp Trp Thr Asp Glu Phe Val Arg Tyr Tyr Lys 485 490 495 Lys Val Ala Phe Asp Gly Ile Trp Ile Asp Met Ser Glu Val Ala Ser 500 505 510 Phe Cys Ile Gly Ser Cys Gly Thr Gly Asn Leu Thr Leu Asn Pro Val 515 520 525 His Pro Pro Trp Gly Leu Pro Gly Glu Pro Gly Ala Leu Val Leu Asp 530 535 540 Tyr Pro Glu Gly Phe Glu Lys Thr Asn Ala Ser Glu Ala Ser Ser Ala 545 550 555 560 Thr Ser Val Tyr Lys Thr Gln Asn Pro Asp Pro Thr Thr Thr Ala Ser 565 570 575 Thr Thr Ser Thr Thr Ser Tyr Leu Arg Thr Thr Pro Thr Pro Gly Val 580 585 590 Arg Asn Ile Asn Tyr Pro Pro Tyr Val Ile Asn Asn Phe His Gly Asp 595 600 605 Ile Gly Thr His Ala Leu Ser Pro Asn Gly Thr His His Gly Gly Thr 610 615 620 Val Asp Tyr Asp Phe His Asn Leu Phe Gly His Gln Ile Leu His Ala 625 630 635 640 Thr Tyr Gln Ala Leu Leu Lys Val Phe Glu Gly Lys Arg Pro Phe Ile 645 650 655 Ile Gly Arg Ser Thr Phe Ala Gly Ser Gly Lys Trp Ala Gly His Trp 660 665 670 Gly Gly Asp Asn Tyr Ser Leu Trp Ala Phe Leu Tyr Phe Ser Ile Pro 675 680 685 Gln Ala Leu Ser Phe Ser Ile Phe Gly Phe Pro Met Phe Gly Val Asp 690 695 700 Thr Cys Gly Phe Asn Gly Asn Thr Asp His Glu Leu Cys Ser Arg Trp 705 710 715 720
    Page 35
    CL6220WOPCT_SequenceListing_ST25.txt
    Met Gln Leu Ser Ala 725 Phe Phe Pro Phe Tyr Arg Asn His Asn Val Arg 730 735 Gly Ala Ile Ser Gln Glu Pro Tyr Val Trp Ser Ser Val Ile Asp Ala 740 745 750 Ser Lys Lys Ala Met Arg Ile Arg Tyr Leu Leu Leu Pro Tyr Met Tyr 755 760 765 Thr Leu Met Ala Gln Ala Ser Leu Ser Gly Asp Thr Val Met Arg Ala 770 775 780 Leu Ser Trp Glu Phe Pro Gln Glu Pro Trp Leu Ala Asp Ala Asp Arg 785 790 795 800 Gln Phe Met Leu Gly Ser Ala Val Met Val Thr Pro Cys Leu Val Gln 805 810 815 Gly Ala Asn Thr Val Asp Gly Val Phe Pro Gly Val Gly Asp Gly Thr 820 825 830 Ile Trp Tyr Asp Trp Tyr Thr Tyr Lys Ala Ala Ser Glu Gly Val Gln 835 840 845 Pro Gly Glu Asn Val Thr Ile Asp Ala Pro Leu Gly His Ile Pro Val 850 855 860 Phe Leu Arg Gly Gly His Val Ile Pro Val Gln Glu Pro Gly Met Thr 865 870 875 880 Thr Thr Glu Ser Arg Gln Asn Glu Trp Ser Val Ile Val Ala Leu Asp 885 890 895 Gly Ala Gly Lys Ala Asn Gly Thr Leu Tyr Leu Asp Asp Gly Glu Ser 900 905 910 Leu Glu Pro Gly Glu Asn Val Lys Trp Val Asp Phe Thr Val Glu Lys 915 920 925 Asn Ser Phe Arg Val Thr Pro Gln Gly Lys Tyr Leu Asp Arg Asn Ser 930 935 940 Leu Ala Asn Val Thr Ile Leu Gly Val Ala Glu Ala Pro Leu Gly Val 945 950 955 960 Ala Ile Asn Ser His Leu Leu Gly Ser Ala Ser Trp Ser Tyr Asp Ser 965 970 975 Glu Gly Lys Phe Leu Ser Val Thr Glu Leu Gln Asp Asn Phe Lys Glu 980 985 990
    Page 36
    CL6220WOPCT_SequenceListing_ST25.txt
    Gly Ala Trp Ala Ser Asn Trp Thr Leu Ser Trp Asn Ser Ala Ser Asn 995 1000 1005
    Ser Gly Ser Ser Pro Val Gln Gly Gly Gly Gly Arg Leu Glu Phe 1010 1015 1020
    Ser Thr Pro Asn Leu Leu His Ala Ala Ala Phe Gly Ile Leu Phe 1025 1030 1035
    Gly Arg Met Phe Val Val 1040 <210> 12 <211> 1022 <212> PRT <213> Neurospora crassa <400> 12
    Gln 1 His Val Ser Val 5 Val Ala Thr Ser Ser Gly Pro 10 Gly Val Leu 15 Ser Gly Thr Val Ala Gly Asp Ser Pro Met Phe Thr Phe Pro Ala Ser Ala 20 25 30 Asp Ile Gly Pro Asn Val Leu Pro Asn Ile Phe Asp Pro Gln Ala Val 35 40 45 Asn Val Gln Ser Val Cys Pro Gly Tyr Thr Ala Ala Asn Ala Gln Lys 50 55 60 Thr Glu Lys Gly Leu Thr Ala Asp Leu Thr Leu Ala Gly Pro Pro Cys 65 70 75 80 Asn Val Tyr Gly Asn Asp Ile Glu His Leu Lys Leu Thr Ile Glu Phe 85 90 95 Gln Ala Asp Asn Arg Ile Asn Val Gln Ile Gln Pro Arg Tyr Thr Gly 100 105 110 Pro Gly Asn Glu Thr Trp Phe Ile Leu Pro Glu Val Leu Val Pro Arg 115 120 125 Pro Glu Ala Glu Pro Asp Ala Asn Ala Ala Arg Ser Lys Leu Glu Ile 130 135 140 Ser Trp Ser Asn Glu Pro Thr Phe Ser Phe Thr Val Lys Arg Lys Glu 145 150 155 160 Thr Gly Asp Val Leu Phe Thr Thr Glu Gly Arg Val Leu Val Tyr Glu 165 170 175
    Page 37
    Asp Gln Phe Ile 180 Glu CL6220WOPCT_SequenceListing_ST25.txt Leu Phe Gly Ser Ser 185 Leu Pro Glu Asn Tyr 190 Asn Tyr Gly Leu Gly Glu Val Met His Gly Phe Arg Leu Gly Asn Asn Leu 195 200 205 Thr Arg Thr Leu Phe Ala Ala Asp Val Gly Asp Asn Leu Asp Ala Asn 210 215 220 Ile Tyr Gly Asn His Pro Ile Tyr Leu Asp Thr Arg Tyr Phe Thr Lys 225 230 235 240 Asp Glu Ser Gly Lys Leu Ser Tyr Val Ser Asp Pro Ala Asp Lys Asn 245 250 255 Ala Lys Tyr Val Ser Tyr Thr Asn Gly Val Phe Leu Arg Asn Ala His 260 265 270 Ala Gln Glu Val Leu Leu Arg Pro Glu Gly Ile Thr Trp Arg Thr Leu 275 280 285 Gly Gly Ser Ile Asp Leu Tyr Phe Phe Glu Gly Pro Phe Ala Gln Asp 290 295 300 Ile Ile Lys Ser Tyr Gln Leu Ser Thr Val Gly Leu Pro Ala Met Gln 305 310 315 320 Gln Tyr Trp Thr Leu Gly Phe His Gln Cys Arg Trp Gly Tyr Ser Asn 325 330 335 Trp Thr Val Val Lys Asp Val Val Asp Asn Phe Arg Lys Phe Gly Ile 340 345 350 Pro Leu Glu Thr Ile Trp Thr Asp Ile Asp Tyr Met Lys Gly Tyr Arg 355 360 365 Asp Phe Glu Asn Asp Pro Asp Gln Phe Ser Tyr Glu Glu Gly Ala Arg 370 375 380 Phe Leu Glu Glu Leu His Lys Asn His Gln His Tyr Val Pro Ile Val 385 390 395 400 Asp Ser Ala Ile Tyr Val Pro Asn Pro Asp Lys Pro Glu Asp Asp Tyr 405 410 415 Glu Pro Tyr His Arg Gly Leu Glu Ala Asp Ala Phe Ile Met Asn Pro 420 425 430 Asp Gly Ser Leu Tyr Ile Gly Ala Val Trp Pro Gly Tyr Thr Val Phe 435 440 445
    Page 38
    CL6220WOPCT_SequenceListing_ST25.txt
    Pro Asp Trp 450 Ile Gly Ala Ala Leu Asn Gly Thr Gly Thr Val Gly Trp 455 460 Trp Thr Asp Glu Phe Val Arg Tyr Tyr Lys Lys Val Ala Phe Asp Gly 465 470 475 480 Ile Trp Ile Asp Met Ser Glu Val Ala Ser Phe Cys Ile Gly Ser Cys 485 490 495 Gly Thr Gly Asn Leu Thr Leu Asn Pro Val His Pro Pro Trp Gly Leu 500 505 510 Pro Gly Glu Pro Gly Ala Leu Val Leu Asp Tyr Pro Glu Gly Phe Glu 515 520 525 Lys Thr Asn Ala Ser Glu Ala Ser Ser Ala Thr Ser Val Tyr Lys Thr 530 535 540 Gln Asn Pro Asp Pro Thr Thr Thr Ala Ser Thr Thr Ser Thr Thr Ser 545 550 555 560 Tyr Leu Arg Thr Thr Pro Thr Pro Gly Val Arg Asn Ile Asn Tyr Pro 565 570 575 Pro Tyr Val Ile Asn Asn Phe His Gly Asp Ile Gly Thr His Ala Leu 580 585 590 Ser Pro Asn Gly Thr His His Gly Gly Thr Val Asp Tyr Asp Phe His 595 600 605 Asn Leu Phe Gly His Gln Ile Leu His Ala Thr Tyr Gln Ala Leu Leu 610 615 620 Lys Val Phe Glu Gly Lys Arg Pro Phe Ile Ile Gly Arg Ser Thr Phe 625 630 635 640 Ala Gly Ser Gly Lys Trp Ala Gly His Trp Gly Gly Asp Asn Tyr Ser 645 650 655 Leu Trp Ala Phe Leu Tyr Phe Ser Ile Pro Gln Ala Leu Ser Phe Ser 660 665 670 Ile Phe Gly Phe Pro Met Phe Gly Val Asp Thr Cys Gly Phe Asn Gly 675 680 685 Asn Thr Asp His Glu Leu Cys Ser Arg Trp Met Gln Leu Ser Ala Phe 690 695 700 Phe Pro Phe Tyr Arg Asn His Asn Val Arg Gly Ala Ile Ser Gln Glu 705 710 715 720
    Page 39
    CL6220WOPCT_SequenceListing_ST25.txt Pro Tyr Val Trp Ser Ser Val Ile Asp Ala Ser Lys Lys Ala Met Arg
    725 730 735
    Ile Arg Tyr Leu 740 Leu Leu Pro Tyr Met Tyr Thr 745 Leu Met Ala 750 Gln Ala Ser Leu Ser Gly Asp Thr Val Met Arg Ala Leu Ser Trp Glu Phe Pro 755 760 765 Gln Glu Pro Trp Leu Ala Asp Ala Asp Arg Gln Phe Met Leu Gly Ser 770 775 780 Ala Val Met Val Thr Pro Cys Leu Val Gln Gly Ala Asn Thr Val Asp 785 790 795 800 Gly Val Phe Pro Gly Val Gly Asp Gly Thr Ile Trp Tyr Asp Trp Tyr 805 810 815 Thr Tyr Lys Ala Ala Ser Glu Gly Val Gln Pro Gly Glu Asn Val Thr 820 825 830 Ile Asp Ala Pro Leu Gly His Ile Pro Val Phe Leu Arg Gly Gly His 835 840 845 Val Ile Pro Val Gln Glu Pro Gly Met Thr Thr Thr Glu Ser Arg Gln 850 855 860 Asn Glu Trp Ser Val Ile Val Ala Leu Asp Gly Ala Gly Lys Ala Asn 865 870 875 880 Gly Thr Leu Tyr Leu Asp Asp Gly Glu Ser Leu Glu Pro Gly Glu Asn 885 890 895 Val Lys Trp Val Asp Phe Thr Val Glu Lys Asn Ser Phe Arg Val Thr 900 905 910 Pro Gln Gly Lys Tyr Leu Asp Arg Asn Ser Leu Ala Asn Val Thr Ile 915 920 925 Leu Gly Val Ala Glu Ala Pro Leu Gly Val Ala Ile Asn Ser His Leu 930 935 940 Leu Gly Ser Ala Ser Trp Ser Tyr Asp Ser Glu Gly Lys Phe Leu Ser 945 950 955 960 Val Thr Glu Leu Gln Asp Asn Phe Lys Glu Gly Ala Trp Ala Ser Asn 965 970 975 Trp Thr Leu Ser Trp Asn Ser Ala Ser Asn Ser Gly Ser Ser Pro Val 980 985 990
    Page 40
    CL6220WOPCT_SequenceListing_ST25.txt Gln Gly Gly Gly Gly Arg Leu Glu Phe Ser Thr Pro Asn Leu Leu His
    995 1000 1005
    Ala Ala Ala Phe Gly Ile Leu Phe Gly Arg Met Phe Val Val 1010 1015 1020 <210> 13 <211> 3293 <212> DNA <213> Rasamsonia composticola <400> 13
    atgcggccaa cttccctcgt caagcacttg gctgcgacca gcctcctctt tctcgcggcg 60 gatgcagctg ctatcgtccg ccgcaacggc gcctctcctt catgccccgg ctataaagcg 120 agtaacgtga agaccgtcga cggtgaaatc gtcagcgcgg atctcaatct cgcgggtccc 180 gcctgcaatg tgtatggcac ggatctggac gatctgaagc tgcaggttga gtaccaatca 240 ggtaagtcgc acagcatcgc cgtctgcagc ttatcggcag catgtgatcg gcgcctggac 300 tagggcttag ggcctggggt tagggctgac tgcttgctgc cgttcgatgt gactggaact 360 gtacggttgg ttgacaacga tctgacatct gcagaacaac gcctccatgt gaagatctac 420 gatgccgccg agcaggtcta ccaggtgccc accgcggtgc ttccccggcc cagcagcgcc 480 aacatccccc cggccaagtc ggacctgaag ttctccatga ccaacgaccc cttctccttt 540 accatcaagc gcagatcaaa cggcgaaatc ctcttcgaca cctccggcca tccgctgatc 600 ttcgagtcgc agtatctggg cctccgtacc aagctgccgg actcgcccaa catctacggc 660 ctgggagagc acaccggttc tttccgcctg cccaccaaga attacacccg cacgctgtgg 720 tcgcgcgatg cgtacggtac gcccaaagac accaacctgt acggcaacca cccggtgtac 780 ttcgactacc gcggcagcaa cggcacccat ggcgtgttcc tgctgaacag caacggcatg 840 gacgtcgata tcgacgtcga ctcggacgga cagtacctgc agtacaacac cctggggggc 900 gtgctggact tctacttcct cagcgggccg gatcccaagg ccgtcgcgac gcagtatgcc 960 gagacggtcg gaaaaccggt catgatgccc tactggggat tcggcttcca caactgcaga 1020 tatggatacc aggacatcta tgaggttgct gagatcattg ccaactacag tgccgcaaac 1080 attccgcttg agacccaatg gactgatatc ggtatgcttt ccatcccggt gccgtggttt 1140 ttgcttctca gcgtggctga ctgttgcaga ctatatggat ctgaggaaag tgtttacgct 1200 ggacccctat cgctatccat tgaagctcgt ccaagaggtt gtctcttatc tccacaagca 1260 caaccagcac tacatcatga tggtggaccc tgcagtggca taccagaact attcagcgtt 1320 caacaacggc gtcgctgccg acgctttcct gaagttctcg aatggctcca tctaccaggg 1380 tgtcgtctgg ccggggccga cggcgttccc ggactggttc gcaccccaga cacaggagtt 1440 ttggaatagc gagttctcga ccttctttga ccccgcccac ggcgtcgaca tcgatgccct 1500 ttggatcgac atgaacgagg cgtccaactt ctgcgacttt ccctgctcga accccgccgc 1560 gtatgcggca gccaacggcg atccgcccac gcctccgccg gtccgcttga gccccccgag 1620
    Page 41
    CL6220WOPCT_SequenceListing_ST25.txt gccgattcct ggatttggcc ctgacttcca gccgacgtgt gtcgccacgg tgtcgttcga 1680 ttgcgatgcg cagacctact ttggcgagaa catcctcatc ctgggtaact cgacgacact 1740 gggagccggc gacgttcaca tggcgccagt catgagcgcg aacaactacc cgatctggca 1800 gctgaccgtc cagatgccgc cgaatgggac gttctcgtac cagtacgttc gcaaggaatc 1860 ggacggcagt tacatctacg aacagacgaa tcgcacggtc acgacgggcg actgcaccag 1920 cggcacgctt aaggtgtccg acaccatcac caccagctct ggaccgcaca agagatccga 1980 attacggccg ctggtgcgct cgccgttccc ggcggaggac ctgaccaggc gccagtctgg 2040 atcgatgttg ggcctgccca acaggaacct gctgaatccg ccatacacca tccacaatgc 2100 ggctggcaac ctgagtgaga agaccatcaa caccgacctg atccatgcgg gcggatatgc 2160 cgagtacgac acgcacaact tgtacggcac gatgatgagc gcgaccagca gggaggcgat 2220 gctgaaccgc agaccagcag tcaggccact tgtgtaagtc atccatcgtc cttaagccag 2280 acacagcatg ttaggggcta acgggcagta gcattacccg gtcgaccttc gctggagccg 2340 gccgacaggt cggccactgg ctcggcgaca atttcgccga ttgggaccac taccggtgga 2400 cgatcgccga gctgcaggaa ttcgcggcgc tgttccagat cccgatggtc ggcagcgaca 2460 tctgcgggta cgacggcaac acgacggaca acctgtgctc gcgctgggtc ttcctcggcg 2520 ccttctcgcc cttcttccgc gaccactcgg acaaccagtc gccgccgcac gagctgtacc 2580 gcactccgca gatcgcggcg gccgcgcgcg ccgccatcga catccgctac cgtctgctcg 2640 actacgcgta cacggtgctg tggacgcaga cccagaccgg cgcgccgatg ctcaacccca 2700 tgttcttcga gtacccggcc gacagcaaca ccgccgacct gcagtaccag ttcttctggg 2760 gcgacagcat catggtcgcg cccgtgaccg acaacgactc gaccaccgtc aacgtctact 2820 tcccgaagga ccagttctac gacttctaca ccggcgcacc tgtgtccggg gagggcaata 2880 ccgtcaccct gaccgacgtc ggcttcgaca ccatcccgct gtacttcaag ggcgggagca 2940 tcgtgcccat gcgcgtgcgc tcggcgaaca cgacggcgga gctgcggcag caggacttcg 3000 tcgtcgtcat cgccccggac agccacggcg acgcgacggg ccagctgtac ctcgacgacg 3060 gcgagagcat caaccagccg cacaccagcg agatccagtt ctcgtaccgc ggaggccatt 3120 tcagcatgac aggcaagttt gactatgatc ccggcaacgt ggtcatcagc cagatcacgc 3180 tgctgggtgc ggacggcgcc ggtaaagggg gttcgtataa cagcaccacc aaggtggcga 3240 cctacaaagt caacgcgaag ttgacgggta aattcgaagc cagcttacac taa 3293 <210> 14 <211> 1035 <212> PRT <213> Rasamsonia composticola <400> 14
    Met Arg Pro Thr Ser Leu Val Lys His Leu Ala Ala Thr Ser Leu Leu
    1 5 10 15
    Page 42
    CL6220WOPCT_SequenceListing_ST25.txt
    Phe Leu Ala Ala 20 Asp Ala Ala Ala Ile Val 25 Arg Arg Asn Gly Ala Ser 30 Pro Ser Cys Pro Gly Tyr Lys Ala Ser Asn Val Lys Thr Val Asp Gly 35 40 45 Glu Ile Val Ser Ala Asp Leu Asn Leu Ala Gly Pro Ala Cys Asn Val 50 55 60 Tyr Gly Thr Asp Leu Asp Asp Leu Lys Leu Gln Val Glu Tyr Gln Ser 65 70 75 80 Gly Pro Gly Val Arg Ala Asp Cys Leu Leu Pro Phe Asp Val Thr Gly 85 90 95 Thr Val Arg Leu Val Asp Asn Asp Leu Thr Ser Ala Glu Gln Arg Leu 100 105 110 His Val Lys Ile Tyr Asp Ala Ala Glu Gln Val Tyr Gln Val Pro Thr 115 120 125 Ala Val Leu Pro Arg Pro Ser Ser Ala Asn Ile Pro Pro Ala Lys Ser 130 135 140 Asp Leu Lys Phe Ser Met Thr Asn Asp Pro Phe Ser Phe Thr Ile Lys 145 150 155 160 Arg Arg Ser Asn Gly Glu Ile Leu Phe Asp Thr Ser Gly His Pro Leu 165 170 175 Ile Phe Glu Ser Gln Tyr Leu Gly Leu Arg Thr Lys Leu Pro Asp Ser 180 185 190 Pro Asn Ile Tyr Gly Leu Gly Glu His Thr Gly Ser Phe Arg Leu Pro 195 200 205 Thr Lys Asn Tyr Thr Arg Thr Leu Trp Ser Arg Asp Ala Tyr Gly Thr 210 215 220 Pro Lys Asp Thr Asn Leu Tyr Gly Asn His Pro Val Tyr Phe Asp Tyr 225 230 235 240 Arg Gly Ser Asn Gly Thr His Gly Val Phe Leu Leu Asn Ser Asn Gly 245 250 255 Met Asp Val Asp Ile Asp Val Asp Ser Asp Gly Gln Tyr Leu Gln Tyr 260 265 270 Asn Thr Leu Gly Gly Val Leu Asp Phe Tyr Phe Leu Ser Gly Pro Asp 275 280 285
    Page 43
    CL6220WOPCT_SequenceListing_ST25.txt
    Pro Lys Ala 290 Val Ala Thr Gln Tyr Ala Glu Thr 295 Val 300 Gly Lys Pro Val Met Met Pro Tyr Trp Gly Phe Gly Phe His Asn Cys Arg Tyr Gly Tyr 305 310 315 320 Gln Asp Ile Tyr Glu Val Ala Glu Ile Ile Ala Asn Tyr Ser Ala Ala 325 330 335 Asn Ile Pro Leu Glu Thr Gln Trp Thr Asp Ile Asp Tyr Met Asp Leu 340 345 350 Arg Lys Val Phe Thr Leu Asp Pro Tyr Arg Tyr Pro Leu Lys Leu Val 355 360 365 Gln Glu Val Val Ser Tyr Leu His Lys His Asn Gln His Tyr Ile Met 370 375 380 Met Val Asp Pro Ala Val Ala Tyr Gln Asn Tyr Ser Ala Phe Asn Asn 385 390 395 400 Gly Val Ala Ala Asp Ala Phe Leu Lys Phe Ser Asn Gly Ser Ile Tyr 405 410 415 Gln Gly Val Val Trp Pro Gly Pro Thr Ala Phe Pro Asp Trp Phe Ala 420 425 430 Pro Gln Thr Gln Glu Phe Trp Asn Ser Glu Phe Ser Thr Phe Phe Asp 435 440 445 Pro Ala His Gly Val Asp Ile Asp Ala Leu Trp Ile Asp Met Asn Glu 450 455 460 Ala Ser Asn Phe Cys Asp Phe Pro Cys Ser Asn Pro Ala Ala Tyr Ala 465 470 475 480 Ala Ala Asn Gly Asp Pro Pro Thr Pro Pro Pro Val Arg Leu Ser Pro 485 490 495 Pro Arg Pro Ile Pro Gly Phe Gly Pro Asp Phe Gln Pro Thr Cys Val 500 505 510 Ala Thr Val Ser Phe Asp Cys Asp Ala Gln Thr Tyr Phe Gly Glu Asn 515 520 525 Ile Leu Ile Leu Gly Asn Ser Thr Thr Leu Gly Ala Gly Asp Val His 530 535 540 Met Ala Pro Val Met Ser Ala Asn Asn Tyr Pro Ile Trp Gln Leu Thr 545 550 555 560
    Page 44
    CL6220WOPCT_SequenceListing_ST25.txt
    Val Gln Met Pro Pro 565 Asn Gly Thr Phe Ser Tyr Gln Tyr Val 570 Arg 575 Lys Glu Ser Asp Gly Ser Tyr Ile Tyr Glu Gln Thr Asn Arg Thr Val Thr 580 585 590 Thr Gly Asp Cys Thr Ser Gly Thr Leu Lys Val Ser Asp Thr Ile Thr 595 600 605 Thr Ser Ser Gly Pro His Lys Arg Ser Glu Leu Arg Pro Leu Val Arg 610 615 620 Ser Pro Phe Pro Ala Glu Asp Leu Thr Arg Arg Gln Ser Gly Ser Met 625 630 635 640 Leu Gly Leu Pro Asn Arg Asn Leu Leu Asn Pro Pro Tyr Thr Ile His 645 650 655 Asn Ala Ala Gly Asn Leu Ser Glu Lys Thr Ile Asn Thr Asp Leu Ile 660 665 670 His Ala Gly Gly Tyr Ala Glu Tyr Asp Thr His Asn Leu Tyr Gly Thr 675 680 685 Met Met Ser Ala Thr Ser Arg Glu Ala Met Leu Asn Arg Arg Pro Ala 690 695 700 Val Arg Pro Leu Val Ile Thr Arg Ser Thr Phe Ala Gly Ala Gly Arg 705 710 715 720 Gln Val Gly His Trp Leu Gly Asp Asn Phe Ala Asp Trp Asp His Tyr 725 730 735 Arg Trp Thr Ile Ala Glu Leu Gln Glu Phe Ala Ala Leu Phe Gln Ile 740 745 750 Pro Met Val Gly Ser Asp Ile Cys Gly Tyr Asp Gly Asn Thr Thr Asp 755 760 765 Asn Leu Cys Ser Arg Trp Val Phe Leu Gly Ala Phe Ser Pro Phe Phe 770 775 780 Arg Asp His Ser Asp Asn Gln Ser Pro Pro His Glu Leu Tyr Arg Thr 785 790 795 800 Pro Gln Ile Ala Ala Ala Ala Arg Ala Ala Ile Asp Ile Arg Tyr Arg 805 810 815 Leu Leu Asp Tyr Ala Tyr Thr Val Leu Trp Thr Gln Thr Gln Thr Gly 820 825 830
    Page 45
    Ala Pro Met 835 Leu Asn CL6220WOPCT_SequenceListi i ng_ST25.txt Ala Asp Ser 845 Asn Pro Met Phe 840 Phe Glu Tyr Pro Thr Ala 850 Asp Leu Gln Tyr Gln 855 Phe Phe Trp Gly Asp 860 Ser Ile Met Val Ala 865 Pro Val Thr Asp Asn Asp 870 Ser Thr Thr Val 875 Asn Val Tyr Phe Pro 880 Lys Asp Gln Phe Tyr 885 Asp Phe Tyr Thr Gly Ala 890 Pro Val Ser Gly 895 Glu Gly Asn Thr Val 900 Thr Leu Thr Asp Val Gly Phe 905 Asp Thr Ile Pro 910 Leu Tyr Phe Lys 915 Gly Gly Ser Ile Val 920 Pro Met Arg Val Arg Ser Ala 925 Asn Thr Thr 930 Ala Glu Leu Arg Gln 935 Gln Asp Phe Val Val 940 Val Ile Ala Pro Asp 945 Ser His Gly Asp Ala Thr 950 Gly Gln Leu Tyr 955 Leu Asp Asp Gly Glu 960 Ser Ile Asn Gln Pro 965 His Thr Ser Glu Ile Gln 970 Phe Ser Tyr Arg 975 Gly Gly His Phe Ser 980 Met Thr Gly Lys Phe Asp Tyr 985 Asp Pro Gly Asn 990 Val Val Ile Ser 995 Gln Ile Thr Leu Leu Gly Ala Asp Gly Ala Gly Lys Gl 1000 1005
    Gly Ser Tyr Asn Ser Thr Thr Lys Val Ala Thr Tyr Lys Val Asn 1010 1015 1020
    Ala Lys Leu Thr Gly Lys Phe Glu Ala Ser Leu His 1025 1030 1035 <210> 15 <211> 1013 <212> PRT <213> Rasamsonia composticola <400> 15
    Ala Ala Ile Val Arg Arg Asn Gly Ala Ser Pro Ser Cys Pro Gly Tyr 1 5 10 15
    Lys Ala Ser Asn Val Lys Thr Val Asp Gly Glu Ile Val Ser Ala Asp 20 25 30
    Leu Asn Leu Ala Gly Pro Ala Cys Asn Val Tyr Gly Thr Asp Leu Asp Page 46
    35 CL6220WOPCT_SequenceListing_ST25.txt 40 45 Asp Leu Lys Leu Gln Val Glu Tyr Gln Ser Gly Pro Gly Val Arg Ala 50 55 60 Asp Cys Leu Leu Pro Phe Asp Val Thr Gly Thr Val Arg Leu Val Asp 65 70 75 80 Asn Asp Leu Thr Ser Ala Glu Gln Arg Leu His Val Lys Ile Tyr Asp 85 90 95 Ala Ala Glu Gln Val Tyr Gln Val Pro Thr Ala Val Leu Pro Arg Pro 100 105 110 Ser Ser Ala Asn Ile Pro Pro Ala Lys Ser Asp Leu Lys Phe Ser Met 115 120 125 Thr Asn Asp Pro Phe Ser Phe Thr Ile Lys Arg Arg Ser Asn Gly Glu 130 135 140 Ile Leu Phe Asp Thr Ser Gly His Pro Leu Ile Phe Glu Ser Gln Tyr 145 150 155 160 Leu Gly Leu Arg Thr Lys Leu Pro Asp Ser Pro Asn Ile Tyr Gly Leu 165 170 175 Gly Glu His Thr Gly Ser Phe Arg Leu Pro Thr Lys Asn Tyr Thr Arg 180 185 190 Thr Leu Trp Ser Arg Asp Ala Tyr Gly Thr Pro Lys Asp Thr Asn Leu 195 200 205 Tyr Gly Asn His Pro Val Tyr Phe Asp Tyr Arg Gly Ser Asn Gly Thr 210 215 220 His Gly Val Phe Leu Leu Asn Ser Asn Gly Met Asp Val Asp Ile Asp 225 230 235 240 Val Asp Ser Asp Gly Gln Tyr Leu Gln Tyr Asn Thr Leu Gly Gly Val 245 250 255 Leu Asp Phe Tyr Phe Leu Ser Gly Pro Asp Pro Lys Ala Val Ala Thr 260 265 270 Gln Tyr Ala Glu Thr Val Gly Lys Pro Val Met Met Pro Tyr Trp Gly 275 280 285 Phe Gly Phe His Asn Cys Arg Tyr Gly Tyr Gln Asp Ile Tyr Glu Val 290 295 300 Ala Glu Ile Ile Ala Asn Tyr Ser Ala Ala Asn Ile Pro Leu Glu Thr
    Page 47
    CL6220WOPCT_SequenceListing_ST25.txt 305 310 315 320
    Gln Trp Thr Asp Ile Asp 325 Tyr Met Asp Leu 330 Arg Lys Val Phe Thr 335 Leu Asp Pro Tyr Arg Tyr Pro Leu Lys Leu Val Gln Glu Val Val Ser Tyr 340 345 350 Leu His Lys His Asn Gln His Tyr Ile Met Met Val Asp Pro Ala Val 355 360 365 Ala Tyr Gln Asn Tyr Ser Ala Phe Asn Asn Gly Val Ala Ala Asp Ala 370 375 380 Phe Leu Lys Phe Ser Asn Gly Ser Ile Tyr Gln Gly Val Val Trp Pro 385 390 395 400 Gly Pro Thr Ala Phe Pro Asp Trp Phe Ala Pro Gln Thr Gln Glu Phe 405 410 415 Trp Asn Ser Glu Phe Ser Thr Phe Phe Asp Pro Ala His Gly Val Asp 420 425 430 Ile Asp Ala Leu Trp Ile Asp Met Asn Glu Ala Ser Asn Phe Cys Asp 435 440 445 Phe Pro Cys Ser Asn Pro Ala Ala Tyr Ala Ala Ala Asn Gly Asp Pro 450 455 460 Pro Thr Pro Pro Pro Val Arg Leu Ser Pro Pro Arg Pro Ile Pro Gly 465 470 475 480 Phe Gly Pro Asp Phe Gln Pro Thr Cys Val Ala Thr Val Ser Phe Asp 485 490 495 Cys Asp Ala Gln Thr Tyr Phe Gly Glu Asn Ile Leu Ile Leu Gly Asn 500 505 510 Ser Thr Thr Leu Gly Ala Gly Asp Val His Met Ala Pro Val Met Ser 515 520 525 Ala Asn Asn Tyr Pro Ile Trp Gln Leu Thr Val Gln Met Pro Pro Asn 530 535 540 Gly Thr Phe Ser Tyr Gln Tyr Val Arg Lys Glu Ser Asp Gly Ser Tyr 545 550 555 560 Ile Tyr Glu Gln Thr Asn Arg Thr Val Thr Thr Gly Asp Cys Thr Ser 565 570 575 Gly Thr Leu Lys Val Ser Asp Thr Ile Thr Thr Ser Ser Gly Pro His
    Page 48
    580 CL6220WOPCT_SequenceListing_ST25.txt 585 590 Lys Arg Ser Glu Leu Arg Pro Leu Val Arg Ser Pro Phe Pro Ala Glu 595 600 605 Asp Leu Thr Arg Arg Gln Ser Gly Ser Met Leu Gly Leu Pro Asn Arg 610 615 620 Asn Leu Leu Asn Pro Pro Tyr Thr Ile His Asn Ala Ala Gly Asn Leu 625 630 635 640 Ser Glu Lys Thr Ile Asn Thr Asp Leu Ile His Ala Gly Gly Tyr Ala 645 650 655 Glu Tyr Asp Thr His Asn Leu Tyr Gly Thr Met Met Ser Ala Thr Ser 660 665 670 Arg Glu Ala Met Leu Asn Arg Arg Pro Ala Val Arg Pro Leu Val Ile 675 680 685 Thr Arg Ser Thr Phe Ala Gly Ala Gly Arg Gln Val Gly His Trp Leu 690 695 700 Gly Asp Asn Phe Ala Asp Trp Asp His Tyr Arg Trp Thr Ile Ala Glu 705 710 715 720 Leu Gln Glu Phe Ala Ala Leu Phe Gln Ile Pro Met Val Gly Ser Asp 725 730 735 Ile Cys Gly Tyr Asp Gly Asn Thr Thr Asp Asn Leu Cys Ser Arg Trp 740 745 750 Val Phe Leu Gly Ala Phe Ser Pro Phe Phe Arg Asp His Ser Asp Asn 755 760 765 Gln Ser Pro Pro His Glu Leu Tyr Arg Thr Pro Gln Ile Ala Ala Ala 770 775 780 Ala Arg Ala Ala Ile Asp Ile Arg Tyr Arg Leu Leu Asp Tyr Ala Tyr 785 790 795 800 Thr Val Leu Trp Thr Gln Thr Gln Thr Gly Ala Pro Met Leu Asn Pro 805 810 815 Met Phe Phe Glu Tyr Pro Ala Asp Ser Asn Thr Ala Asp Leu Gln Tyr 820 825 830 Gln Phe Phe Trp Gly Asp Ser Ile Met Val Ala Pro Val Thr Asp Asn 835 840 845 Asp Ser Thr Thr Val Asn Val Tyr Phe Pro Lys Asp Gln Phe Tyr Asp
    Page 49
    850 CL6220WOPCT_SequenceListing_ST25. txt 855 860 Phe 865 Tyr Thr Gly Ala Pro Val 870 Ser Gly Glu Gly Asn Thr Val 875 Thr Leu 880 Thr Asp Val Gly Phe 885 Asp Thr Ile Pro Leu Tyr Phe Lys Gly 890 Gly Ser 895 Ile Val Pro Met Arg 900 Val Arg Ser Ala Asn Thr Thr Ala Glu 905 910 Leu Arg Gln Gln Asp Phe Val 915 Val Val Ile Ala Pro Asp Ser His Gly 920 925 Asp Ala Thr Gly Gln Leu Tyr 930 Leu Asp 935 Asp Gly Glu Ser Ile Asn Gln 940 Pro His Thr 945 Ser Glu Ile Gln Phe Ser 950 Tyr Arg Gly Gly His Phe Ser 955 Met Thr 960 Gly Lys Phe Asp Tyr 965 Asp Pro Gly Asn Val Val Ile Ser Gln 970 Ile Thr 975 Leu Leu Gly Ala Asp 980 Gly Ala Gly Lys Gly Gly Ser Tyr Asn 985 990 Ser Thr Thr Lys Val Ala Thr 995 Tyr Lys Val Asn Ala Lys Leu Thr Gly Lys Phe 1000 1005
    Glu Ala Ser Leu His 1010 <210> 16 <211> 3162 <212> DNA <213> Rasamsonia composticola <400> 16
    atggcaggct ccgccgccct tgttgccagc ctcgtctggc ttgctcaggc cttcgacgct 60 cttgcaggac cggtcagcag tacgactgcc gcagcaccat ctgctcaatt caccgtcccg 120 gccgctgcgg atgttggggc caacttgctt gccaacatcg acgatcccaa tgccgtcaac 180 gcccaggatg tctgtcccgg ttacacggcg tcgaacgtgc agaacaccga gtctgggttt 240 gtggcgaccc tgacgctggc ggggaaacca tgtaatgtgt acggaacgga cgtggagtcc 300 ctgaacctga cggttgagta ccaagctgcg gatcgactga acatcaatat cgtcccgacg 360 cacgtcgatt cttcaaacca gtcgtggtat ctgcttcccg aaaatgtagt gcccaaaccg 420 ggggtcgatg caggagccca agtcccggag agtgatctcg tcttcagctg gtcgaatgaa 480 ccctccttca atttcaaggt gatccggaaa gccacaggcg acattctctt cgacacggag 540 ggttctgtcc tggtgttcga aaaccagttc atcgagtttg cgagcgctct gccggagaac 600
    Page 50
    CL6220WOPCT_SequenceListing_ST25.txt
    tacaatctct acggtctggg agagcgtatc catggcctgc gactggggaa caacttcacc 660 gccacgacgt atgccgcgga tagcgcagac cctattgacc ggtgagtatc tgagatcgac 720 tgctcagtct gctctgttgg atctgaaaga agttataaaa ctgacctagc tcaggaacat 780 ctacgggacc catccctttt atctggacac ccggtactac gaggttgatt ccgagcatgg 840 gaggttcacg ttggtgacgg acaacgagac cgatttctcc aaggaatatc tgtcgctctc 900 gcatggagtt ttcctgagaa atgcccacgg acaggaggtg ctgctgcgtc ctcagagcat 960 cacctggcgg acactcggtg gcagcattga tctttacttc tacgccggtc cgacccaggc 1020 cgatgttacc cgcagctacc agaccagcac cgttggcctc ccggcaatgc agcagtactt 1080 caccctgggc tatcatcagt gccgctgggg atacagaaac tggtcggagc tagctgatgt 1140 agtggccaat ttcgagaaat tcgagatccc attggaaaat atctggtaag gcatacgcta 1200 tctgaaagag ttgctgggaa agtgatctga caacttcgtc tctccaggtc ggatattgat 1260 tacatgaacg agtaccgcga ctttgagaac gacccggttc gcttctccta cagcgaggga 1320 gccaaattcc tggaccagct ccacaagagt ggccgtcact acatcccgat tgtggacgcc 1380 gcgatctatg accccaaccc taacaatgac tccgacgcgt aagtctagtc ttgtagggag 1440 gtgataggga gtggagctga cttctcgatt aggtatgcga catatgatcg aggttctaag 1500 gacgatatct ggttgaagaa tcccgacggc agcgtgtaca tcggagccgt ctggcctggc 1560 tacacagtgt tcaccgattg gcaccatcca aaagccaacg agtggtgggc aaacgagctg 1620 gctctgtggc acgaaaaggt cgcttttgac ggaatctggc tggacatgaa cgaggtctcg 1680 tccttctgcg ttggcagctg tggaacaggg aacctgaccc tgaatcccgt gcacccgaac 1740 ttcgcgctcc cgggagagcc tggagctgtc atctacgact accccgagga cttcaacgtg 1800 acgaatgcca cggcggcggc gtctgcatct gccgcgtcct cgagccaagc tgctgcgaca 1860 gcgacagcta cttcttcgtc cacgactacc agctacctgg tgaccacgcc cactcctgga 1920 gtgcggaatg tcaactaccc tccctatgtg attaatcacg tgcaggaggg tcacgatctc 1980 gctgttcacg ccgtctcgcc caacgcaacc catgtcgatg gtgtgcagga gtacgacgtg 2040 cacaatctct ggggctacca ggagacaaat gcaacctacc atgccctgct gagcatcttc 2100 cccgggaaga gaccgttcat catctcccgt tccacgttcg ccggcagcgg cagatgggcc 2160 ggacactggg gtggcgacaa cgcctcgaaa tgggcgtaca tgttcttttc tatcccgcag 2220 gcgctatcgt tctcgctgtt cggcatcccc atgttcggcg tcgacacctg cgggttcaac 2280 ggcaactcgg acgaagagct gtgcaaccgc tggatgcagc tctccgcctt cttccccttc 2340 taccgcaacc acaacgtcct gtcggccatc ccgcaggagc cctatgtctg ggcatccgtc 2400 atcgaggcga gcaagtcggc aatgaggatc cgctacaccc tgctccctta cctctacaca 2460 ctgttctacc tcgcccacac cacggggtcg accgtcatgc gtgccttggc gtgggagttc 2520 cccaacgacc cgtccctcgc tgccgtggac cggcagttcc tcctgggccc gtcgctgatg 2580 gtcgtccccg tgctcgagcc gcaggtcgat accgtcaagg gcgtcttccc gggcgttgcc 2640
    Page 51
    CL6220WOPCT_SequenceListmg_ST25.txt cagggccaag tctggtacga ctggtacacg cagaccgcgt tcgacgcgca gccaggcgtg aacacgacca tctccgcgcc gctgggccac ctccccatgc agcagccggc actggtgacg ctggtcgcgc tgggcagcga cggcacggcc agcatcacac ctccggcgtc cctgcacgtc ttcgcgacgg cccgcggtgc gttcaaggac ggcgtcccag ccgcgccgtc gtctgcagtc tcggtgtcgt acaatgccac ctccaaagtc cgtgacggag cctggagcag tgactgggtt atccccgtgt tcgtccgcgg cgggagcgtg cgggacgtgc gcaacagccc ctggtcgctg tcgggacagc tgtacgtgga cgacggcgag gacttcgtgg cggccaactt ctcgaccctc agcaacacgc tggctaacgt cacggtgctg acttggaaca acgagacggt tccttcggag ctcgtggtca atggactgca gagtcttacc ctgaagtggt aa
    2700
    2760
    2820
    2880
    2940
    3000
    3060
    3120
    3162 <210> 17 <211> 990 <212> PRT <213> Rasamsonia composticola
    <400> 17 Met 1 Ala Gly Ser Ala 5 Ala Leu Val Ala Phe Asp Ala 20 Leu Ala Gly Pro Pro Ser Ala 35 Gln Phe Thr Val Pro 40 Leu Leu 50 Ala Asn Ile Asp Asp 55 Pro Cys 65 Pro Gly Tyr Thr Ala 70 Ser Asn Val Ala Thr Leu Thr 85 Leu Ala Gly Asp Val Glu Ser 100 Leu Asn Leu Thr Leu Asn Ile 115 Asn Ile Val Pro Thr 120 Trp Tyr 130 Leu Leu Pro Glu Asn 135 Val Gly 145 Ala Gln Val Pro Glu 150 Ser Asp Pro Ser Phe Asn Phe Lys Val Ile
    Ala Ser 10 Leu Val Trp Leu Ala 15 Gln Val 25 Ser Ser Thr Thr Ala 30 Ala Ala Ala Ala Ala Asp Val 45 Gly Ala Asn Asn Ala Val Asn 60 Ala Gln Asp Val Val Gln Asn 75 Thr Glu Ser Gly Phe 80 Lys Pro 90 Cys Asn Val Tyr Gly 95 Thr Val 105 Glu Tyr Gln Ala Ala 110 Asp Arg His Val Asp Ser Ser 125 Asn Gln Ser Val Pro Lys Pro 140 Gly Val Asp Ala Leu Val Phe 155 Ser Trp Ser Asn Glu 160 Arg Lys Ala Thr Gly Asp Ile Leu
    Page 52
    CL6220WOPCT_SequenceListing_ST25.txt 165 170 175
    Phe Asp Thr Glu 180 Gly Ser Val Leu Val 185 Phe Glu Asn Gln Phe 190 Ile Glu Phe Ala Ser Ala Leu Pro Glu Asn Tyr Asn Leu Tyr Gly Leu Gly Glu 195 200 205 Arg Ile His Gly Leu Arg Leu Gly Asn Asn Phe Thr Ala Thr Thr Tyr 210 215 220 Ala Ala Asp Ser Ala Asp Pro Ile Asp Arg Asn Ile Tyr Gly Thr His 225 230 235 240 Pro Phe Tyr Leu Asp Thr Arg Tyr Tyr Glu Val Asp Ser Glu His Gly 245 250 255 Arg Phe Thr Leu Val Thr Asp Asn Glu Thr Asp Phe Ser Lys Glu Tyr 260 265 270 Leu Ser Leu Ser His Gly Val Phe Leu Arg Asn Ala His Gly Gln Glu 275 280 285 Val Leu Leu Arg Pro Gln Ser Ile Thr Trp Arg Thr Leu Gly Gly Ser 290 295 300 Ile Asp Leu Tyr Phe Tyr Ala Gly Pro Thr Gln Ala Asp Val Thr Arg 305 310 315 320 Ser Tyr Gln Thr Ser Thr Val Gly Leu Pro Ala Met Gln Gln Tyr Phe 325 330 335 Thr Leu Gly Tyr His Gln Cys Arg Trp Gly Tyr Arg Asn Trp Ser Glu 340 345 350 Leu Ala Asp Val Val Ala Asn Phe Glu Lys Phe Glu Ile Pro Leu Glu 355 360 365 Asn Ile Trp Ser Asp Ile Asp Tyr Met Asn Glu Tyr Arg Asp Phe Glu 370 375 380 Asn Asp Pro Val Arg Phe Ser Tyr Ser Glu Gly Ala Lys Phe Leu Asp 385 390 395 400 Gln Leu His Lys Ser Gly Arg His Tyr Ile Pro Ile Val Asp Ala Ala 405 410 415 Ile Tyr Asp Pro Asn Pro Asn Asn Asp Ser Asp Ala Tyr Ala Thr Tyr 420 425 430 Asp Arg Gly Ser Lys Asp Asp Ile Trp Leu Lys Asn Pro Asp Gly Ser
    Page 53
    CL6220WOPCT_SequenceListing_ST25.txt
    435 440 445 Val Tyr Ile Gly Ala Val Trp Pro Gly Tyr Thr Val Phe Thr Asp Trp 450 455 460 His His Pro Lys Ala Asn Glu Trp Trp Ala Asn Glu Leu Ala Leu Trp 465 470 475 480 His Glu Lys Val Ala Phe Asp Gly Ile Trp Leu Asp Met Asn Glu Val 485 490 495 Ser Ser Phe Cys Val Gly Ser Cys Gly Thr Gly Asn Leu Thr Leu Asn 500 505 510 Pro Val His Pro Asn Phe Ala Leu Pro Gly Glu Pro Gly Ala Val Ile 515 520 525 Tyr Asp Tyr Pro Glu Asp Phe Asn Val Thr Asn Ala Thr Ala Ala Ala 530 535 540 Ser Ala Ser Ala Ala Ser Ser Ser Gln Ala Ala Ala Thr Ala Thr Ala 545 550 555 560 Thr Ser Ser Ser Thr Thr Thr Ser Tyr Leu Val Thr Thr Pro Thr Pro 565 570 575 Gly Val Arg Asn Val Asn Tyr Pro Pro Tyr Val Ile Asn His Val Gln 580 585 590 Glu Gly His Asp Leu Ala Val His Ala Val Ser Pro Asn Ala Thr His 595 600 605 Val Asp Gly Val Gln Glu Tyr Asp Val His Asn Leu Trp Gly Tyr Gln 610 615 620 Glu Thr Asn Ala Thr Tyr His Ala Leu Leu Ser Ile Phe Pro Gly Lys 625 630 635 640 Arg Pro Phe Ile Ile Ser Arg Ser Thr Phe Ala Gly Ser Gly Arg Trp 645 650 655 Ala Gly His Trp Gly Gly Asp Asn Ala Ser Lys Trp Ala Tyr Met Phe 660 665 670 Phe Ser Ile Pro Gln Ala Leu Ser Phe Ser Leu Phe Gly Ile Pro Met 675 680 685 Phe Gly Val Asp Thr Cys Gly Phe Asn Gly Asn Ser Asp Glu Glu Leu 690 695 700 Cys Asn Arg Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn Pag e 54
    CL6220WOPCT_SequenceListing_ST25.txt 705 710 715 720
    His Asn Val Leu Ser 725 Ala Ile Pro Gln Glu 730 Pro Tyr Val Trp Ala 735 Ser Val Ile Glu Ala Ser Lys Ser Ala Met Arg Ile Arg Tyr Thr Leu Leu 740 745 750 Pro Tyr Leu Tyr Thr Leu Phe Tyr Leu Ala His Thr Thr Gly Ser Thr 755 760 765 Val Met Arg Ala Leu Ala Trp Glu Phe Pro Asn Asp Pro Ser Leu Ala 770 775 780 Ala Val Asp Arg Gln Phe Leu Leu Gly Pro Ser Leu Met Val Val Pro 785 790 795 800 Val Leu Glu Pro Gln Val Asp Thr Val Lys Gly Val Phe Pro Gly Val 805 810 815 Ala Gln Gly Gln Val Trp Tyr Asp Trp Tyr Thr Gln Thr Ala Phe Asp 820 825 830 Ala Gln Pro Gly Val Asn Thr Thr Ile Ser Ala Pro Leu Gly His Ile 835 840 845 Pro Val Phe Val Arg Gly Gly Ser Val Leu Pro Met Gln Gln Pro Ala 850 855 860 Leu Val Thr Arg Asp Val Arg Asn Ser Pro Trp Ser Leu Leu Val Ala 865 870 875 880 Leu Gly Ser Asp Gly Thr Ala Ser Gly Gln Leu Tyr Val Asp Asp Gly 885 890 895 Glu Ser Ile Thr Pro Pro Ala Ser Leu His Val Asp Phe Val Ala Ala 900 905 910 Asn Phe Ser Thr Leu Phe Ala Thr Ala Arg Gly Ala Phe Lys Asp Ser 915 920 925 Asn Thr Leu Ala Asn Val Thr Val Leu Gly Val Pro Ala Ala Pro Ser 930 935 940 Ser Ala Val Thr Trp Asn Asn Glu Thr Val Pro Ser Glu Ser Val Ser 945 950 955 960 Tyr Asn Ala Thr Ser Lys Val Leu Val Val Asn Gly Leu Gln Ser Leu 965 970 975 Thr Arg Asp Gly Ala Trp Ser Ser Asp Trp Val Leu Lys Trp
    Page 55
    CL6220WOPCT_SequenceListing_ST25.txt 980 985 990 <210> 18 <211> 973 <212> PRT <213> Rasamsonia composticola <400> 18
    Phe Asp 1 Ala Leu Ala 5 Gly Pro Val Ser Ser Thr Thr Ala Ala Ala Pro 10 15 Ser Ala Gln Phe Thr Val Pro Ala Ala Ala Asp Val Gly Ala Asn Leu 20 25 30 Leu Ala Asn Ile Asp Asp Pro Asn Ala Val Asn Ala Gln Asp Val Cys 35 40 45 Pro Gly Tyr Thr Ala Ser Asn Val Gln Asn Thr Glu Ser Gly Phe Val 50 55 60 Ala Thr Leu Thr Leu Ala Gly Lys Pro Cys Asn Val Tyr Gly Thr Asp 65 70 75 80 Val Glu Ser Leu Asn Leu Thr Val Glu Tyr Gln Ala Ala Asp Arg Leu 85 90 95 Asn Ile Asn Ile Val Pro Thr His Val Asp Ser Ser Asn Gln Ser Trp 100 105 110 Tyr Leu Leu Pro Glu Asn Val Val Pro Lys Pro Gly Val Asp Ala Gly 115 120 125 Ala Gln Val Pro Glu Ser Asp Leu Val Phe Ser Trp Ser Asn Glu Pro 130 135 140 Ser Phe Asn Phe Lys Val Ile Arg Lys Ala Thr Gly Asp Ile Leu Phe 145 150 155 160 Asp Thr Glu Gly Ser Val Leu Val Phe Glu Asn Gln Phe Ile Glu Phe 165 170 175 Ala Ser Ala Leu Pro Glu Asn Tyr Asn Leu Tyr Gly Leu Gly Glu Arg 180 185 190 Ile His Gly Leu Arg Leu Gly Asn Asn Phe Thr Ala Thr Thr Tyr Ala 195 200 205 Ala Asp Ser Ala Asp Pro Ile Asp Arg Asn Ile Tyr Gly Thr His Pro 210 215 220 Phe Tyr Leu Asp Thr Arg Tyr Tyr Glu Val Asp Ser Glu His Gly Arg 225 230 235 240
    Page 56
    CL6220WOPCT_SequenceListing_ST25.txt
    Phe Thr Leu Val Thr Asp 245 Asn Glu Thr Asp 250 Phe Ser Lys Glu Tyr 255 Leu Ser Leu Ser His Gly Val Phe Leu Arg Asn Ala His Gly Gln Glu Val 260 265 270 Leu Leu Arg Pro Gln Ser Ile Thr Trp Arg Thr Leu Gly Gly Ser Ile 275 280 285 Asp Leu Tyr Phe Tyr Ala Gly Pro Thr Gln Ala Asp Val Thr Arg Ser 290 295 300 Tyr Gln Thr Ser Thr Val Gly Leu Pro Ala Met Gln Gln Tyr Phe Thr 305 310 315 320 Leu Gly Tyr His Gln Cys Arg Trp Gly Tyr Arg Asn Trp Ser Glu Leu 325 330 335 Ala Asp Val Val Ala Asn Phe Glu Lys Phe Glu Ile Pro Leu Glu Asn 340 345 350 Ile Trp Ser Asp Ile Asp Tyr Met Asn Glu Tyr Arg Asp Phe Glu Asn 355 360 365 Asp Pro Val Arg Phe Ser Tyr Ser Glu Gly Ala Lys Phe Leu Asp Gln 370 375 380 Leu His Lys Ser Gly Arg His Tyr Ile Pro Ile Val Asp Ala Ala Ile 385 390 395 400 Tyr Asp Pro Asn Pro Asn Asn Asp Ser Asp Ala Tyr Ala Thr Tyr Asp 405 410 415 Arg Gly Ser Lys Asp Asp Ile Trp Leu Lys Asn Pro Asp Gly Ser Val 420 425 430 Tyr Ile Gly Ala Val Trp Pro Gly Tyr Thr Val Phe Thr Asp Trp His 435 440 445 His Pro Lys Ala Asn Glu Trp Trp Ala Asn Glu Leu Ala Leu Trp His 450 455 460 Glu Lys Val Ala Phe Asp Gly Ile Trp Leu Asp Met Asn Glu Val Ser 465 470 475 480 Ser Phe Cys Val Gly Ser Cys Gly Thr Gly Asn Leu Thr Leu Asn Pro 485 490 495 Val His Pro Asn Phe Ala Leu Pro Gly Glu Pro Gly Ala Val Ile Tyr 500 505 510
    Page 57
    CL6220WOPCT_SequenceListing_ST25.txt
    Asp Tyr Pro 515 Glu Asp Phe Asn Val 520 Thr Asn Ala Thr Ala Ala Ala 525 Ser Ala Ser Ala Ala Ser Ser Ser Gln Ala Ala Ala Thr Ala Thr Ala Thr 530 535 540 Ser Ser Ser Thr Thr Thr Ser Tyr Leu Val Thr Thr Pro Thr Pro Gly 545 550 555 560 Val Arg Asn Val Asn Tyr Pro Pro Tyr Val Ile Asn His Val Gln Glu 565 570 575 Gly His Asp Leu Ala Val His Ala Val Ser Pro Asn Ala Thr His Val 580 585 590 Asp Gly Val Gln Glu Tyr Asp Val His Asn Leu Trp Gly Tyr Gln Glu 595 600 605 Thr Asn Ala Thr Tyr His Ala Leu Leu Ser Ile Phe Pro Gly Lys Arg 610 615 620 Pro Phe Ile Ile Ser Arg Ser Thr Phe Ala Gly Ser Gly Arg Trp Ala 625 630 635 640 Gly His Trp Gly Gly Asp Asn Ala Ser Lys Trp Ala Tyr Met Phe Phe 645 650 655 Ser Ile Pro Gln Ala Leu Ser Phe Ser Leu Phe Gly Ile Pro Met Phe 660 665 670 Gly Val Asp Thr Cys Gly Phe Asn Gly Asn Ser Asp Glu Glu Leu Cys 675 680 685 Asn Arg Trp Met Gln Leu Ser Ala Phe Phe Pro Phe Tyr Arg Asn His 690 695 700 Asn Val Leu Ser Ala Ile Pro Gln Glu Pro Tyr Val Trp Ala Ser Val 705 710 715 720 Ile Glu Ala Ser Lys Ser Ala Met Arg Ile Arg Tyr Thr Leu Leu Pro 725 730 735 Tyr Leu Tyr Thr Leu Phe Tyr Leu Ala His Thr Thr Gly Ser Thr Val 740 745 750 Met Arg Ala Leu Ala Trp Glu Phe Pro Asn Asp Pro Ser Leu Ala Ala 755 760 765 Val Asp Arg Gln Phe Leu Leu Gly Pro Ser Leu Met Val Val Pro Val 770 775 780
    Page 58
    CL6220WOPCT_SequenceListing_ST25.txt
    Leu 785 Glu Pro Gln Val Asp 790 Thr Val Gln Gly Gln Val Trp 805 Tyr Asp Trp Gln Pro Gly Val 820 Asn Thr Thr Ile Val Phe Val 835 Arg Gly Gly Ser Val 840 Val Thr 850 Arg Asp Val Arg Asn 855 Ser Gly 865 Ser Asp Gly Thr Ala 870 Ser Gly Ser Ile Thr Pro Pro 885 Ala Ser Leu Phe Ser Thr Leu 900 Phe Ala Thr Ala Thr Leu Ala 915 Asn Val Thr Val Leu 920 Ala Val 930 Thr Trp Asn Asn Glu 935 Thr Asn 945 Ala Thr Ser Lys Val 950 Leu Val Arg Asp Gly Ala Trp 965 Ser Ser Asp
    Lys Gly Val 795 Phe Pro Gly Val Ala 800 Tyr Thr 810 Gln Thr Ala Phe Asp 815 Ala Ser 825 Ala Pro Leu Gly His 830 Ile Pro Leu Pro Met Gln Gln 845 Pro Ala Leu Pro Trp Ser Leu 860 Leu Val Ala Leu Gln Leu Tyr 875 Val Asp Asp Gly Glu 880 His Val 890 Asp Phe Val Ala Ala 895 Asn Arg 905 Gly Ala Phe Lys Asp 910 Ser Asn Gly Val Pro Ala Ala 925 Pro Ser Ser Val Pro Ser Glu 940 Ser Val Ser Tyr Val Asn Gly 955 Leu Gln Ser Leu Thr 960 Trp Val 970 Leu Lys Trp
    <210> 19 <211> 1815 <212> DNA <213> Bifidobacterium longum <400> 19 atgaccgcaa ataatctcaa tgacgactgg ccgcgcagct tcaaggacgt taacggcgac aagatggact acctgaagaa cctcggcgtc tccgatctgg cggacggcgg ctacgacgta ggcaccatgg aagacttcga cgcgatggcc atcgtggaca tcgtgcccaa ccacaccgct tggaagcagg ccgtcgtcta ccagatctac ggactcggcg acatcgccgg cgtcaccgag gacgcaatct ggctctcccc gttctacccc atcgactacc gcaacgtcga cccgcgccta aaggccgcgc acgaggccgg catcaaggtg gacaagcacg tgttcttcca ggaggccctc
    Page 59
    120
    180
    240
    300
    360
    gccgccgagc ctggctcccc CL6220WOPCT_SequenceListing_ST25.txt ggcgcgcgac cgctacatct tccgcgacgg ccgcggcgag 420 catggcgaac tgcccccgaa cgactggcaa tccttcttcg gcggcccggc ctgggcgcgc 480 gtggccgacg gccagtggta tctgcacctg ttcgataagg cgcagccgga cgtcaactgg 540 aagaatccgg acatccacga ggagttcaag aagaccctgc gtttctggtc cgaccacggc 600 accgacggct tccgcatcga cgtggcgcac ggcctggcca aggacctcga atccaagccg 660 ttggaagagc tcggccgcga atacagcgtg gtcggcgtgc tgaatcatga cttcagccac 720 ccgctgttcg accgtcgcga ggtgcacgat atctaccgcg aatggcgcaa ggtgttcaac 780 gagtacgacc cgccgcgctt tgccgtggcc gaggcgtggg tggtgcccga gcaccagcac 840 ctgtacgctt cgatggacga gctgggccag tccttcaact tcgacttcgc gcaagccaac 900 tggtatgccg acgagttccg cgaggccatc gccgccggac tcaaggcggc ggccgaaacc 960 ggcggttcca ccaccacgtg ggtcatgaac aatcacgacg tgccgcgcag cccctcccgt 1020 tacggcctgc cgcagatcaa gggcgcaccc taccaccagc tgccgcacga ctggctgctg 1080 cgcaacggca ctacctatcc cgaggatcgc gagcttggca ctcgccgcgc ccgcgccgcc 1140 gctttgatgg agctcggcct gcccggcgcc gcctatatct atcagggcga ggagctgggc 1200 ctgtttgagg tggccgatat tccgtgggat cacttggagg atccgaccgc tttccacact 1260 gctcaggcca cgatggacaa gggccgcgac ggctgccgcg tgccgctgcc gtggaccgcc 1320 gccgatgagc cggccttggc cgatttcagc cgtccgactc cggccgatga cggtaccggc 1380 gagaaccatg tgccgctgtg cgccgccggc cagttcggca cgggcgcttc cttcggcttc 1440 tcgccggcta cgcgcgctga gggcgtgacg ccggccgccg acccgcacct gccgcaaccg 1500 ctgtggttca aggattacgc ggtggacgtg gagcaagccg acccggactc gatgctcgcg 1560 ctgtatcgcg ccgcactggc gattcgccag gagtcgctga ccgccacgcg cgacaccacg 1620 gccgagcagg tggacatggg cgacgatgtg gtggcgtaca cccgcgcggc ggttggcggg 1680 cgggtgttca cctcaatcac caacttcggc aatgctccgg tcgcgctgcc cgatggctcc 1740 gtggtgctgg cgtccggccc gctgaccccc gaagcccagc tccccaccga cacttctgcg 1800 tgggttgttc agtag 1815
    <210> 20 <211> 604 <212> PRT <213> Bifidobacterium longum <400> 20
    Met Thr Ala Asn Asn Leu Asn Asp Asp Trp Trp Lys Gln Ala Val Val 1 5 10 15 Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Val Asn Gly Asp Gly Leu 20 25 30 Gly Asp Ile Ala Gly Val Thr Glu Lys Met Asp Tyr Leu Lys Asn Leu
    35 40 45
    Page 60
    CL6220WOPCT_SequenceListing_ST25.txt
    Gly Val 50 Asp Ala Ile Trp Leu 55 Ser Pro Phe Tyr Pro 60 Ser Asp Leu Ala Asp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu 65 70 75 80 Gly Thr Met Glu Asp Phe Asp Ala Met Ala Lys Ala Ala His Glu Ala 85 90 95 Gly Ile Lys Val Ile Val Asp Ile Val Pro Asn His Thr Ala Asp Lys 100 105 110 His Val Phe Phe Gln Glu Ala Leu Ala Ala Glu Pro Gly Ser Pro Ala 115 120 125 Arg Asp Arg Tyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly Glu Leu 130 135 140 Pro Pro Asn Asp Trp Gln Ser Phe Phe Gly Gly Pro Ala Trp Ala Arg 145 150 155 160 Val Ala Asp Gly Gln Trp Tyr Leu His Leu Phe Asp Lys Ala Gln Pro 165 170 175 Asp Val Asn Trp Lys Asn Pro Asp Ile His Glu Glu Phe Lys Lys Thr 180 185 190 Leu Arg Phe Trp Ser Asp His Gly Thr Asp Gly Phe Arg Ile Asp Val 195 200 205 Ala His Gly Leu Ala Lys Asp Leu Glu Ser Lys Pro Leu Glu Glu Leu 210 215 220 Gly Arg Glu Tyr Ser Val Val Gly Val Leu Asn His Asp Phe Ser His 225 230 235 240 Pro Leu Phe Asp Arg Arg Glu Val His Asp Ile Tyr Arg Glu Trp Arg 245 250 255 Lys Val Phe Asn Glu Tyr Asp Pro Pro Arg Phe Ala Val Ala Glu Ala 260 265 270 Trp Val Val Pro Glu His Gln His Leu Tyr Ala Ser Met Asp Glu Leu 275 280 285 Gly Gln Ser Phe Asn Phe Asp Phe Ala Gln Ala Asn Trp Tyr Ala Asp 290 295 300 Glu Phe Arg Glu Ala Ile Ala Ala Gly Leu Lys Ala Ala Ala Glu Thr
    305 310 315 320
    Page 61
    CL6220WOPCT_SequenceListing_ST25.txt
    Gly Gly Ser Thr Thr Thr Trp Val 325 Met Asn Asn 330 His Asp Val Pro 335 Arg Ser Pro Ser Arg Tyr Gly Leu Pro Gln Ile Lys Gly Ala Pro Tyr His 340 345 350 Gln Leu Pro His Asp Trp Leu Leu Arg Asn Gly Thr Thr Tyr Pro Glu 355 360 365 Asp Arg Glu Leu Gly Thr Arg Arg Ala Arg Ala Ala Ala Leu Met Glu 370 375 380 Leu Gly Leu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly Glu Glu Leu Gly 385 390 395 400 Leu Phe Glu Val Ala Asp Ile Pro Trp Asp His Leu Glu Asp Pro Thr 405 410 415 Ala Phe His Thr Ala Gln Ala Thr Met Asp Lys Gly Arg Asp Gly Cys 420 425 430 Arg Val Pro Leu Pro Trp Thr Ala Ala Asp Glu Pro Ala Leu Ala Asp 435 440 445 Phe Ser Arg Pro Thr Pro Ala Asp Asp Gly Thr Gly Glu Asn His Val 450 455 460 Pro Leu Cys Ala Ala Gly Gln Phe Gly Thr Gly Ala Ser Phe Gly Phe 465 470 475 480 Ser Pro Ala Thr Arg Ala Glu Gly Val Thr Pro Ala Ala Asp Pro His 485 490 495 Leu Pro Gln Pro Leu Trp Phe Lys Asp Tyr Ala Val Asp Val Glu Gln 500 505 510 Ala Asp Pro Asp Ser Met Leu Ala Leu Tyr Arg Ala Ala Leu Ala Ile 515 520 525 Arg Gln Glu Ser Leu Thr Ala Thr Arg Asp Thr Thr Ala Glu Gln Val 530 535 540 Asp Met Gly Asp Asp Val Val Ala Tyr Thr Arg Ala Ala Val Gly Gly 545 550 555 560 Arg Val Phe Thr Ser Ile Thr Asn Phe Gly Asn Ala Pro Val Ala Leu 565 570 575 Pro Asp Gly Ser Val Val Leu Ala Ser Gly Pro Leu Thr Pro Glu Ala 580 585 590
    Page 62
    CL6220WOPCT_SequenceListing_ST25.txt
    Gln Leu Pro Thr Asp Thr Ser Ala Trp Val Val Gln 595 600 <210> 21 <211> 1812 <212> DNA <213> Artificial sequence <220>
    <223> BloGlu1_codon-optimized <400> 21
    gtgacagcaa ataatcttaa cgatgattgg tggaaacaag cagtggttta ccaaatctac 60 ccgagatcat ttaaagacgt taatggcgac ggactgggag atattgcggg cgtgacagag 120 aaaatggact atctgaagaa tctgggagtg gatgcgattt ggctgtcacc gttctatccg 180 agcgatctgg ctgacggagg ctacgatgtc attgattata gaaatgtgga tccgagactt 240 ggcacgatgg aggatttcga cgcgatggcg aaggcagccc atgaagcagg cattaaggtt 300 attgtcgata ttgttccgaa tcatacggcg gacaaacatg tgttttttca agaggcactt 360 gccgctgaac ctggaagccc ggctagagat agatacattt tcagagatgg cagaggcgaa 420 cacggcgagc ttcctcctaa cgactggcaa tcattcttcg gaggccctgc atgggcaaga 480 gtggcggacg gccaatggta cctgcatctt tttgataagg cacagccgga tgttaattgg 540 aaaaatcctg atattcacga agagtttaag aaaacactta gattctggtc agatcatggc 600 acggacggat ttagaattga tgtcgcacac ggccttgcga aagatctgga gtcaaaaccg 660 cttgaggagc ttggaagaga atatagcgtg gttggagttc tgaatcatga cttcagccac 720 cctctgtttg acagaagaga ggttcatgac atttatagag aatggagaaa agttttcaat 780 gaatatgatc cgccgagatt tgcggttgct gaggcctggg ttgtgcctga gcatcaacat 840 ctgtacgctt caatggacga gcttggccag tcattcaact ttgattttgc ccaagcaaat 900 tggtatgcag atgaatttag agaggctatt gctgctggcc tgaaagcagc ggctgaaacg 960 ggaggatcaa cgacaacatg ggttatgaat aatcacgatg ttcctagatc accgtcaaga 1020 tatggcctgc ctcagattaa gggcgcaccg tatcaccagc ttccgcacga ttggctgctt 1080 agaaacggca cgacgtaccc ggaagataga gagctgggaa caagaagagc aagagcagca 1140 gctctgatgg aactgggact gcctggcgca gcatacatct atcaaggcga agaacttgga 1200 cttttcgaag ttgcagacat tccgtgggat catctggaag atcctacagc atttcacaca 1260 gcccaggcga caatggataa gggcagagac ggatgtagag tcccgctgcc gtggacagct 1320 gccgatgagc ctgcactggc agacttctca agaccgacac cggccgacga cggaacaggc 1380 gagaaccatg tgccgctttg tgcagccggc cagtttggca caggcgctag ctttggattt 1440 tcaccggcga cgagagcgga aggcgtcaca cctgctgcag atcctcacct gccgcaacct 1500 ctgtggttca aggattatgc tgttgacgtt gagcaagcag acccggactc aatgcttgca 1560 ctgtatagag cggccctggc tattagacaa gaaagcctta cggcaacgag agacacgaca Page 63 1620
    CL6220WOPCT_SequenceListing_ST25.txt gcggagcaag ttgatatggg cgatgatgtt gttgcttata caagagccgc agttggcgga agagtcttta cgtcaattac aaattttggc aatgcaccgg ttgcacttcc ggatggctca gttgttctgg catcaggccc gcttacacct gaagcacaac tgcctacaga tacgtcagca tgggtcgttc aa
    1680
    1740
    1800
    1812 <210> 22 <211> 604 <212> PRT <213> Bifidobacterium longum <400> 22
    Met 1 Thr Ala Asn Asn 5 Leu Asn Asp Asp Trp Trp Lys Gln Ala Val Val 10 15 Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Val Asn Gly Asp Gly Leu 20 25 30 Gly Asp Ile Ala Gly Val Thr Glu Lys Met Asp Tyr Leu Lys Asn Leu 35 40 45 Gly Val Asp Ala Ile Trp Leu Ser Pro Phe Tyr Pro Ser Asp Leu Ala 50 55 60 Asp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu 65 70 75 80 Gly Thr Met Glu Asp Phe Asp Ala Met Ala Lys Ala Ala His Glu Ala 85 90 95 Gly Ile Lys Val Ile Val Asp Ile Val Pro Asn His Thr Ala Asp Lys 100 105 110 His Val Phe Phe Gln Glu Ala Leu Ala Ala Glu Pro Gly Ser Pro Ala 115 120 125 Arg Asp Arg Tyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly Glu Leu 130 135 140 Pro Pro Asn Asp Trp Gln Ser Phe Phe Gly Gly Pro Ala Trp Ala Arg 145 150 155 160 Val Ala Asp Gly Gln Trp Tyr Leu His Leu Phe Asp Lys Ala Gln Pro 165 170 175 Asp Val Asn Trp Lys Asn Pro Asp Ile His Glu Glu Phe Lys Lys Thr 180 185 190 Leu Arg Phe Trp Ser Asp His Gly Thr Asp Gly Phe Arg Ile Asp Val 195 200 205
    Page 64
    CL6220WOPCT_SequenceListing_ST25.txt
    Ala His 210 Gly Leu Ala Lys Asp 215 Leu Glu Ser Lys Pro 220 Leu Glu Glu Leu Gly Arg Glu Tyr Ser Val Val Gly Val Leu Asn His Asp Phe Ser His 225 230 235 240 Pro Leu Phe Asp Arg Arg Glu Val His Asp Ile Tyr Arg Glu Trp Arg 245 250 255 Lys Val Phe Asn Glu Tyr Asp Pro Pro Arg Phe Ala Val Ala Glu Ala 260 265 270 Trp Val Val Pro Glu His Gln His Leu Tyr Ala Ser Met Asp Glu Leu 275 280 285 Gly Gln Ser Phe Asn Phe Asp Phe Ala Gln Ala Asn Trp Tyr Ala Asp 290 295 300 Glu Phe Arg Glu Ala Ile Ala Ala Gly Leu Lys Ala Ala Ala Glu Thr 305 310 315 320 Gly Gly Ser Thr Thr Thr Trp Val Met Asn Asn His Asp Val Pro Arg 325 330 335 Ser Pro Ser Arg Tyr Gly Leu Pro Gln Ile Lys Gly Ala Pro Tyr His 340 345 350 Gln Leu Pro His Asp Trp Leu Leu Arg Asn Gly Thr Thr Tyr Pro Glu 355 360 365 Asp Arg Glu Leu Gly Thr Arg Arg Ala Arg Ala Ala Ala Leu Met Glu 370 375 380 Leu Gly Leu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly Glu Glu Leu Gly 385 390 395 400 Leu Phe Glu Val Ala Asp Ile Pro Trp Asp His Leu Glu Asp Pro Thr 405 410 415 Ala Phe His Thr Ala Gln Ala Thr Met Asp Lys Gly Arg Asp Gly Cys 420 425 430 Arg Val Pro Leu Pro Trp Thr Ala Ala Asp Glu Pro Ala Leu Ala Asp 435 440 445 Phe Ser Arg Pro Thr Pro Ala Asp Asp Gly Thr Gly Glu Asn His Val 450 455 460 Pro Leu Cys Ala Ala Gly Gln Phe Gly Thr Gly Ala Ser Phe Gly Phe
    465 470 475 480
    Page 65
    CL6220WOPCT_SequenceListing_ST25.txt
    Ser Pro Ala Thr Arg 485 Ala Glu Gly Val Thr 490 Pro Ala Ala Asp Pro 495 His Leu Pro Gln Pro Leu Trp Phe Lys Asp Tyr Ala Val Asp Val Glu Gln 500 505 510 Ala Asp Pro Asp Ser Met Leu Ala Leu Tyr Arg Ala Ala Leu Ala Ile 515 520 525 Arg Gln Glu Ser Leu Thr Ala Thr Arg Asp Thr Thr Ala Glu Gln Val 530 535 540 Asp Met Gly Asp Asp Val Val Ala Tyr Thr Arg Ala Ala Val Gly Gly 545 550 555 560 Arg Val Phe Thr Ser Ile Thr Asn Phe Gly Asn Ala Pro Val Ala Leu 565 570 575 Pro Asp Gly Ser Val Val Leu Ala Ser Gly Pro Leu Thr Pro Glu Ala 580 585 590 Gln Leu Pro Thr Asp Thr Ser Ala Trp Val Val Gln 595 600
    <210> 23 <211> 1812 <212> DNA <213> Artificial sequence <220>
    <223> BloGlu2_codon-optimized <400> 23
    gtgacggcaa acaatctgaa tgatgattgg tggaaacaag cagtggtcta tcagatttat 60 cctagatcat ttaaggatgt taatggcgac ggcctgggag atattgcagg cgtgacggaa 120 aaaatggatt atctgaagaa tctgggcgtt gacgccattt ggctgtcacc gttttacccg 180 agcgacctgg ccgatggcgg ctatgacgtg attgattata gaaatgttga cccgagactg 240 ggcacgatgg acgattttga cgcaatggct gaagccgcac acgaggctgg cattaaagtt 300 attgttgata ttgtcccgaa ccacacagca gacaagcacg tttttttcaa agaagcgctg 360 gcaagcgaac ctggctcacc ggcgagagac agatacattt ttagagacgg aagaggagag 420 catggcgaac tgcctccgaa cgattggcaa tcattctttg gcggacctgc ttgggctaga 480 gtcccggacg gccaatggta ccttcacctg ttcgataaag ctcagccgga tgtgaattgg 540 aaaaatcctg atatccatga agagtttaag aagacgctta gattttggtc agatcatgga 600 acggatggat tcagaattga tgttgcacac ggacttgcaa aggatcttga atcaaaacct 660 cttgaagagc ttggaagaga atactcagtc gttggcgttc ttaatcacga cttttcacac 720 ccgctttttg acagaagaga ggtgcatgat atttacagag aatggagaaa agttttcaat 780
    Page 66
    CL6220WOPCT_SequenceListing_ST25.txt
    gaatatacac cgccgagatt cgcagtcgcc gaggcgtggg tggtgcctga acatcagcat 840 ctttatgcat caatggatga acttggccag tcatttaact tcgatttcgc tcaagcaaat 900 tggtacgctg atgaatttag aaaagcaatc gctgccggcc tgaaagcagc ggcagaaaca 960 ggaggaagca cgacaacatg ggtcatgaac aatcatgacg tccctagatc accgtcaaga 1020 tacggccttc ctcaggttaa aggcgcaccg tatcaccaac ttccgcacga ctggctgctg 1080 agagatggca cgacgtatcc ggaggataga gaactgggaa caagaagagc aagagcggct 1140 gccctgatgg agctgggact gcctggagca gcctatatct atcaaggcga ggaacttgga 1200 ctgtttgagg ttgctgacat tccttgggat agactggaag atccgacagc gtttcataca 1260 gctcaggcga caatggacaa gggcagagat ggatgcagag ttccgcttcc gtggacggcg 1320 tcagacgaac cggcacttgc agacttctca agacctgccc ctgcagacga cggcacaggc 1380 gagaatcatg ttcctctgtg cgctgctgga caattcggaa caggcgcttc attcggcttt 1440 agccctgccg ttagagcaga cggagttaca cctgcggccg acccgcatct gcctcaaccg 1500 ctttggttta aagattatgc agtggatgtc gagcaagcag atccggactc aatgtatacg 1560 ctgtatcacg ctgcactggc aattagacaa gaatcactga cagctacaag agatacaaca 1620 gctgagcagg tggacatggg agcagatgtg gttgcatata gaagagccgc ggtcgaagga 1680 agaacattta cgtcagttac gaactttggc acagctccgg tttcactgcc ggaaggctca 1740 gtggtgctga cgagcggacc gcttacgcct gacggacagc tgccgacaga cacaagcgca 1800 tgggttatta ag 1812
    <210> 24 <211> 604 <212> PRT <213> Bifidobacterium longum <400> 24
    Met Thr Ala Asn Asn Leu Asn Asp Asp Trp Trp Lys Gln Ala Val Val 1 5 10 15 Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Val Asn Gly Asp Gly Leu 20 25 30 Gly Asp Ile Ala Gly Val Thr Glu Lys Met Asp Tyr Leu Lys Asn Leu 35 40 45 Gly Val Asp Ala Ile Trp Leu Ser Pro Phe Tyr Pro Ser Asp Leu Ala 50 55 60 Asp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu 65 70 75 80 Gly Thr Met Asp Asp Phe Asp Ala Met Ala Glu Ala Ala His Glu Ala 85 90 95
    Page 67
    CL6220WOPCT_SequenceListing_ST25.txt
    Gly Ile Lys Val 100 Ile Val Asp Ile Val 105 Pro Asn His Thr Ala 110 Asp Lys His Val Phe Phe Lys Glu Ala Leu Ala Ser Glu Pro Gly Ser Pro Ala 115 120 125 Arg Asp Arg Tyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly Glu Leu 130 135 140 Pro Pro Asn Asp Trp Gln Ser Phe Phe Gly Gly Pro Ala Trp Ala Arg 145 150 155 160 Val Pro Asp Gly Gln Trp Tyr Leu His Leu Phe Asp Lys Ala Gln Pro 165 170 175 Asp Val Asn Trp Lys Asn Pro Asp Ile His Glu Glu Phe Lys Lys Thr 180 185 190 Leu Arg Phe Trp Ser Asp His Gly Thr Asp Gly Phe Arg Ile Asp Val 195 200 205 Ala His Gly Leu Ala Lys Asp Leu Glu Ser Lys Pro Leu Glu Glu Leu 210 215 220 Gly Arg Glu Tyr Ser Val Val Gly Val Leu Asn His Asp Phe Ser His 225 230 235 240 Pro Leu Phe Asp Arg Arg Glu Val His Asp Ile Tyr Arg Glu Trp Arg 245 250 255 Lys Val Phe Asn Glu Tyr Thr Pro Pro Arg Phe Ala Val Ala Glu Ala 260 265 270 Trp Val Val Pro Glu His Gln His Leu Tyr Ala Ser Met Asp Glu Leu 275 280 285 Gly Gln Ser Phe Asn Phe Asp Phe Ala Gln Ala Asn Trp Tyr Ala Asp 290 295 300 Glu Phe Arg Lys Ala Ile Ala Ala Gly Leu Lys Ala Ala Ala Glu Thr 305 310 315 320 Gly Gly Ser Thr Thr Thr Trp Val Met Asn Asn His Asp Val Pro Arg 325 330 335 Ser Pro Ser Arg Tyr Gly Leu Pro Gln Val Lys Gly Ala Pro Tyr His 340 345 350 Gln Leu Pro His Asp Trp Leu Leu Arg Asp Gly Thr Thr Tyr Pro Glu 355 360 365
    Page 68
    CL6220WOPCT_SequenceListing_ST25.txt
    Asp Arg Glu Leu Gly Thr Arg Arg Ala Arg Ala Ala Ala Leu Met Glu 370 375 380 Leu Gly Leu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly Glu Glu Leu Gly 385 390 395 400 Leu Phe Glu Val Ala Asp Ile Pro Trp Asp Arg Leu Glu Asp Pro Thr 405 410 415 Ala Phe His Thr Ala Gln Ala Thr Met Asp Lys Gly Arg Asp Gly Cys 420 425 430 Arg Val Pro Leu Pro Trp Thr Ala Ser Asp Glu Pro Ala Leu Ala Asp 435 440 445 Phe Ser Arg Pro Ala Pro Ala Asp Asp Gly Thr Gly Glu Asn His Val 450 455 460 Pro Leu Cys Ala Ala Gly Gln Phe Gly Thr Gly Ala Ser Phe Gly Phe 465 470 475 480 Ser Pro Ala Val Arg Ala Asp Gly Val Thr Pro Ala Ala Asp Pro His 485 490 495 Leu Pro Gln Pro Leu Trp Phe Lys Asp Tyr Ala Val Asp Val Glu Gln 500 505 510 Ala Asp Pro Asp Ser Met Tyr Thr Leu Tyr His Ala Ala Leu Ala Ile 515 520 525 Arg Gln Glu Ser Leu Thr Ala Thr Arg Asp Thr Thr Ala Glu Gln Val 530 535 540 Asp Met Gly Ala Asp Val Val Ala Tyr Arg Arg Ala Ala Val Glu Gly 545 550 555 560 Arg Thr Phe Thr Ser Val Thr Asn Phe Gly Thr Ala Pro Val Ser Leu 565 570 575 Pro Glu Gly Ser Val Val Leu Thr Ser Gly Pro Leu Thr Pro Asp Gly 580 585 590 Gln Leu Pro Thr Asp Thr Ser Ala Trp Val Ile Lys 595 600
    <210> 25 <211> 1815 <212> DNA <213> Bifidobacterium longum <400> 25 atgaccgcca acaacctcaa tgacgactgg tggaagcagg ccgtcgtcta ccagatctac
    Page 69
    ccgcgcagct tcaaggacgt CL6220WOPCT_SequenceListing_ST25 caacggcgac ggactcggcg acatcgccgg .txt cgtcaccgag 120 aagatggatt acctgaagaa cctcggcgtc gacgcgatct ggctctcccc gttctacccc 180 tcggatctgg cggacggcgg ctacgacgtg atcgactacc gcaacgtcga cccgcgccta 240 ggcaccatgg acgacttcga cgcgatggcc gaggccgcgc acgaggccgg catcaaagtg 300 atcgtggaca tcgtgcccaa ccacaccgcc gacaagcacg tgttcttcaa ggaagccctc 360 gcctccgaac ctggctcccc cgcacgcgat cgctatatct tccgtgacgg ccgcggcgag 420 cacggcgaac tgccgccgaa cgactggcag tccttcttcg gcggcccggc ctgggcgcgc 480 gtgcccgacg gccagtggta cctgcatctg ttcgacaagg cgcagccgga cgtcaactgg 540 aagaatccgg acatccacga ggagttcaaa aagaccctgc gcttctggtc cgaccacggc 600 accgacggct tccgcatcga cgtagcgcac ggcctggcca aagaccttga atccaagccg 660 ctggaggagc tcggccgcga atacagcgtg gtcggcgtgc tgaaccacga cttcagccac 720 ccgctgttcg accgccgcga ggtgcacgac atctaccgcg aatggcgcaa ggtgttcaac 780 gaatacactc cgccgcgctt cgccgtggcc gaggcgtggg tggtgcccga gcaccagcac 840 ctgtacgctt cgatggacga gctgggccag tccttcaact tcgacttcgc gcaggccaac 900 tggtatgccg acgagttccg caaggccatc gccgccgggc tcaaggcggc ggccgaaacc 960 ggcggctcca ccaccacgtg ggtcatgaac aatcatgacg tgccgcgcag cccctcccgc 1020 tatggtctgc cgcaggtcaa gggcgcgccg tatcaccagc tgccacacga ctggctgctg 1080 cgcgacggca ccacctaccc ggagaaccgc gaactcggca cccgccgtgc ccgtgccgcc 1140 gcgctgatgg agctcggcct gcccggtgcc gcatacatct atcagggcga ggaactgggc 1200 ctgtttgagg tggccgatat tccgtgggat cacttggagg atccgaccgc cttccacacc 1260 acccgcaaca cgatggacaa gggccgcgac ggctgccgcg tgccgctgcc gtggaccgcc 1320 gccgatgagc cggccttggc cgatttcagc cgtccggctc cggccgatga cggtaccggc 1380 gaaaaccatg tgccgctgtg cgccgccggc cagttcggca cgggcgcttc cttcggcttc 1440 tcccctgcgg ttcgcgccga tggcgtgacg ccggccgccg acccgcacct gccgcagccg 1500 ctgtggttca aggattacgc ggtggacgtg gagcaagccg acccggactc gatgctcgcg 1560 ctgtatcgcg ccgcactggc gattcgccag gagtcgctga ccgccacgcg cgacaccacg 1620 gccgagcagg tggacatggg cgacgatgtg gtggcgtaca cccgcgcggc ggttggcggg 1680 cgggtgttca cctcaatcac caacttcggc aatgccccgg tcgcgctgcc cgatggctcc 1740 gtggtgctgg cgtccggccc gctgaccccc gaaggccagc tccccaccga cacttctgcg 1800 tgggttatca agtag <210> 26 <211> 604 <212> PRT <213> Bifidobacterium longum <400> 26 1815
    Page 70
    CL6220WOPCT_SequenceListing_ST25.txt Met Thr Ala Asn Asn Leu Asn Asp Asp Trp Trp Lys Gln Ala Val Val
    1 5 10 15 Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Val Asn Gly Asp Gly Leu 20 25 30 Gly Asp Ile Ala Gly Val Thr Glu Lys Met Asp Tyr Leu Lys Asn Leu 35 40 45 Gly Val Asp Ala Ile Trp Leu Ser Pro Phe Tyr Pro Ser Asp Leu Ala 50 55 60 Asp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu 65 70 75 80 Gly Thr Met Asp Asp Phe Asp Ala Met Ala Glu Ala Ala His Glu Ala 85 90 95 Gly Ile Lys Val Ile Val Asp Ile Val Pro Asn His Thr Ala Asp Lys 100 105 110 His Val Phe Phe Lys Glu Ala Leu Ala Ser Glu Pro Gly Ser Pro Ala 115 120 125 Arg Asp Arg Tyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly Glu Leu 130 135 140 Pro Pro Asn Asp Trp Gln Ser Phe Phe Gly Gly Pro Ala Trp Ala Arg 145 150 155 160 Val Pro Asp Gly Gln Trp Tyr Leu His Leu Phe Asp Lys Ala Gln Pro 165 170 175 Asp Val Asn Trp Lys Asn Pro Asp Ile His Glu Glu Phe Lys Lys Thr 180 185 190 Leu Arg Phe Trp Ser Asp His Gly Thr Asp Gly Phe Arg Ile Asp Val 195 200 205 Ala His Gly Leu Ala Lys Asp Leu Glu Ser Lys Pro Leu Glu Glu Leu 210 215 220 Gly Arg Glu Tyr Ser Val Val Gly Val Leu Asn His Asp Phe Ser His 225 230 235 240 Pro Leu Phe Asp Arg Arg Glu Val His Asp Ile Tyr Arg Glu Trp Arg 245 250 255 Lys Val Phe Asn Glu Tyr Thr Pro Pro Arg Phe Ala Val Ala Glu Ala 260 265 270
    Page 71
    CL6220WOPCT_SequenceListing_ST25.txt
    Trp Val Val 275 Pro Glu His Gln His 280 Leu Tyr Ala Ser Met 285 Asp Glu Leu Gly Gln Ser Phe Asn Phe Asp Phe Ala Gln Ala Asn Trp Tyr Ala Asp 290 295 300 Glu Phe Arg Lys Ala Ile Ala Ala Gly Leu Lys Ala Ala Ala Glu Thr 305 310 315 320 Gly Gly Ser Thr Thr Thr Trp Val Met Asn Asn His Asp Val Pro Arg 325 330 335 Ser Pro Ser Arg Tyr Gly Leu Pro Gln Val Lys Gly Ala Pro Tyr His 340 345 350 Gln Leu Pro His Asp Trp Leu Leu Arg Asp Gly Thr Thr Tyr Pro Glu 355 360 365 Asn Arg Glu Leu Gly Thr Arg Arg Ala Arg Ala Ala Ala Leu Met Glu 370 375 380 Leu Gly Leu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly Glu Glu Leu Gly 385 390 395 400 Leu Phe Glu Val Ala Asp Ile Pro Trp Asp His Leu Glu Asp Pro Thr 405 410 415 Ala Phe His Thr Thr Arg Asn Thr Met Asp Lys Gly Arg Asp Gly Cys 420 425 430 Arg Val Pro Leu Pro Trp Thr Ala Ala Asp Glu Pro Ala Leu Ala Asp 435 440 445 Phe Ser Arg Pro Ala Pro Ala Asp Asp Gly Thr Gly Glu Asn His Val 450 455 460 Pro Leu Cys Ala Ala Gly Gln Phe Gly Thr Gly Ala Ser Phe Gly Phe 465 470 475 480 Ser Pro Ala Val Arg Ala Asp Gly Val Thr Pro Ala Ala Asp Pro His 485 490 495 Leu Pro Gln Pro Leu Trp Phe Lys Asp Tyr Ala Val Asp Val Glu Gln 500 505 510 Ala Asp Pro Asp Ser Met Leu Ala Leu Tyr Arg Ala Ala Leu Ala Ile 515 520 525 Arg Gln Glu Ser Leu Thr Ala Thr Arg Asp Thr Thr Ala Glu Gln Val 530 535 540
    Page 72
    CL6220WOPCT_SequenceListing_ST25.txt
    Asp Met Gly Asp Asp Val Val Ala Tyr Thr Arg Ala Ala Val Gly Gly 545 550 555 560 Arg Val Phe Thr Ser Ile Thr Asn Phe Gly Asn Ala Pro Val Ala Leu 565 570 575 Pro Asp Gly Ser Val Val Leu Ala Ser Gly Pro Leu Thr Pro Glu Gly 580 585 590 Gln Leu Pro Thr Asp Thr Ser Ala Trp Val Ile Lys
    595 600 <210> 27 <211> 1812 <212> DNA <213> Artificial sequence <220>
    <223> BloGlu3_codon-optimized <400> 27
    gtgacagcaa acaacctgaa tgacgactgg tggaaacagg ccgtcgtgta tcaaatttac 60 cctagatcat ttaaggacgt taatggcgat ggactgggag atattgcagg cgtcacggag 120 aagatggatt accttaaaaa cctgggcgtt gatgctattt ggctgtcacc gttttacccg 180 tcagacctgg ctgatggcgg ctatgacgtt atcgattata gaaatgtgga tcctagactg 240 ggcacaatgg atgattttga cgcaatggcc gaagccgcgc atgaagcggg aattaaagtt 300 attgttgata tcgttcctaa tcatacagca gataagcatg tttttttcaa agaagcactg 360 gcctcagaac cgggctcacc tgcaagagat agatacattt ttagagacgg aagaggcgaa 420 cacggcgagc tgcctccgaa tgactggcaa tcattttttg gaggacctgc atgggcaaga 480 gtccctgacg gacaatggta tctgcacctt ttcgataaag cacaaccgga tgttaattgg 540 aaaaaccctg acatccatga agaatttaag aaaacactga gattttggtc agatcacggc 600 acagacggat ttagaattga tgtggcacat ggacttgcaa aagatctgga aagcaaaccg 660 ctggaggaac tgggaagaga gtacagcgtt gttggcgttc ttaatcatga cttttcacat 720 ccgctgtttg acagaagaga agtgcatgac atctacagag aatggagaaa ggtgtttaac 780 gaatatacac cgccgagatt tgcagtcgca gaagcatggg tggtgcctga acaccaacat 840 ctgtatgcat caatggacga gctgggccaa tcattcaatt tcgactttgc acaagctaat 900 tggtatgcag atgagttcag aaaagctatc gctgcgggcc tgaaagcagc agctgagacg 960 ggcggatcaa cgacaacatg ggtcatgaat aatcacgatg ttccgagatc accgtcaaga 1020 tatggcctgc cgcaggttaa gggcgcaccg tatcatcagc ttccgcacga ttggcttctg 1080 agagacggca cgacgtaccc ggagaataga gagctgggaa cgagaagagc aagagcagcg 1140 gcgcttatgg aacttggact gcctggagcg gcttacattt atcagggcga ggagcttggc 1200 ctttttgagg tggcagatat tccttgggat catcttgaag atccgacagc atttcacaca 1260 acaagaaata cgatggataa aggcagagat ggctgcagag tgcctctgcc gtggacagct Page 73 1320
    CL6220WOPCT_SequenceListing_ST25.txt gccgatgagc ctgcccttgc cgactttagc gagaaccacg ttcctctgtg tgcagcgggc tcaccggccg ttagagccga tggcgttaca ctgtggttta aagattatgc agttgacgtt ctgtatagag ctgcacttgc tattagacaa gcggaacagg tcgacatggg cgatgatgtc agagttttta caagcattac aaattttggc gtcgttctgg cttcaggacc tctgacaccg tgggttatta ag agaccggcac ctgccgacga cggaacggga cagtttggca cgggagcatc atttggattc ccggctgcgg acccgcatct gcctcagccg gaacaagccg atcctgattc aatgctggca gaatcactta cggcaacgag agatacaacg gtcgcatata cgagagcggc cgtgggagga aacgcacctg tcgcactgcc ggatggcagc gagggacaac tgcctacaga cacgtcagct
    1380
    1440
    1500
    1560
    1620
    1680
    1740
    1800
    1812 <210> 28 <211> 585 <212> PRT <213> Bifidobacterium pseudolongum <400> 28
    Met Thr 1 Leu Asn Asn 5 Thr His Asp Asp Trp Trp Lys Gln Ala Val Val 10 15 Tyr Gln Val Tyr Pro Arg Ser Phe Arg Asp Ala Asn Gly Asp Gly Leu 20 25 30 Gly Asp Ile Ala Gly Ile Thr Ser Arg Ile Pro Tyr Leu Arg Gln Leu 35 40 45 Gly Val Asp Ala Leu Trp Leu Ser Pro Phe Tyr Pro Ser Glu Leu Ala 50 55 60 Asp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu 65 70 75 80 Gly Thr Leu Asp Asp Phe Asp Ala Met Val Ala Ala Ala His Ser Ala 85 90 95 Gly Leu Lys Val Val Val Asp Ile Val Pro Asn His Thr Ser Asn Met 100 105 110 His Pro Trp Phe Gln Glu Ala Leu Ala Ser Ala Pro Gly Ser Pro Ala 115 120 125 Arg Asp Arg Tyr Ile Phe Arg Asp Gly Glu Gly Ala His Gly Glu Leu 130 135 140 Pro Pro Asn Asn Trp Gln Ser Leu Phe Gly Gly Pro Ala Trp Glu Ala 145 150 155 160 Ala Gly Asp Gly Gln Trp Tyr Leu His Leu Phe Thr Lys Glu Gln Pro
    Page 74
    CL6220WOPCT_SequenceListing_ST25.txt
    165 170 175 Asp Leu Asn Trp Lys Asn Pro Asp Val His Glu Asp Phe Arg Thr Thr 180 185 190 Leu Arg Phe Trp Ser Asp Arg Gly Val Asp Gly Phe Arg Ile Asp Val 195 200 205 Ala His Gly Leu Ala Lys Asp Leu Asp Ser Glu Pro Leu Lys Asp Leu 210 215 220 Glu Arg Phe Pro Val Gly Gly Asn Pro Val Pro Gly His Pro Leu Trp 225 230 235 240 Asp Arg Pro Glu Val His Glu Ile Tyr Arg Glu Trp Asn Lys Val Phe 245 250 255 Asn Glu Tyr Asp Pro Pro Arg Phe Ala Val Gly Glu Ala Trp Val Pro 260 265 270 Ala Glu His Gln His Leu Tyr Ala Ser Lys Asp Glu Leu Gly Gln Val 275 280 285 Phe Asn Phe Glu Phe Ala Lys Ala Asn Trp Phe Ala Asp Asp Phe Arg 290 295 300 Leu Ala Ile Glu Glu Gly Leu Ala Ser Ala Asp Glu Ser Lys Ser Thr 305 310 315 320 Thr Thr Trp Val Met Ser Asn His Asp Val Pro Arg His Val Ser Arg 325 330 335 Tyr Gly Leu Pro Gln Val His Thr Arg Gly Tyr His Glu Leu Pro Asn 340 345 350 Asp Trp Leu Leu Arg Asn Gly Thr Thr Tyr Ile Glu Asp Arg Glu Leu 355 360 365 Gly Thr Arg Arg Ala Arg Ala Ala Ile Leu Met Glu Leu Gly Leu Pro 370 375 380 Gly Ser Val Tyr Val Tyr Gln Gly Glu Glu Leu Gly Leu Pro Glu Val 385 390 395 400 Ala Thr Ile Pro Trp Asp His Leu Glu Asp Pro Val Ala Phe Asn Thr 405 410 415 Asp His Ser Asp Ala Ala Lys Gly Arg Asp Gly Cys Arg Val Pro Leu 420 425 430 Pro Trp Ser Ala Gln Asp Met Pro Gln Pro Ala Pro Trp Asp Pro Glu Pag e 75
    435 CL6220WOPCT_SequenceListing_ST25.txt 440 445 Phe Gly Thr Gly Ala Ser Phe Gly Phe Ser Glu His Ala Gly Gly Arg 450 455 460 Ala Ser Ala Asp Pro His Leu Pro Gln Pro Leu Trp Tyr Ala Gly Tyr 465 470 475 480 Ala Ala Asp Met Glu Asp Thr Asp Pro Ala Ser Met Leu Asn Leu Tyr 485 490 495 Arg Arg Ala Met His Trp Arg Gln Glu His Leu Thr Pro Thr Gly Asp 500 505 510 Thr Ser Leu Thr Trp Leu Ser Pro Gln Ser Phe Ala Asp Cys Gly Asp 515 520 525 Asp Val Val Ala Tyr Ala Arg Pro Leu Ala Asp Asp Ser Gly Asp Arg 530 535 540 Phe Val Cys Ile Val Asn Phe Gly Ala Ala Ser Ile Glu Leu Pro His 545 550 555 560 Gly Asp Val Met Met Arg Ser Ile Pro Phe Asp Gly Tyr Gln Leu Pro 565 570 575 Ala Asp Ala Ala Val Trp Met Arg Ile 580 585
    <210> 29 <211> 1755 <212> DNA <213> Artificial sequence <220> <223> BpsGlu1_codon-optimized <400> 29 gtgacactga ataatacaca cgatgactgg tggaaacaag ccgttgtgta tcaagtttat 60 ccgagatcat ttagagacgc taacggcgat ggactgggag acatcgcagg cattacgagc 120 agaatcccgt atcttagaca acttggcgtg gacgcgcttt ggctttcacc gttttatcct 180 agcgagctgg cagacggcgg atatgatgtt attgattaca gaaatgttga cccgagactt 240 ggcacactgg acgactttga tgctatggtg gcggctgccc atagcgcggg actgaaagtt 300 gttgttgata tcgttccgaa tcatacaagc aacatgcacc cttggttcca agaggcgctg 360 gcgagcgcac cgggatcacc tgcgagagat agatacattt tcagagatgg agaaggcgca 420 cacggcgagc ttccgcctaa caattggcaa agcctgtttg gaggaccggc ctgggaagca 480 gcaggcgacg gacaatggta cctgcacctt tttacaaaag aacagcctga tctgaattgg 540 aaaaatcctg acgttcacga ggatttcaga acgacactta gattctggtc agacagaggc 600
    Page 76
    gttgatggct ttagaatcga CL6220WOPCT_SequenceListing_ST25 cgttgcacat ggacttgcga aagacctgga .txt tagcgaaccg 660 ctgaaggatc ttgagagatt cccggtcgga ggaaatccgg ttccgggcca tccgctgtgg 720 gatagacctg aagtgcacga gatttacaga gagtggaata aagtctttaa tgaatacgac 780 ccgcctagat tcgctgttgg cgaagcatgg gtgcctgccg aacatcaaca tctttatgcc 840 tcaaaggatg agctgggaca agtgttcaac ttcgaatttg ctaaggcaaa ttggtttgca 900 gatgacttta gactggcaat tgaggagggc ctggcctcag cggacgagtc aaaatcaacg 960 acaacatggg ttatgtcaaa tcacgacgtg ccgagacacg tgagcagata tggccttccg 1020 caagttcata cgagaggcta tcacgagctg cctaacgatt ggctgctgag aaatggcaca 1080 acatatattg aagatagaga actgggcaca agaagagcaa gagccgcgat cctgatggaa 1140 cttggacttc ctggatcagt ttatgtttat caaggcgagg agcttggcct gcctgaggtg 1200 gcaacaattc cttgggacca cctggaagat ccggtcgcct tcaacacaga tcactcagat 1260 gcagcaaaag gaagagacgg atgtagagtg ccgctgccgt ggtcagctca ggatatgccg 1320 cagcctgcgc cgtgggatcc ggaatttggc acgggcgcat catttggatt ctcagagcat 1380 gccggaggca gagcttcagc agacccgcat ctgccgcaac ctctgtggta tgcaggctat 1440 gctgccgata tggaggatac ggatcctgcg tcaatgctga atctgtatag aagagctatg 1500 cactggagac aggagcatct tacacctaca ggcgatacat cactgacatg gctgtcaccg 1560 caatcattcg cagattgcgg agatgatgtc gtggcatacg caagaccgct ggctgacgat 1620 agcggcgaca gatttgtttg cattgttaac ttcggagcag cgagcatcga actgcctcac 1680 ggagacgtta tgatgagatc aatccctttc gatggatacc agcttcctgc ggacgctgcg 1740 gtgtggatga gaatt 1755
    <210> 30 <211> 585 <212> PRT <213> Artificial sequence <220>
    <223> BpsGlu1 protein_encoded by optimized sequence <400> 30
    Met Thr Leu Asn Asn Thr His Asp Asp Trp Trp Lys Gln Ala Val Val 1 5 10 15 Tyr Gln Val Tyr Pro Arg Ser Phe Arg Asp Ala Asn Gly Asp Gly Leu 20 25 30 Gly Asp Ile Ala Gly Ile Thr Ser Arg Ile Pro Tyr Leu Arg Gln Leu 35 40 45 Gly Val Asp Ala Leu Trp Leu Ser Pro Phe Tyr Pro Ser Glu Leu Ala 50 55 60 Asp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu
    Page 77
    CL6220WOPCT_SequenceListing_ST25.txt
    65 70 75 80 Gly Thr Leu Asp Asp Phe Asp Ala Met Val Ala Ala Ala His Ser Ala 85 90 95 Gly Leu Lys Val Val Val Asp Ile Val Pro Asn His Thr Ser Asn Met 100 105 110 His Pro Trp Phe Gln Glu Ala Leu Ala Ser Ala Pro Gly Ser Pro Ala 115 120 125 Arg Asp Arg Tyr Ile Phe Arg Asp Gly Glu Gly Ala His Gly Glu Leu 130 135 140 Pro Pro Asn Asn Trp Gln Ser Leu Phe Gly Gly Pro Ala Trp Glu Ala 145 150 155 160 Ala Gly Asp Gly Gln Trp Tyr Leu His Leu Phe Thr Lys Glu Gln Pro 165 170 175 Asp Leu Asn Trp Lys Asn Pro Asp Val His Glu Asp Phe Arg Thr Thr 180 185 190 Leu Arg Phe Trp Ser Asp Arg Gly Val Asp Gly Phe Arg Ile Asp Val 195 200 205 Ala His Gly Leu Ala Lys Asp Leu Asp Ser Glu Pro Leu Lys Asp Leu 210 215 220 Glu Arg Phe Pro Val Gly Gly Asn Pro Val Pro Gly His Pro Leu Trp 225 230 235 240 Asp Arg Pro Glu Val His Glu Ile Tyr Arg Glu Trp Asn Lys Val Phe 245 250 255 Asn Glu Tyr Asp Pro Pro Arg Phe Ala Val Gly Glu Ala Trp Val Pro 260 265 270 Ala Glu His Gln His Leu Tyr Ala Ser Lys Asp Glu Leu Gly Gln Val 275 280 285 Phe Asn Phe Glu Phe Ala Lys Ala Asn Trp Phe Ala Asp Asp Phe Arg 290 295 300 Leu Ala Ile Glu Glu Gly Leu Ala Ser Ala Asp Glu Ser Lys Ser Thr 305 310 315 320 Thr Thr Trp Val Met Ser Asn His Asp Val Pro Arg His Val Ser Arg 325 330 335 Tyr Gly Leu Pro Gln Val His Thr Arg Gly Tyr His Glu Leu Pro Asn Pag ie 78
    340 CL6220WOPCT_SequenceListing_ST25.txt 345 350 Asp Trp Leu 355 Leu Arg Asn Gly Thr Thr Tyr Ile Glu Asp 360 365 Arg Glu Leu Gly Thr Arg 370 Arg Ala Arg Ala Ala Ile Leu Met Glu Leu 375 380 Gly Leu Pro Gly Ser Val 385 Tyr Val Tyr Gln Gly Glu Glu Leu Gly Leu 390 395 Pro Glu Val 400 Ala Thr Ile Pro Trp Asp His Leu Glu Asp Pro Val Ala 405 410 Phe Asn Thr 415 Asp His Ser Asp 420 Ala Ala Lys Gly Arg Asp Gly Cys Arg 425 Val Pro Leu 430 Pro Trp Ser 435 Ala Gln Asp Met Pro Gln Pro Ala Pro Trp 440 445 Asp Pro Glu Phe Gly Thr 450 Gly Ala Ser Phe Gly Phe Ser Glu His Ala 455 460 Gly Gly Arg Ala Ser Ala 465 Asp Pro His Leu Pro Gln Pro Leu Trp Tyr 470 475 Ala Gly Tyr 480 Ala Ala Asp Met Glu Asp Thr Asp Pro Ala Ser Met Leu 485 490 Asn Leu Tyr 495 Arg Arg Ala Met 500 His Trp Arg Gln Glu His Leu Thr Pro 505 Thr Gly Asp 510 Thr Ser Leu 515 Thr Trp Leu Ser Pro Gln Ser Phe Ala Asp 520 525 Cys Gly Asp Asp Val Val 530 Ala Tyr Ala Arg Pro Leu Ala Asp Asp Ser 535 540 Gly Asp Arg Phe Val Cys 545 Ile Val Asn Phe Gly Ala Ala Ser Ile Glu 550 555 Leu Pro His 560 Gly Asp Val Met Met Arg Ser Ile Pro Phe Asp Gly Tyr 565 570 Ala Asp Ala Ala Val Trp Met Arg Ile 580 585 <210> 31 <211> 1806 <212> DNA <213> Bifidobacterium thermophilum Gln Leu Pro 575
    Page 79
    <400> 31 atggcagaac gcaagtcccc CL6220WOPCT_SequenceListing_ST25 ccagtccgcg caagaatcca cggcatcgga .txt tcgcgccgca 60 gcggcctggt ggcatcaagc ggtggtctat caggtctatc cgcgttcgtt caaggacacc 120 accggttccg gcctcggcga catcgccggt gttaccagcc gcatcggcta cctcaagcaa 180 ctgggtgttg acgcgatttg gctcagcccc ttctatccga gccaactcgc cgatggcggg 240 tacgatgtcg atgattaccg caacgtcgac ccgaaactcg gtacgatgga cgatttcgac 300 aaactcgcca aaaccgcgca cgaggccggt atcaagattg tcgtcgatat cgttcccaac 360 cattcttcca acctgcaccc ctggttcaag gccgcgcttg cagctggtcc gggctcgccg 420 gaacgtgacc gctacatctt ccgtgacggc cgcggtgagc atggcgaact gccgcccacc 480 gactgggtgt cccatttcgg cggccccgcg tggacgcgcg tgcctgacgg ccagtggtat 540 ctgcacctgt tcaccgtgga gcagcccgac tggaactgga agaacccgga tgtgcaggcg 600 gacttcatca agaccctgcg tttttggctt gatcacggcg ctgacggctt ccgtgtcgat 660 gttgcgcacg gcctgtgcaa ggacctcgac cgcgacaatc tcgaccagtg gagcgtcacc 720 ccgccaagcc tgcccgccga cggcagccat ccgctgtacg accgcgacga cgtgcatcag 780 atttatcgcg agtggcgcaa ggtgttcaac gaatatgatc cgccggcatt cgccgtggcc 840 gaggcgtggg ttaatcccgc gcggcagtat ctgtatgcgt ccgacgatga gctcggtcag 900 gtgttcaact tcgagttcgc gaagaagaac tgggtgcgtg acgacatgca tcaggcgatc 960 gaggaaggtc tcgaggcggc gcgtcgttct ggctctaccg ccacgtgggt gatgagtaat 1020 cacgatgttc cacgtcacgc cagccgttac gcactgccgc aagtgccgag cacgcggcat 1080 catcagttgg cgcatgactg gctgctgcgt gacggcacca gctatcacga ggatcgcgaa 1140 gccggtacgc gccgcgcccg ggccgcgatt ttgatggagc ttgcgttgcc cggttcggcg 1200 tacctgtacc agggcgaaga gcttggtctg ttcgaggtcg ctgatattcc gtggaacaag 1260 ctcgaggatc cgaccgcgcg caatagcgaa cgtgcggcca aggacaaggg gcgcgacggg 1320 tgccgcgtcc ccctgccgtg ggtcgccgcc gacggggtcg aggggtcgtt cggcttctcg 1380 cctcgtgtga aatccgtggg cgctggcgtt tccgccgatc aggccgggca gccgtcggag 1440 cctgcacacc tgccgcaacc cgcatggttc gctgatttcg ccgccgaccg tgagagcgcg 1500 cagccggagt cgatgttgaa cctgtaccgc agggcgttgg cgttgcgcca tgagctgatg 1560 ccggccgaca caacgctgac ttggctggat gaagaccgcc cgtctgatgc gccggatggc 1620 gctgacggtc agcacggcgg cgtgattgct taccgccggt ccaacggctg ggcgagtgtg 1680 accaatttcg gtgcggaacc tgtcgcattg ccggcgggcg aggtgctgct cacctccggc 1740 gagctgtgct ccgacggccg gctgccgcaa gataccaccg tttggctgcg gttgaaccag 1800
    gactga 1806 <210> 32 <211> 601 <212> PRT <213> Bifidobacterium thermophilum
    Page 80
    CL6220WOPCT_SequenceListing_ST25.txt <400> 32
    Met Ala 1 Glu Arg Lys Ser 5 Pro Gln Ser Ala Gln Glu Ser Thr Ala Ser 10 15 Asp Arg Ala Ala Ala Ala Trp Trp His Gln Ala Val Val Tyr Gln Val 20 25 30 Tyr Pro Arg Ser Phe Lys Asp Thr Thr Gly Ser Gly Leu Gly Asp Ile 35 40 45 Ala Gly Val Thr Ser Arg Ile Gly Tyr Leu Lys Gln Leu Gly Val Asp 50 55 60 Ala Ile Trp Leu Ser Pro Phe Tyr Pro Ser Gln Leu Ala Asp Gly Gly 65 70 75 80 Tyr Asp Val Asp Asp Tyr Arg Asn Val Asp Pro Lys Leu Gly Thr Met 85 90 95 Asp Asp Phe Asp Lys Leu Ala Lys Thr Ala His Glu Ala Gly Ile Lys 100 105 110 Ile Val Val Asp Ile Val Pro Asn His Ser Ser Asn Leu His Pro Trp 115 120 125 Phe Lys Ala Ala Leu Ala Ala Gly Pro Gly Ser Pro Glu Arg Asp Arg 130 135 140 Tyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly Glu Leu Pro Pro Thr 145 150 155 160 Asp Trp Val Ser His Phe Gly Gly Pro Ala Trp Thr Arg Val Pro Asp 165 170 175 Gly Gln Trp Tyr Leu His Leu Phe Thr Val Glu Gln Pro Asp Trp Asn 180 185 190 Trp Lys Asn Pro Asp Val Gln Ala Asp Phe Ile Lys Thr Leu Arg Phe 195 200 205 Trp Leu Asp His Gly Ala Asp Gly Phe Arg Val Asp Val Ala His Gly 210 215 220 Leu Cys Lys Asp Leu Asp Arg Asp Asn Leu Asp Gln Trp Ser Val Thr 225 230 235 240 Pro Pro Ser Leu Pro Ala Asp Gly Ser His Pro Leu Tyr Asp Arg Asp 245 250 255 Asp Val His Gln Ile Tyr Arg Glu Trp Arg Lys Val Phe Asn Glu Tyr Page 81
    260 CL6220WOPCT_SequenceListing_ST25.txt 265 270 Asp Pro Pro Ala Phe Ala Val Ala Glu Ala Trp Val Asn Pro Ala Arg 275 280 285 Gln Tyr Leu Tyr Ala Ser Asp Asp Glu Leu Gly Gln Val Phe Asn Phe 290 295 300 Glu Phe Ala Lys Lys Asn Trp Val Arg Asp Asp Met His Gln Ala Ile 305 310 315 320 Glu Glu Gly Leu Glu Ala Ala Arg Arg Ser Gly Ser Thr Ala Thr Trp 325 330 335 Val Met Ser Asn His Asp Val Pro Arg His Ala Ser Arg Tyr Ala Leu 340 345 350 Pro Gln Val Pro Ser Thr Arg His His Gln Leu Ala His Asp Trp Leu 355 360 365 Leu Arg Asp Gly Thr Ser Tyr His Glu Asp Arg Glu Ala Gly Thr Arg 370 375 380 Arg Ala Arg Ala Ala Ile Leu Met Glu Leu Ala Leu Pro Gly Ser Ala 385 390 395 400 Tyr Leu Tyr Gln Gly Glu Glu Leu Gly Leu Phe Glu Val Ala Asp Ile 405 410 415 Pro Trp Asn Lys Leu Glu Asp Pro Thr Ala Arg Asn Ser Glu Arg Ala 420 425 430 Ala Lys Asp Lys Gly Arg Asp Gly Cys Arg Val Pro Leu Pro Trp Val 435 440 445 Ala Ala Asp Gly Val Glu Gly Ser Phe Gly Phe Ser Pro Arg Val Lys 450 455 460 Ser Val Gly Ala Gly Val Ser Ala Asp Gln Ala Gly Gln Pro Ser Glu 465 470 475 480 Pro Ala His Leu Pro Gln Pro Ala Trp Phe Ala Asp Phe Ala Ala Asp 485 490 495 Arg Glu Ser Ala Gln Pro Glu Ser Met Leu Asn Leu Tyr Arg Arg Ala 500 505 510 Leu Ala Leu Arg His Glu Leu Met Pro Ala Asp Thr Thr Leu Thr Trp 515 520 525 Leu Asp Glu Asp Arg Pro Ser Asp Ala Pro Asp Gly Ala Asp Gly Gln
    Page 82
    CL6220WOPCT_SequenceListing_ST25.txt
    530 535 540 His Gly Gly Val Ile Ala Tyr Arg Arg Ser Asn Gly Trp Ala Ser Val 545 550 555 560 Thr Asn Phe Gly Ala Glu Pro Val Ala Leu Pro Ala Gly Glu Val Leu 565 570 575 Leu Thr Ser Gly Glu Leu Cys Ser Asp Gly Arg Leu Pro Gln Asp Thr 580 585 590 Thr Val Trp Leu Arg Leu Asn Gln Asp
    595 600 <210> 33 <211> 1803 <212> DNA <213> Artificial sequence <220>
    <223> BthGlu1_codon-optimized <400> 33
    gtggctgaaa gaaaatcacc gcaaagcgca caggaaagca cggcctcaga cagagcagcc 60 gcagcctggt ggcatcaggc cgttgtttat caagtttatc ctagatcatt caaggataca 120 acgggctcag gactgggcga tattgcagga gttacaagca gaatcggcta ccttaagcaa 180 cttggagtcg atgctatttg gctgtcaccg ttttacccgt cacaactggc cgatggcggc 240 tacgatgttg atgactacag aaacgtggat cctaaactgg gcacaatgga tgattttgat 300 aagctggcaa aaacagcaca tgaggcggga atcaagattg ttgttgatat cgttccgaat 360 catagctcaa atctgcatcc gtggtttaaa gcagcactgg cagccggacc gggcagcccg 420 gagagagata gatacatttt cagagacggc agaggcgaac atggcgaact gcctcctaca 480 gactgggttt cacattttgg aggaccggct tggacaagag ttcctgatgg ccagtggtac 540 ctgcatcttt ttacggttga acagcctgat tggaattgga aaaaccctga cgtccaggcg 600 gacttcatta aaacacttag attctggctg gaccatggag cggatggctt tagagttgac 660 gttgcacatg gcctttgtaa ggaccttgac agagacaacc ttgatcagtg gtcagttacg 720 ccgccttcac tgcctgctga cggatcacac ccgctttatg atagagacga cgttcatcag 780 atttatagag aatggagaaa agtttttaac gaatacgatc ctcctgcgtt tgcagtggca 840 gaagcgtggg ttaatccggc tagacaatat ctttatgcaa gcgacgacga gctgggacaa 900 gttttcaact ttgaatttgc caaaaagaat tgggtcagag atgatatgca tcaagcgatt 960 gaagaaggcc tggaagctgc tagaagaagc ggcagcacag caacatgggt tatgtcaaac 1020 catgatgtgc cgagacacgc gtcaagatac gcacttcctc aagttccgag cacaagacat 1080 caccaacttg ctcatgactg gcttctgaga gacggaacgt cataccacga ggatagagag 1140 gctggaacaa gaagagccag agccgcaatt ctgatggagc tggccctgcc gggatcagca 1200
    Page 83
    CL6220WOPCT_SequenceListing_ST25.txt
    tatctgtatc agggcgaaga actgggactg tttgaggttg cagacattcc gtggaataag 1260 ctggaagatc cgacagcaag aaattcagaa agagcggcga aagacaaagg aagagatgga 1320 tgtagagtgc cgctgccttg ggtcgctgcc gacggagttg agggctcatt tggattctca 1380 cctagagtta agagcgtcgg agcgggagtt tcagcagacc aggccggaca accgagcgaa 1440 cctgcacatc tgcctcagcc ggcatggttc gccgatttcg cagccgacag agaatcagca 1500 caaccggagt caatgcttaa cctttacaga agagcgcttg ctctgagaca tgaacttatg 1560 cctgccgata cgacactgac atggcttgac gaagatagac cttcagacgc accggacgga 1620 gcagacggac agcatggagg cgttattgca tatagaagat caaacggctg ggcaagcgtt 1680 acaaatttcg gagctgaacc tgtcgcgctt cctgctggcg aggttcttct tacgagcgga 1740 gaactgtgta gcgatggcag acttccgcag gacacaacag tgtggcttag actgaatcag 1800
    gat 1803 <210> 34 <211> 662 <212> PRT <213> Bifidobacterium breve <400> 34
    Met 1 Tyr Phe His Ile Asn His 5 Leu His Asp Thr Val 10 Val Ile Asn 15 Val Ile Ser Lys His Gly Phe Thr Val Ala Val Arg Val Leu Leu Asn Pro 20 25 30 Ile Thr Thr Asn Pro Gln Gln Ser Gly Ala Thr His His Val Ser His 35 40 45 Thr Ile Thr His Ala Gln Lys Gly Ile Gly Met Thr Ala Asn Asn Leu 50 55 60 Asn Asp Asp Trp Trp Lys Gln Ala Val Val Tyr Gln Ile Tyr Pro Arg 65 70 75 80 Ser Phe Lys Asp Val Asn Gly Asp Gly Ile Gly Asp Ile Ala Gly Val 85 90 95 Thr Glu Lys Met Asp Tyr Leu Lys Asn Leu Gly Val Asp Ala Ile Trp 100 105 110 Leu Ser Pro Phe Tyr Pro Ser Asp Leu Ala Asp Gly Gly Tyr Asp Val 115 120 125 Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu Gly Thr Met Asp Asp Phe 130 135 140 Asp Ala Met Ala Lys Ala Ala His Glu Ala Gly Ile Lys Val Ile Val 145 150 155 160 Page 84
    CL6220WOPCT_SequenceListing_ST25.txt
    Asp Ile Val Pro Asn 165 His Thr Ala Asp Lys 170 His Val Phe Phe Lys 175 Glu Ala Leu Ala Ala Glu Pro Gly Ser Pro Ala Arg Asp Arg Tyr Ile Phe 180 185 190 Arg Asp Gly Arg Gly Glu His Gly Glu Leu Pro Pro Asn Asp Trp Gln 195 200 205 Ser Phe Phe Gly Gly Pro Ala Trp Ala Arg Val Ala Asp Gly Gln Trp 210 215 220 Tyr Leu His Leu Phe Asp Lys Ala Gln Pro Asp Val Asn Trp Lys Asn 225 230 235 240 Pro Asp Ile His Glu Glu Phe Lys Lys Thr Leu Arg Phe Trp Ser Asp 245 250 255 His Gly Thr Asp Gly Phe Arg Ile Asp Val Ala His Gly Leu Ala Lys 260 265 270 Asp Leu Glu Ser Lys Pro Leu Glu Glu Leu Gly Arg Glu Tyr Ser Val 275 280 285 Val Gly Val Leu Asn His Asp Phe Ser His Pro Leu Phe Asp Arg Arg 290 295 300 Glu Val His Asp Ile Tyr Arg Glu Trp Arg Lys Val Phe Asn Glu Tyr 305 310 315 320 Asp Pro Pro Arg Phe Ala Val Ala Glu Ala Trp Val Val Pro Glu His 325 330 335 Gln His Leu Tyr Ala Ser Met Asp Glu Leu Gly Gln Ser Phe Asn Phe 340 345 350 Asp Phe Ala Gln Ala Ser Trp Tyr Ala Asp Glu Phe Arg Ala Ala Ile 355 360 365 Ala Ala Gly Leu Lys Ala Ala Ala Glu Thr Gly Gly Ser Thr Thr Thr 370 375 380 Trp Val Met Asn Asn His Asp Val Pro Arg Ser Pro Ser Arg Tyr Gly 385 390 395 400 Leu Pro Gln Val Lys Gly Ala Pro Tyr His Gln Leu Pro His Asp Trp 405 410 415 Leu Leu Arg Asn Gly Thr Thr Tyr Pro Glu Asp Arg Glu Leu Gly Thr 420 425 430
    Page 85
    CL6220WOPCT_SequenceListing_ST25.txt
    Arg Arg Ala 435 Arg Ala Ala Ala Leu 440 Met Glu Leu Gly Leu 445 Pro Gly Ala Ala Tyr Ile Tyr Gln Gly Glu Glu Leu Gly Leu Phe Glu Val Ala Asp 450 455 460 Ile Pro Trp Asp Arg Leu Glu Asp Pro Thr Ala Phe His Thr Ala Gln 465 470 475 480 Ala Thr Met Asp Lys Gly Arg Asp Gly Cys Arg Val Pro Ile Pro Trp 485 490 495 Thr Ala Ala Asn Glu Pro Thr Leu Ala Asp Phe Ser Arg Pro Ile Pro 500 505 510 Ala Asp Asp Gly Thr Gly Glu Asn His Val Pro Leu Cys Ala Ala Gly 515 520 525 Gln Phe Gly Thr Gly Ala Ser Phe Gly Phe Ser Pro Ala Thr Arg Ala 530 535 540 Glu Gly Val Thr Pro Ala Ala Asp Pro His Leu Pro Gln Pro Leu Trp 545 550 555 560 Phe Lys Asp Tyr Ala Val Asp Val Glu Gln Ala Asp Pro Asp Ser Met 565 570 575 Leu Ala Leu Tyr His Ala Ala Leu Ala Ile Arg Gln Glu Ser Leu Thr 580 585 590 Ala Thr Arg Asp Thr Thr Ala Glu Gln Val Asp Met Gly Pro Asp Val 595 600 605 Val Ala Tyr Thr Arg Ala Ala Val Gly Gly Arg Thr Phe Thr Ser Ile 610 615 620 Thr Asn Phe Gly Thr Glu Pro Val Glu Leu Pro Gly Gly Ser Val Val 625 630 635 640 Leu Thr Ser Gly Pro Leu Thr Pro Asp Gly Gln Leu Pro Thr Asp Thr 645 650 655 Ser Ala Trp Val Ile Lys
    <210> 35 <211> 1812 <212> DNA <213> Artificial sequence
    660 <220>
    Page 86
    CL6220WOPCT_SequenceListing_ST25.txt <223> BbrGlu2_codon-optimized <400> 35
    gtgacagcaa ataaccttaa tgacgactgg tggaaacaag cagtggttta ccaaatctat 60 cctagatcat tcaaggatgt taatggcgat ggaattggcg atattgcagg cgttacggaa 120 aaaatggatt acctgaaaaa tctgggagtt gacgctatct ggcttagccc tttttacccg 180 agcgatctgg ccgacggagg ctatgatgtg attgactata gaaatgttga tccgagactg 240 ggaacgatgg atgatttcga tgcgatggct aaggcggcac atgaagcagg cattaaagtt 300 attgttgata ttgtcccgaa tcatacagct gacaaacatg tctttttcaa agaagcactt 360 gcagcagaac cgggctcacc tgccagagac agatacatct ttagagatgg aagaggagaa 420 cacggcgaac ttccgcctaa tgactggcaa tcattctttg gcggaccggc atgggctaga 480 gtcgcagatg gccaatggta ccttcatctt ttcgacaagg cgcaacctga tgtcaattgg 540 aaaaaccctg acattcacga agaattcaag aaaacgctga gattttggtc agatcatggc 600 acggatggct tcagaatcga tgtggcacac ggacttgcaa aagatctgga aagcaaaccg 660 ctggaggaac ttggcagaga atatagcgtg gttggcgttc tgaaccatga cttttcacat 720 ccgctgttcg atagaagaga agttcatgat atctacagag agtggagaaa agtttttaac 780 gaatatgacc cgccgagatt tgccgttgca gaagcatggg tggtgcctga acaccaacac 840 ctgtatgcat caatggacga gctgggacaa tcattcaact ttgattttgc acaggcatca 900 tggtatgccg atgagtttag agctgccatt gccgcaggac ttaaggccgc agcggaaaca 960 ggaggctcaa cgacaacgtg ggtgatgaat aatcacgacg ttcctagaag cccgtcaaga 1020 tatggcctgc cgcaagttaa aggagcacct taccaccagc ttccgcacga ttggcttctg 1080 agaaatggaa caacatatcc ggaagataga gagcttggca cgagaagagc aagagcagcg 1140 gcactgatgg aactgggcct tcctggcgca gcatacattt atcagggcga agaactggga 1200 ctttttgaag tggcggatat tccgtgggat agacttgagg acccgacagc attccacaca 1260 gcacaagcga caatggataa aggaagagac ggatgcagag tgcctattcc ttggacagct 1320 gcaaatgaac ctacactggc agacttcagc agaccgattc cggcagacga cggaacgggc 1380 gaaaaccatg tcccgctgtg cgctgcagga caattcggca caggagcgtc attcggcttt 1440 agcccggcta caagagccga aggagttaca ccggctgcag atccgcatct gcctcaacct 1500 ctttggttta aagattatgc agttgatgtc gagcaggccg acccggattc aatgctggca 1560 ctgtatcatg cagcactggc gattagacaa gaatcactta cagcaacaag agacacgacg 1620 gcggaacaag tggatatggg ccctgacgtt gttgcatata cgagagcggc cgtgggcggc 1680 agaacattta catcaattac aaattttgga acggagcctg ttgaactgcc gggaggatca 1740 gttgttctga caagcggacc tctgacaccg gacggccaac tgcctacgga cacatcagct 1800 tgggtcatca aa 1812
    <210> 36 <211> 604
    Page 87
    CL6220WOPCT_SequenceListing_ST25.txt <212> PRT <213> Artificial sequence <220>
    <223> BbrGlu2 protein_encoded by optimized sequence <400> 36
    Met 1 Thr Ala Asn Asn 5 Leu Asn Asp Asp Trp Trp Lys Gln Ala Val Val 10 15 Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Val Asn Gly Asp Gly Ile 20 25 30 Gly Asp Ile Ala Gly Val Thr Glu Lys Met Asp Tyr Leu Lys Asn Leu 35 40 45 Gly Val Asp Ala Ile Trp Leu Ser Pro Phe Tyr Pro Ser Asp Leu Ala 50 55 60 Asp Gly Gly Tyr Asp Val Ile Asp Tyr Arg Asn Val Asp Pro Arg Leu 65 70 75 80 Gly Thr Met Asp Asp Phe Asp Ala Met Ala Lys Ala Ala His Glu Ala 85 90 95 Gly Ile Lys Val Ile Val Asp Ile Val Pro Asn His Thr Ala Asp Lys 100 105 110 His Val Phe Phe Lys Glu Ala Leu Ala Ala Glu Pro Gly Ser Pro Ala 115 120 125 Arg Asp Arg Tyr Ile Phe Arg Asp Gly Arg Gly Glu His Gly Glu Leu 130 135 140 Pro Pro Asn Asp Trp Gln Ser Phe Phe Gly Gly Pro Ala Trp Ala Arg 145 150 155 160 Val Ala Asp Gly Gln Trp Tyr Leu His Leu Phe Asp Lys Ala Gln Pro 165 170 175 Asp Val Asn Trp Lys Asn Pro Asp Ile His Glu Glu Phe Lys Lys Thr 180 185 190 Leu Arg Phe Trp Ser Asp His Gly Thr Asp Gly Phe Arg Ile Asp Val 195 200 205 Ala His Gly Leu Ala Lys Asp Leu Glu Ser Lys Pro Leu Glu Glu Leu 210 215 220 Gly Arg Glu Tyr Ser Val Val Gly Val Leu Asn His Asp Phe Ser His 225 230 235 240
    Page 88
    CL6220WOPCT_SequenceListing_ST25.txt
    Pro Leu Phe Asp Arg 245 Arg Glu Val His Asp 250 Ile Tyr Arg Glu Trp 255 Arg Lys Val Phe Asn Glu Tyr Asp Pro Pro Arg Phe Ala Val Ala Glu Ala 260 265 270 Trp Val Val Pro Glu His Gln His Leu Tyr Ala Ser Met Asp Glu Leu 275 280 285 Gly Gln Ser Phe Asn Phe Asp Phe Ala Gln Ala Ser Trp Tyr Ala Asp 290 295 300 Glu Phe Arg Ala Ala Ile Ala Ala Gly Leu Lys Ala Ala Ala Glu Thr 305 310 315 320 Gly Gly Ser Thr Thr Thr Trp Val Met Asn Asn His Asp Val Pro Arg 325 330 335 Ser Pro Ser Arg Tyr Gly Leu Pro Gln Val Lys Gly Ala Pro Tyr His 340 345 350 Gln Leu Pro His Asp Trp Leu Leu Arg Asn Gly Thr Thr Tyr Pro Glu 355 360 365 Asp Arg Glu Leu Gly Thr Arg Arg Ala Arg Ala Ala Ala Leu Met Glu 370 375 380 Leu Gly Leu Pro Gly Ala Ala Tyr Ile Tyr Gln Gly Glu Glu Leu Gly 385 390 395 400 Leu Phe Glu Val Ala Asp Ile Pro Trp Asp Arg Leu Glu Asp Pro Thr 405 410 415 Ala Phe His Thr Ala Gln Ala Thr Met Asp Lys Gly Arg Asp Gly Cys 420 425 430 Arg Val Pro Ile Pro Trp Thr Ala Ala Asn Glu Pro Thr Leu Ala Asp 435 440 445 Phe Ser Arg Pro Ile Pro Ala Asp Asp Gly Thr Gly Glu Asn His Val 450 455 460 Pro Leu Cys Ala Ala Gly Gln Phe Gly Thr Gly Ala Ser Phe Gly Phe 465 470 475 480 Ser Pro Ala Thr Arg Ala Glu Gly Val Thr Pro Ala Ala Asp Pro His 485 490 495 Leu Pro Gln Pro Leu Trp Phe Lys Asp Tyr Ala Val Asp Val Glu Gln 500 505 510
    Page 89
    CL6220WOPCT_SequenceListing_ST25.txt Ala Asp Pro Asp Ser Met Leu Ala Leu Tyr His Ala Ala Leu Ala Ile
    515 520 525
    Arg Gln Glu Ser Leu Thr Ala Thr Arg Asp Thr Thr Ala Glu Gln Val 530 535 540
    Asp Met Gly Pro Asp Val Val Ala Tyr Thr Arg Ala Ala Val Gly Gly
    545 550 555 560
    Arg Thr Phe Thr Ser Ile Thr Asn Phe Gly Thr Glu Pro Val Glu Leu
    565 570 575
    Pro Gly Gly Ser Val Val Leu Thr Ser Gly Pro Leu Thr Pro Asp Gly 580 585 590
    Gln Leu Pro Thr Asp Thr Ser Ala Trp Val Ile Lys 595 600 <210> 37 <211> 1821 <212> DNA <213> Bifidobacterium breve <400> 37
    atgactactt tcaaccgcgc aataattcct gacgccattc gcaccaacgg agccacgccc 60 aacccgtggt ggtcgaatgc ggtggtgtac cagatctacc cgcggtcgtt ccaggacacg 120 aacggcgatg gtctcggcga cctgaagggc atcacctccc gcctcgacta tcttgccgat 180 ctcggcgtgg atgtgctctg gctctccccg gtctacaggt ccccgcaaga cgacaacggc 240 tacgacatct ccgactaccg ggacatcgac ccgctgttcg gcacgcttga cgacatggac 300 gagctgctgg ccgaagcgca caagcgcggc ctcaagatcg tgatggacct ggtggtcaac 360 cacacctccg acgagcacgc gtggttcgag gcgtcgaagg acaaggacga cccgcacgcc 420 gactggtact ggtggcgtcc cgcccgcccc ggccacgagc cgggcacgcc cggcgccgag 480 ccgaaccagt ggggctccta cttcggcggc tccgcatggg agtacagccc ggagcgcggc 540 gagtactacc tgcaccagtt ctcgaagaag cagcctgatc tcaactggga gaacccggcc 600 gtgcgccgtg cagtgtacga catgatgaat tggtggctcg atcgcggcat cgacggcttc 660 cgtatggacg tcatcaccct tatctccaag cgtacggatg caaacggcag gctccccggc 720 gagtacggtt ccgagctcca tgacctgccg gtgggggagg agggctactc cagcccgaat 780 ccgttctgtg ccgacggtcc gcgtcaggac gagttcctcg ccgagatgcg ccgcgaggtg 840 ttcgacgggc gtgacggctt cctcaccgtc ggcgaggccc ccgggatcac cgccgaacgc 900 aacgagcaca tcaccaatcc ggccaatggg gagctggata tgctgttcct gttcgaacat 960 gtcgattttg attgtgatgg cgtcaagtgg aagcctctgc cgctcgattt gccgggattc 1020 aagcggatca tggccggata tcagactgct gtggagaacg tgggctgggc aagcttgttc 1080 actggtaacc acgatcagcc acgtgtggtc tctcgttggg gcgatgactc ctcggaggaa 1140
    Page 90
    CL6220WOPCT_SequenceListmg_ST25.txt tcccgcgtgc gctcggccaa agcgcttggc tacgtatatc agggtgagga gctgggcatg taccgcgacc ttgaatctct caatgcctat tcgccggaat cgatgatggc gggtatcgcc atgcaatggg atggttctgc ctatgccggt tggatttccg tcaacccgaa tcatgctgaa gactcggtgt atgccttcta caagaagctc gcggctggcg agtggcggct gattgatgcg acgcttggca acgagcgatt gctggttgtg ccgcgtgaat ccaccgagct gattgccggc acgtacgacg cccctcacac tgtggtctcc gctgctgccg tccagctgta a ctgatgttgc acatgcatcg cggcaccccg accaatgctc acttcaccag cctcgatcag cgtcagaggg tcgaggaagc caaggtacaa gcgcgcggtc gcgacaattc gcgtacccca ttcaccgcac cggatgcagc gacggagccg atcaatgcgg ccggcgaatt cgacgatcct atcgccttgc gccacaacag ttcgattgtg gatgacgcgc atgtatatgc gttcacccgc gttaacctgt ccggccgaac cgtcgacttg ggcgtcactg agccagatat cattctctcc ctcgccaacc gtgagcttga cccgtgggag
    1200
    1260
    1320
    1380
    1440
    1500
    1560
    1620
    1680
    1740
    1800
    1821 <210> 38 <211> 419 <212> DNA <213> Bifidobacterium breve <220>
    <221> misc _feature <222> (4). .(4) <223> n is a, c, g, or t <220> <221> misc _feature <222> (11) ..(11) <223> n is a, c, g, or t <220> <221> misc _feature <222> (15) ..(15) <223> n is a, c, g, or t <220> <221> misc _feature <222> (19) ..(19) <223> n is a, c, g, or t <220> <221> misc _feature <222> (29) ..(29) <223> n is a, c, g, or t <220> <221> misc _feature <222> (56) ..(56) <223> n is a, c, g, or t <220> <221> misc _feature <222> (84) ..(84) <223> n is a, c, g, or t <220> <221> misc _feature
    Page 91
    CL6220WOPCT_SequenceListing_ST25.txt <222> (115)..(115) <223> n is a, c, g, <220>
    <221> misc_feature <222> (136)..(136) <223> n is a, c, g, <220>
    <221> misc_feature <222> (138)..(138) <223> n is a, c, g, <220>
    <221> misc_feature <222> (149)..(149) <223> n is a, c, g, <220>
    <221> misc_feature <222> (168)..(168) <223> n is a, c, g, <220>
    <221> misc_feature <222> (183)..(183) <223> n is a, c, g, <220>
    <221> misc_feature <222> (208)..(208) <223> n is a, c, g, <220>
    <221> misc_feature <222> (211)..(211) <223> n is a, c, g, <220>
    <221> misc_feature <222> (213)..(213) <223> n is a, c, g, <220>
    <221> misc_feature <222> (239)..(239) <223> n is a, c, g, <220>
    <221> misc_feature <222> (247)..(247) <223> n is a, c, g, <220>
    <221> misc_feature <222> (284)..(284) <223> n is a, c, g, <220>
    <221> misc_feature <222> (294)..(294) <223> n is a, c, g, <220>
    <221> misc_feature <222> (315)..(315) <223> n is a, c, g, or t or t or t or t or t or t or t or t or t or t or t or t or t or t
    Page 92
    CL6220WOPCT_SequenceListing_ST25.txt <220>
    <221> misc_feature <222> (337)..(338) <223> n is a, c, g, or t <220>
    <221> misc_feature <222> (341)..(341) <223> n is a, c, g, or t <220>
    <221> misc_feature <222> (358)..(358) <223> n is a, c, g, or t <220>
    <221> misc_feature <222> (380)..(380) <223> n is a, c, g, or t <220>
    <221> misc_feature <222> (385)..(385) <223> n is a, c, g, or t <220>
    <221> misc_feature <222> (412)..(412) <223> n is a, c, g, or t <400> 38
    mttnradart ngatnwwsna vvyyrsdtng dggdkgtsrd yadgvdvwsv yrsddngyds 60 dyrddgtddm daahkrgkvm dvvnhtsdha waskdkddha dwywwrargh gtganwgsyg 120 gsawysrgyy hskkdnwnav rravydmmnw wdrgdgrmdv tskrtdangr gygshdvggy 180 ssncadgrda mrrvdgrdgt vgagtarnht nangdmhvdd cdgvkwkdgk rmagytavnv 240 gwastgnhdr vvsrwgddss srvrsakagm hmhrgtyvyg gmtnahtsdy rdsnayrrva 300 kvssmmagaa rgrdnsrtmw dgsayagtad aatwsvnnha naagdddsvy aykkarhnss 360 vaagwrdadd ahvyatrtgn rvvvnsgrtv drstaggvtd stydahtvvs anrdwaaav 419 <210> 39 <211> 1818 <212> DNA <213> Artificial sequence <220> <223> BbrGlu5_codon-optimized <400> 39 gtgacaacat tcaatagagc aattattccg gatgcgatta gaacaaatgg agccacaccg 60 aatccgtggt ggtcaaacgc agtggtctat caaatctatc cgagatcatt tcaagacaca 120 aacggagacg gccttggcga tcttaaggga atcacatcaa gactggatta tctggctgac 180 cttggagttg atgttctgtg gctgagcccg gtttatagat cacctcaaga cgacaatggc 240 tatgacatca gcgactatag agacattgat cctctgtttg gcacactgga tgatatggac 300 gagctgcttg cagaagcaca taaaagagga cttaaaatcg ttatggacct ggttgtgaac 360 catacatcag atgaacacgc atggtttgaa gcatcaaaag Page 93 ataaagacga tccgcacgct 420
    CL6220WOPCT_SequenceListing_ST25.txt
    gactggtatt ggtggagacc tgctagaccg ggccatgaac cgggaacacc tggcgcagag 480 ccgaaccaat ggggctcata ttttggcgga tcagcatggg agtatagccc ggaaagaggc 540 gaatactatc ttcatcagtt ctcaaaaaaa caaccggatc tgaattggga aaatccggcg 600 gtcagaagag cggtgtacga tatgatgaac tggtggctgg atagaggaat tgatggattt 660 agaatggatg ttattacact gatttcaaaa agaacagacg ccaatggaag acttccggga 720 gaatatggat cagaactgca cgaccttcct gtgggcgaag agggctattc atcacctaat 780 ccgttttgcg ccgacggccc gagacaagat gaattccttg ccgaaatgag aagagaagtt 840 tttgacggaa gagatggctt tctgacagtc ggcgaagcac ctggaattac agcagaaaga 900 aacgaacaca ttacaaaccc tgcaaacggc gaacttgata tgctgttcct gtttgaacat 960 gtggactttg attgcgatgg cgttaaatgg aaaccgcttc cgctggatct tcctggcttt 1020 aaaagaatta tggcaggcta tcagacagca gttgaaaatg tcggatgggc atcactgttt 1080 acaggcaatc atgaccaacc gagagttgtc agcagatggg gcgatgactc atcagaggag 1140 agcagagtta gaagcgccaa agcactgggc cttatgctgc acatgcacag aggcacaccg 1200 tatgtttatc aaggcgagga acttggaatg acaaatgctc attttacgtc acttgaccag 1260 tacagagatc ttgagtcact taatgcttat agacaaagag ttgaagaagc caaggttcag 1320 tcacctgaaa gcatgatggc cggcattgca gctagaggca gagataattc aagaacgccg 1380 atgcaatggg atggaagcgc atacgcaggc tttacggcac ctgacgcagc tacggaaccg 1440 tggatttcag ttaatccgaa tcatgcagaa attaacgcag caggagaatt tgatgacccg 1500 gattcagtct atgcattcta caaaaaactg attgcactga gacataatag cagcattgtt 1560 gcagcgggcg aatggagact tatcgatgca gacgatgcac acgtttatgc gtttacaaga 1620 acacttggca acgagagact gcttgtcgtg gttaatctga gcggcagaac agttgatctg 1680 ccgagagagt caacagagct tattgctggc ggcgtgacag aaccggacat tattctttca 1740 acatatgacg cccctcatac agtggtttca ctggcaaata gagagctgga cccgtgggaa 1800 gctgcggcag tgcagctg 1818
    <210> 40 <211> 1570 <212> PRT <213> Streptococcus sp C150 <400> 40
    Met 1 Lys Lys Asn Trp Val 5 Thr Ile Gly Val 10 Thr Ala Leu Ser Met 15 Val Thr Val Ala Gly Gly Thr Leu Leu Glu Asp Gln Gln Val Gln Ala Asp 20 25 30 Glu Gln Asn Ala Ala Asn Gln Ser Gly Asp Ser Ser Gln Asp Leu Leu
    35 40 45
    Page 94
    CL6220WOPCT_SequenceListing_ST25.txt
    Arg Glu Ala 50 Ser Ala Thr Thr 55 Asn Asp Thr Ala Thr 60 Thr Val Ala Pro Thr Ile Ser Ala Asp Ala Asn Thr Ala Ser Val Asn Ile Pro Val Ala 65 70 75 80 Asp Ala Thr Asn Thr Thr Thr Ala Ala Thr Asp Arg Ala Ala Ala Pro 85 90 95 Thr Thr Thr Ala Ala Thr Val Asp Thr Asn Ser Gly Gln Ala Ala Pro 100 105 110 Ser Thr Asn Val Gln Ala Ala Ala Ala Asp Thr Ser Ala Thr Pro Thr 115 120 125 Asp Thr Asn Thr Asn Thr Asn Ala Ser Val Thr Ala Thr Asp Arg Ala 130 135 140 Val Ala Thr Asp Thr Ala Asn Thr Glu Ala Arg Thr Pro Ser Arg Arg 145 150 155 160 Ala Leu Ala Glu Thr Arg Glu Ala Asn Thr Asn Thr Ser Thr Gly Ile 165 170 175 Gln Trp Ile Asn Gly Lys Gln Tyr Tyr Val Asn Ser Asp Gly Ser Val 180 185 190 Arg Lys Asn Phe Val Phe Glu Gln Asp Gly Lys Ser Tyr Tyr Phe Asp 195 200 205 Ala Glu Thr Gly Ala Leu Ala Thr Lys Ser Gln Asp Glu Phe Ser Thr 210 215 220 Glu Pro Ile Lys Ala Ala Val Asp Phe Ser Ser Gly Asn Gln Leu Tyr 225 230 235 240 Lys Asn Asp Asn Lys Ser Leu Asp Gln Leu Asp Thr Phe Ile Thr Ala 245 250 255 Asp Ala Trp Tyr Arg Pro Lys Ser Ile Leu Lys Asp Gly Lys Thr Trp 260 265 270 Thr Ala Ser Thr Glu Ala Asp Lys Arg Pro Leu Leu Met Val Trp Trp 275 280 285 Pro Asp Lys Ser Thr Gln Val Asn Tyr Leu Asn Tyr Met Gln Asn Gln 290 295 300 Gly Leu Gly Ala Gly Ser Phe Ser Thr Asn Ser Ser Gln Glu Ser Leu
    305 310 315 320
    Page 95
    CL6220WOPCT_SequenceListing_ST25.txt
    Asn Leu Ala Ala Lys Ala 325 Val Gln Thr Lys 330 Ile Glu Glu Arg Ile 335 Ala Arg Glu Gly Asn Thr Asn Trp Leu Arg Thr Ser Ile Asp Gln Phe Ile 340 345 350 Lys Thr Gln Pro Gly Trp Asn Ser Ser Thr Glu Asn Ser Ser Tyr Asp 355 360 365 His Leu Gln Gly Gly Gln Leu Leu Phe Asn Asn Ser Lys Gly Asp Thr 370 375 380 Gly Asn Arg Thr Ser Tyr Ala Asn Ser Asp Tyr Arg Leu Leu Asn Arg 385 390 395 400 Thr Pro Thr Asn Gln Ser Gly Thr Arg Lys Tyr Phe Lys Asp Asn Ser 405 410 415 Ile Gly Gly Leu Glu Phe Leu Leu Ala Asn Asp Ile Asp Asn Ser Asn 420 425 430 Pro Ala Val Gln Ala Glu Gln Leu Asn Trp Leu His Phe Met Met Asn 435 440 445 Ile Gly Ser Ile Met Ala Asn Asp Pro Thr Ala Asn Phe Asp Gly Leu 450 455 460 Arg Val Asp Ala Leu Asp Asn Val Asp Ala Asp Leu Leu Gln Ile Ala 465 470 475 480 Ser Asp Tyr Phe Lys Ala Val Tyr Gly Val Asp Lys Ser Glu Ala Asn 485 490 495 Ala Ile Lys His Leu Ser Tyr Leu Glu Ala Trp Ser Ala Asn Asp Pro 500 505 510 Tyr Tyr Asn Lys Asp Thr Lys Gly Ala Gln Leu Pro Ile Asp Asn Ala 515 520 525 Leu Arg Asn Ala Leu Thr Asn Leu Leu Met Arg Asp Lys Asn Thr Arg 530 535 540 Met Gln Leu Gly Asp Met Thr Ala Phe Met Asn Ser Ser Leu Asn Pro 545 550 555 560 Arg Gly Ala Asn Asp Lys Asn Gly Glu Arg Met Ala Asn Tyr Ile Phe 565 570 575 Thr Arg Ala His Asp Thr Glu Ala Gln Thr Ile Ile Gln Arg Ile Ile 580 585 590
    Page 96
    CL6220WOPCT_SequenceListing_ST25.txt
    Arg Asp Arg Ile Asn Pro Asn Leu 600 Phe Gly Tyr Asn Phe 605 Thr Arg Asp 595 Glu Ile Lys Lys Ala Phe Glu Ile Tyr Asn Ala Asp Ile Asn Thr Ala 610 615 620 His Lys Thr Tyr Ala Ser Tyr Asn Leu Pro Ser Val Tyr Ala Leu Met 625 630 635 640 Leu Thr Asn Lys Asp Ser Val Thr Arg Val Tyr Tyr Gly Asp Leu Tyr 645 650 655 Arg Glu Asp Gly His Tyr Met Ala Lys Lys Thr Pro Tyr Phe Asp Ala 660 665 670 Ile Asp Thr Leu Leu Arg Ala Arg Ile Lys Tyr Val Ala Gly Gly Gln 675 680 685 Asp Met Glu Val Lys Lys Val Gly Asn Asp Gly Leu Leu Thr Ser Val 690 695 700 Arg Tyr Gly Lys Gly Ala Asn Asn Ser Thr Asp Trp Gly Thr Thr Glu 705 710 715 720 Thr Arg Thr Gln Gly Met Gly Val Ile Leu Thr Asn Asn Tyr Asp Phe 725 730 735 Arg Leu Gly Ser Asn Glu Thr Val Thr Met Asn Met Gly Arg Ala His 740 745 750 Arg Asn Gln Leu Tyr Arg Pro Leu Leu Leu Thr Thr Lys Asp Gly Leu 755 760 765 Ala Thr Tyr Leu Asn Asp Ser Asp Val Pro Ser Asn Leu Leu Lys Arg 770 775 780 Thr Asp Trp Asn Gly Asn Leu Thr Phe Asn Ala Asn Asp Val Phe Gly 785 790 795 800 Val Glu Asn Val Gln Val Ser Gly Tyr Leu Gly Val Trp Val Pro Val 805 810 815 Gly Ala Lys Ala Asn Gln Asp Ala Arg Thr Gln Pro Ser Asn Arg Ala 820 825 830 Asn Ser Asp Gly Gln Val Tyr Lys Ser Ser Ala Ala Leu Asp Ser Gln 835 840 845 Val Met Tyr Glu Ala Phe Ser Asn Phe Gln Ala Phe Ala Asp Asp Gln
    850 855 860
    Page 97
    CL6220WOPCT_SequenceListing_ST25.txt
    Pro 865 Glu Leu Tyr Met Asn Arg Val 870 Leu Ala Lys 875 Asn Thr Asp Leu Leu 880 Lys Ala Trp Gly Val Thr Ser Val Gly Leu Pro Pro Gln Tyr Val Ser 885 890 895 Ser Lys Asp Gly Thr Phe Leu Asp Ser Thr Ile Asp Asn Gly Tyr Ala 900 905 910 Phe Asp Asp Arg Tyr Asp Met Ala Leu Ser Gln Asn Asn Lys Tyr Gly 915 920 925 Ser Leu Glu Asp Leu Leu Asn Val Leu Arg Ala Leu His Lys Asp Gly 930 935 940 Ile Gln Ala Ile Ala Asp Trp Val Pro Asp Gln Ile Tyr Asn Leu Pro 945 950 955 960 Gly Lys Glu Val Val Asn Ala Thr Arg Val Asn Gly Tyr Gly Tyr His 965 970 975 Gln Gln Gly Tyr Gln Ile Val Asp Gln Ala Tyr Val Ala Asn Thr Arg 980 985 990
    Thr Asp Gly Thr Asp Tyr Gln Gly Arg Tyr Gly Gly Ala Phe Leu Asp 995 1000 1005
    Glu Leu 1010 Lys Ala Lys Tyr Pro 1015 Ser Ile Phe Asn Arg 1020 Val Gln Ile Ser Asn Gly Lys Gln Leu Pro Thr Asn Glu Lys Ile Thr Lys Trp 1025 1030 1035 Ser Ala Lys Tyr Phe Asn Gly Thr Asn Ile Leu Gly Arg Gly Ile 1040 1045 1050 Asn Tyr Val Leu Arg Asp Asp Lys Thr Asn Gln Tyr Phe Asn Thr 1055 1060 1065 Ser Ala Asn Gly Gln Leu Leu Pro Thr Pro Leu Arg Asp Thr Gly 1070 1075 1080 Ala Ile Thr Ser Thr Gln Val Phe Gln Arg Arg Gly Gln Asp Val 1085 1090 1095 Tyr Phe Leu Arg Asp Asn Gln Val Ile Lys Asn Glu Phe Val Gln 1100 1105 1110 Asp Gly Asn Gly Asn Trp Tyr Tyr Phe Gly Ala Asp Gly Lys Met 1115 1120 1125
    Page 98
    CL6220WOPCT_SequenceListing_ST25.txt
    Thr Lys 1130 Gly Ala Gln Asn Ile 1135 Asn Ser Lys Asp Tyr 1140 Tyr Phe Phe Asp Asn Gly Val Gln Leu Arg Asn Ala Leu Arg Arg Ala Ser Asn 1145 1150 1155 Gly Tyr Thr Tyr Tyr Tyr Gly Leu Asp Gly Ala Met Ile Lys Asn 1160 1165 1170 Ala Phe Val Asp Phe Asp Asp Lys His Gln Gln Val Arg Ala Phe 1175 1180 1185 Thr Thr Gln Gly Thr Met Val Val Gly Asn Leu His Trp Ser Gly 1190 1195 1200 His His Phe Tyr Phe Asp Arg Glu Thr Gly Ile Gln Ala Lys Asp 1205 1210 1215 Arg Ile Val Arg Thr Asp Asp Gly Lys Leu His Tyr Tyr Val Ala 1220 1225 1230 Gln Thr Gly Asp Met Gly Arg Asn Val Phe Ala Thr Asp Ser Arg 1235 1240 1245 Thr Gly Lys Arg Tyr Tyr Phe Asp Ala Asp Gly Asn Thr Val Thr 1250 1255 1260 Gly Ser Arg Val Ile Asp Gly Lys Thr Tyr Tyr Phe Asn Gln Asp 1265 1270 1275 Gly Ser Val Gly Thr Ala Tyr Ser Asn Arg Ala Asp Ser Ile Ile 1280 1285 1290 Phe Glu Asn Gly Lys Ala Arg Tyr Ile Thr Pro Ala Gly Glu Ile 1295 1300 1305 Gly Arg Ser Ile Phe Val Tyr Asn Pro Ala Thr Lys Ala Trp Asn 1310 1315 1320 Tyr Phe Asp Lys Glu Gly Asn Arg Val Thr Gly Arg Gln Tyr Ile 1325 1330 1335 Asp Gly Asn Leu Tyr Tyr Phe Lys Glu Asp Gly Ser Gln Val Lys 1340 1345 1350 Gly Ala Ile Val Glu Glu Asn Gly Ile Lys Tyr Tyr Tyr Glu Pro 1355 1360 1365 Gly Ser Gly Ile Leu Ala Ser Gly Arg Tyr Leu Gln Val Gly Asp 1370 1375 1380
    Page 99
    CL6220WOPCT_SequenceListing_ST25.txt
    Asp Gln 1385 Trp Ile Tyr Phe Lys 1390 His Asp Gly Ser Leu 1395 Ala Ile Gly Gln Val Arg Ala Asp Gly Gly Tyr Leu Lys Tyr Phe Asp Lys Asn 1400 1405 1410 Gly Ile Gln Val Lys Gly Gln Thr Ile Val Glu Asp Gly His Thr 1415 1420 1425 Tyr Tyr Tyr Asp Ala Asp Ser Gly Ala Leu Val Thr Ser Ser Phe 1430 1435 1440 Ala Glu Ile Ala Pro Asn Gln Trp Ala Tyr Phe Asn Thr Glu Gly 1445 1450 1455 Gln Ala Leu Lys Gly Lys Trp Thr Ile Asn Gly Lys Glu Tyr Tyr 1460 1465 1470 Phe Asp Gln Asn Gly Ile Gln Tyr Lys Gly Lys Ala Val Lys Val 1475 1480 1485 Gly Ser Arg Tyr Lys Tyr Tyr Asp Glu Asn Asp Gly Gln Pro Val 1490 1495 1500 Thr Asn Arg Phe Ala Gln Ile Glu Pro Asn Val Trp Ala Tyr Phe 1505 1510 1515 Gly Ala Asp Gly Tyr Ala Val Thr Gly Glu Gln Val Ile Asn Gly 1520 1525 1530 Gln His Leu Tyr Phe Asp Gln Ser Gly Arg Gln Val Lys Gly Ala 1535 1540 1545 Tyr Val Thr Val Asn Gly Gln Arg Arg Tyr Tyr Asp Ala Asn Thr 1550 1555 1560 Gly Glu Tyr Ile Pro Gly Arg
    1565 1570 <210> 41 <211> 4179 <212> DNA <213> Streptococcus sp C150 <400> 41
    atcaatggca aacagtacta tgtaaattcg gacggtagcg tgcgtaagaa tttcgttttt 60 gaacaggatg gtaagagcta ctactttgac gcggaaactg gcgcgctggc cactaaaagc 120 caagatgaat ttagcacgga gccgattaaa gcagcagtgg acttctctag cggcaaccag 180 ctgtacaaaa atgacaacaa atcgctggat cagctggata cgtttatcac cgctgacgca 240 tggtaccgcc ctaagtctat tctgaaggat ggcaaaacct ggaccgcgtc taccgaagct Page 100 300
    CL6220WOPCT_SequenceListing_ST25.txt
    gataagcgtc cgttgctgat ggtgtggtgg ccggacaagt ccacccaagt taactacctg 360 aactacatgc agaaccaggg tttgggtgcg ggtagcttca gcaccaatag cagccaagaa 420 tccctgaatc tggctgcgaa agcagttcag accaagatcg aagaacgcat cgcacgtgag 480 ggtaacacca attggctgcg taccagcatt gaccaattca ttaagacgca gccaggctgg 540 aacagcagca ctgagaatag cagctatgat cacttgcagg gtggtcaact gctgttcaat 600 aacagcaaag gtgatacggg taaccgcacc agctatgcga atagcgacta tcgtctgctg 660 aaccgtaccc caactaatca aagcggcacc cgtaagtact ttaaggataa ttccatcggt 720 ggtctggaat ttctgctggc aaacgacatc gacaacagca accctgccgt tcaggcggag 780 cagctgaact ggctgcactt catgatgaac attggttcta tcatggcgaa tgacccgacg 840 gcgaactttg atggtttgcg tgtggacgcg ttggataacg tggatgcgga cctgttgcag 900 atcgcgagcg attacttcaa ggcagtctac ggtgttgata aatccgaggc gaatgcgatc 960 aagcacctga gctatctgga ggcgtggagc gccaatgacc cgtattacaa caaggatacc 1020 aaaggcgcgc aactgccgat tgacaacgcg ctgcgcaacg cactgaccaa cctgttgatg 1080 cgtgacaaga atacgcgcat gcagctgggt gacatgacgg cgtttatgaa tagctctctg 1140 aacccacgtg gtgcgaatga caaaaacggc gagcgtatgg cgaattacat tttcacccgc 1200 gcacacgata ccgaggcgca gaccatcatt cagcgtatta tccgcgatcg tatcaatccg 1260 aacctgtttg gctacaattt cacccgcgat gaaatcaaaa aggcgtttga gatctacaac 1320 gcggacatta acacggcgca taagacgtac gcgagctaca atctgccgtc cgtctacgca 1380 ctgatgctga cgaataagga cagcgtgacc cgtgtgtatt acggtgacct gtatcgtgag 1440 gacggtcact acatggccaa gaaaacgcct tatttcgatg caatcgatac cctgctgcgt 1500 gcgcgcatca aatacgtggc gggtggtcaa gacatggagg tgaagaaagt tggtaatgac 1560 ggcttgctga cgagcgtccg ctatggcaag ggtgcgaaca atagcaccga ctggggcacg 1620 actgaaaccc gtacccaagg tatgggcgtt atcctgacga acaactatga tttccgcctg 1680 ggcagcaacg aaaccgtcac gatgaacatg ggccgtgcgc atcgcaatca gctgtatcgt 1740 ccgctgctgc tgacgaccaa ggatggtctg gccacgtacc tgaatgatag cgacgtgcct 1800 tcgaatttgc tgaaacgcac ggactggaat ggtaacttga cctttaatgc caacgatgtg 1860 tttggtgtag agaacgtcca ggtcagcggt tacctgggtg tttgggtacc ggttggtgct 1920 aaagctaacc aggatgcgcg tacccaaccg agcaaccgtg cgaacagcga tggtcaggtc 1980 tataagtcgt ctgcggcatt ggacagccag gtcatgtatg aggcgtttag caattttcag 2040 gcatttgcgg acgatcaacc ggaactgtac atgaaccgcg ttctggcgaa gaacaccgat 2100 ctgctgaaag cgtggggcgt tactagcgtt ggcttgccgc cacaatacgt tagcagcaaa 2160 gacggcacct tcctggatag cactattgat aacggctatg cgttcgatga tcgttacgac 2220 atggcgctga gccagaacaa caaatacggt tctctggagg acttgctgaa cgttctgcgc 2280 gctctgcaca aagacggtat tcaggcgatt gcggactggg tcccggatca aatctacaat 2340
    Page 101
    CL6220WOPCT_SequenceListing_ST25.txt
    ttgccgggta aagaggttgt taatgcgacg cgtgttaacg gttacggtta ccatcagcag 2400 ggctaccaga ttgttgacca ggcgtacgtt gcaaacaccc gtacggatgg taccgattat 2460 cagggtcgtt acggtggtgc ttttctggac gaactgaagg cgaagtaccc gagcattttc 2520 aatcgtgtcc agattagcaa cggtaaacag ctgccaacca atgagaaaat cacgaaatgg 2580 tccgcgaaat acttcaatgg cacgaacatc ctgggccgtg gtattaacta tgtgctgcgc 2640 gacgacaaga ccaatcagta tttcaacacc agcgcaaacg gccaactgct gccgacgcca 2700 ctgcgcgaca ccggtgccat caccagcacg caagttttcc agcgtcgtgg ccaagacgtc 2760 tattttctgc gtgataacca ggttatcaaa aacgagtttg tgcaagatgg taacggtaat 2820 tggtactact tcggtgccga cggtaaaatg acgaagggtg cacaaaacat caatagcaag 2880 gattactatt tcttcgataa tggcgtccag ctgcgtaatg cgctgcgtcg cgcgtccaat 2940 ggttacacct actattatgg cctggacggt gccatgatca agaacgcttt cgtcgatttt 3000 gatgataagc accaacaggt gcgtgcgttt actacgcagg gcacgatggt ggtcggtaat 3060 ttgcactgga gcggtcacca cttctatttt gaccgcgaaa cgggtatcca agccaaagac 3120 cgcattgtgc gtaccgatga tggcaagctg cactattatg tcgcacaaac cggcgatatg 3180 ggccgcaatg tgtttgcgac cgacagccgc acgggcaagc gctattactt tgatgcggac 3240 ggcaacaccg ttacgggctc ccgtgtcatc gacggcaaga cctactactt caaccaggac 3300 ggttcggtcg gtaccgcgta cagcaatcgt gcggatagca ttatctttga gaatggcaag 3360 gctcgctata tcactccggc tggcgagatt ggccgttcca tttttgtcta caacccggcg 3420 accaaagcgt ggaattactt cgacaaggaa ggtaaccgtg tcaccggtcg tcagtatatt 3480 gacggcaatc tgtactactt taaagaggac ggctcccaag tgaaaggtgc gattgttgaa 3540 gagaacggta tcaagtacta ctacgaaccg ggcagcggta tcctggcgag cggtcgttat 3600 ctgcaagtcg gtgacgacca atggatctac ttcaaacacg acggtagcct ggcgatcggt 3660 caggttcgtg cagacggtgg ttacttgaaa tactttgata agaatggcat ccaggtcaag 3720 ggccaaacca ttgtggagga tggtcatacc tattactacg atgccgactc cggtgctctg 3780 gtgacctcta gcttcgcgga gattgctccg aaccagtggg cctacttcaa taccgagggc 3840 caagccctga agggcaaatg gaccatcaat ggtaaagagt actattttga tcagaacggc 3900 attcagtata aaggcaaggc agttaaggtc ggcagccgtt acaaatacta tgacgagaat 3960 gacggtcaac cggtcactaa ccgttttgcc cagattgagc cgaacgtctg ggcgtacttt 4020 ggtgccgatg gctacgcagt tactggcgaa caggtgatta atggccagca cctgtacttc 4080 gatcagtcgg gtcgtcaggt taaaggtgcg tacgtcaccg tgaatggtca acgtcgttac 4140 tacgacgcaa acacgggtga atacattccg ggtcgttaa 4179
    <210> 42 <211> 1392 <212> PRT <213> Streptococcus sp C150
    Page 102
    CL6220WOPCT_SequenceListing_ST25.txt
    <400> 42 Ile Asn Gly Lys Gln Tyr Tyr Val Asn Ser Asp Gly Ser Val Arg Lys 1 5 10 15 Asn Phe Val Phe Glu Gln Asp Gly Lys Ser Tyr Tyr Phe Asp Ala Glu 20 25 30 Thr Gly Ala Leu Ala Thr Lys Ser Gln Asp Glu Phe Ser Thr Glu Pro 35 40 45 Ile Lys Ala Ala Val Asp Phe Ser Ser Gly Asn Gln Leu Tyr Lys Asn 50 55 60 Asp Asn Lys Ser Leu Asp Gln Leu Asp Thr Phe Ile Thr Ala Asp Ala 65 70 75 80 Trp Tyr Arg Pro Lys Ser Ile Leu Lys Asp Gly Lys Thr Trp Thr Ala 85 90 95 Ser Thr Glu Ala Asp Lys Arg Pro Leu Leu Met Val Trp Trp Pro Asp 100 105 110 Lys Ser Thr Gln Val Asn Tyr Leu Asn Tyr Met Gln Asn Gln Gly Leu 115 120 125 Gly Ala Gly Ser Phe Ser Thr Asn Ser Ser Gln Glu Ser Leu Asn Leu 130 135 140 Ala Ala Lys Ala Val Gln Thr Lys Ile Glu Glu Arg Ile Ala Arg Glu 145 150 155 160 Gly Asn Thr Asn Trp Leu Arg Thr Ser Ile Asp Gln Phe Ile Lys Thr 165 170 175 Gln Pro Gly Trp Asn Ser Ser Thr Glu Asn Ser Ser Tyr Asp His Leu 180 185 190 Gln Gly Gly Gln Leu Leu Phe Asn Asn Ser Lys Gly Asp Thr Gly Asn 195 200 205 Arg Thr Ser Tyr Ala Asn Ser Asp Tyr Arg Leu Leu Asn Arg Thr Pro 210 215 220 Thr Asn Gln Ser Gly Thr Arg Lys Tyr Phe Lys Asp Asn Ser Ile Gly 225 230 235 240 Gly Leu Glu Phe Leu Leu Ala Asn Asp Ile Asp Asn Ser Asn Pro Ala 245 250 255 Val Gln Ala Glu Gln Leu Asn Trp Leu His Phe Met Met Asn Ile Gly
    Page 103
    260 CL6220WOPCT_SequenceListing_ST25.txt 265 270 Ser Ile Met Ala Asn 275 Asp Pro Thr Ala Asn Phe Asp Gly 280 285 Leu Arg Val Asp Ala 290 Leu Asp Asn Val Asp Ala Asp Leu Leu Gln Ile 295 300 Ala Ser Asp Tyr Phe 305 Lys Ala Val Tyr Gly Val Asp Lys Ser Glu Ala 310 315 Asn Ala Ile 320 Lys His Leu Ser Tyr 325 Leu Glu Ala Trp Ser Ala Asn Asp 330 Pro Tyr Tyr 335 Asn Lys Asp Thr Lys 340 Gly Ala Gln Leu Pro Ile Asp Asn 345 Ala Leu Arg 350 Asn Ala Leu Thr Asn 355 Leu Leu Met Arg Asp Lys Asn Thr 360 365 Arg Met Gln Leu Gly 370 Asp Met Thr Ala Phe Met Asn Ser Ser Leu Asn 375 380 Pro Arg Gly Ala Asn 385 Asp Lys Asn Gly Glu Arg Met Ala Asn Tyr Ile 390 395 Phe Thr Arg 400 Ala His Asp Thr Glu 405 Ala Gln Thr Ile Ile Gln Arg Ile 410 Ile Arg Asp 415 Arg Ile Asn Pro Asn 420 Leu Phe Gly Tyr Asn Phe Thr Arg 425 Asp Glu Ile 430 Lys Lys Ala Phe Glu 435 Ile Tyr Asn Ala Asp Ile Asn Thr 440 445 Ala His Lys Thr Tyr 450 Ala Ser Tyr Asn Leu Pro Ser Val Tyr Ala Leu 455 460 Met Leu Thr Asn Lys 465 Asp Ser Val Thr Arg Val Tyr Tyr Gly Asp Leu 470 475 Tyr Arg Glu 480 Asp Gly His Tyr Met 485 Ala Lys Lys Thr Pro Tyr Phe Asp 490 Ala Ile Asp 495 Thr Leu Leu Arg Ala 500 Arg Ile Lys Tyr Val Ala Gly Gly 505 Gln Asp Met 510 Glu Val Lys Lys Val 515 Gly Asn Asp Gly Leu Leu Thr Ser 520 525 Val Arg Tyr Gly Lys Gly Ala Asn Asn Ser Thr Asp Trp Gly Thr Thr Page 104 Glu Thr Arg
    CL6220WOPCT_SequenceListing_ST25.txt 530 535 540
    Thr Gln 545 Gly Met Gly Val 550 Ile Leu Thr Asn Asn 555 Tyr Asp Phe Arg Leu 560 Gly Ser Asn Glu Thr Val Thr Met Asn Met Gly Arg Ala His Arg Asn 565 570 575 Gln Leu Tyr Arg Pro Leu Leu Leu Thr Thr Lys Asp Gly Leu Ala Thr 580 585 590 Tyr Leu Asn Asp Ser Asp Val Pro Ser Asn Leu Leu Lys Arg Thr Asp 595 600 605 Trp Asn Gly Asn Leu Thr Phe Asn Ala Asn Asp Val Phe Gly Val Glu 610 615 620 Asn Val Gln Val Ser Gly Tyr Leu Gly Val Trp Val Pro Val Gly Ala 625 630 635 640 Lys Ala Asn Gln Asp Ala Arg Thr Gln Pro Ser Asn Arg Ala Asn Ser 645 650 655 Asp Gly Gln Val Tyr Lys Ser Ser Ala Ala Leu Asp Ser Gln Val Met 660 665 670 Tyr Glu Ala Phe Ser Asn Phe Gln Ala Phe Ala Asp Asp Gln Pro Glu 675 680 685 Leu Tyr Met Asn Arg Val Leu Ala Lys Asn Thr Asp Leu Leu Lys Ala 690 695 700 Trp Gly Val Thr Ser Val Gly Leu Pro Pro Gln Tyr Val Ser Ser Lys 705 710 715 720 Asp Gly Thr Phe Leu Asp Ser Thr Ile Asp Asn Gly Tyr Ala Phe Asp 725 730 735 Asp Arg Tyr Asp Met Ala Leu Ser Gln Asn Asn Lys Tyr Gly Ser Leu 740 745 750 Glu Asp Leu Leu Asn Val Leu Arg Ala Leu His Lys Asp Gly Ile Gln 755 760 765 Ala Ile Ala Asp Trp Val Pro Asp Gln Ile Tyr Asn Leu Pro Gly Lys 770 775 780 Glu Val Val Asn Ala Thr Arg Val Asn Gly Tyr Gly Tyr His Gln Gln 785 790 795 800 Gly Tyr Gln Ile Val Asp Gln Ala Tyr Val Ala Asn Thr Arg Thr Asp
    Page 105
    CL6220WOPCT_SequenceListing_ST25.txt 805 810 815
    Gly Thr Asp Tyr Gln 820 Gly Arg Tyr Gly 825 Gly Ala Phe Leu Asp 830 Glu Leu Lys Ala Lys Tyr Pro Ser Ile Phe Asn Arg Val Gln Ile Ser Asn Gly 835 840 845 Lys Gln Leu Pro Thr Asn Glu Lys Ile Thr Lys Trp Ser Ala Lys Tyr 850 855 860 Phe Asn Gly Thr Asn Ile Leu Gly Arg Gly Ile Asn Tyr Val Leu Arg 865 870 875 880 Asp Asp Lys Thr Asn Gln Tyr Phe Asn Thr Ser Ala Asn Gly Gln Leu 885 890 895 Leu Pro Thr Pro Leu Arg Asp Thr Gly Ala Ile Thr Ser Thr Gln Val 900 905 910 Phe Gln Arg Arg Gly Gln Asp Val Tyr Phe Leu Arg Asp Asn Gln Val 915 920 925 Ile Lys Asn Glu Phe Val Gln Asp Gly Asn Gly Asn Trp Tyr Tyr Phe 930 935 940 Gly Ala Asp Gly Lys Met Thr Lys Gly Ala Gln Asn Ile Asn Ser Lys 945 950 955 960 Asp Tyr Tyr Phe Phe Asp Asn Gly Val Gln Leu Arg Asn Ala Leu Arg 965 970 975 Arg Ala Ser Asn Gly Tyr Thr Tyr Tyr Tyr Gly Leu Asp Gly Ala Met 980 985 990
    Ile Lys Asn Ala Phe Val Asp Phe Asp Asp Lys His Gln Gln Val Arg 995 1000 1005
    Ala Phe Thr Thr Gln Gly Thr 1015 Met Val Val Gly Asn 1020 Leu His Trp 1010 Ser Gly His His Phe Tyr Phe Asp Arg Glu Thr Gly Ile Gln Ala 1025 1030 1035 Lys Asp Arg Ile Val Arg Thr Asp Asp Gly Lys Leu His Tyr Tyr 1040 1045 1050 Val Ala Gln Thr Gly Asp Met Gly Arg Asn Val Phe Ala Thr Asp 1055 1060 1065 Ser Arg Thr Gly Lys Arg Tyr Tyr Phe Asp Ala Asp Gly Asn Thr
    Page 106
    CL6220WOPCT_SequenceListing_ST25.txt 1070 1075 1080
    Val Thr 1085 Gly Ser Arg Val Ile 1090 Asp Gly Lys Thr Tyr 1095 Tyr Phe Asn Gln Asp Gly Ser Val Gly Thr Ala Tyr Ser Asn Arg Ala Asp Ser 1100 1105 1110 Ile Ile Phe Glu Asn Gly Lys Ala Arg Tyr Ile Thr Pro Ala Gly 1115 1120 1125 Glu Ile Gly Arg Ser Ile Phe Val Tyr Asn Pro Ala Thr Lys Ala 1130 1135 1140 Trp Asn Tyr Phe Asp Lys Glu Gly Asn Arg Val Thr Gly Arg Gln 1145 1150 1155 Tyr Ile Asp Gly Asn Leu Tyr Tyr Phe Lys Glu Asp Gly Ser Gln 1160 1165 1170 Val Lys Gly Ala Ile Val Glu Glu Asn Gly Ile Lys Tyr Tyr Tyr 1175 1180 1185 Glu Pro Gly Ser Gly Ile Leu Ala Ser Gly Arg Tyr Leu Gln Val 1190 1195 1200 Gly Asp Asp Gln Trp Ile Tyr Phe Lys His Asp Gly Ser Leu Ala 1205 1210 1215 Ile Gly Gln Val Arg Ala Asp Gly Gly Tyr Leu Lys Tyr Phe Asp 1220 1225 1230 Lys Asn Gly Ile Gln Val Lys Gly Gln Thr Ile Val Glu Asp Gly 1235 1240 1245 His Thr Tyr Tyr Tyr Asp Ala Asp Ser Gly Ala Leu Val Thr Ser 1250 1255 1260 Ser Phe Ala Glu Ile Ala Pro Asn Gln Trp Ala Tyr Phe Asn Thr 1265 1270 1275 Glu Gly Gln Ala Leu Lys Gly Lys Trp Thr Ile Asn Gly Lys Glu 1280 1285 1290 Tyr Tyr Phe Asp Gln Asn Gly Ile Gln Tyr Lys Gly Lys Ala Val 1295 1300 1305 Lys Val Gly Ser Arg Tyr Lys Tyr Tyr Asp Glu Asn Asp Gly Gln 1310 1315 1320 Pro Val Thr Asn Arg Phe Ala Gln Ile Glu Pro Asn Val Trp Ala
    Page 107
    CL6220WOPCT_SequenceListing_ST25.txt 1325 1330 1335
    Tyr Phe 1340 Gly Ala Asp Gly Tyr 1345 Ala Val Thr Gly Glu 1350 Gln Val Ile Asn Gly Gln His Leu Tyr Phe Asp Gln Ser Gly Arg Gln Val Lys 1355 1360 1365 Gly Ala Tyr Val Thr Val Asn Gly Gln Arg Arg Tyr Tyr Asp Ala 1370 1375 1380 Asn Thr Gly Glu Tyr Ile Pro Gly Arg
    1385 1390 <210> 43 <211> 1455 <212> PRT <213> Streptococcus mutans <400> 43
    Met 1 Glu Lys Lys Val 5 Arg Phe Lys Leu Arg 10 Lys Val Lys Lys Arg 15 Trp Val Thr Val Ser Val Ala Ser Ala Val Val Thr Leu Thr Ser Leu Ser 20 25 30 Gly Ser Leu Val Lys Ala Asp Ser Thr Asp Asp Arg Gln Gln Ala Val 35 40 45 Thr Glu Ser Gln Ala Ser Leu Val Thr Thr Ser Glu Ala Ala Lys Glu 50 55 60 Thr Leu Thr Ala Thr Asp Thr Ser Thr Ala Thr Ser Ala Thr Ser Gln 65 70 75 80 Leu Thr Ala Thr Val Thr Asp Asn Val Ser Thr Thr Asn Gln Ser Thr 85 90 95 Asn Thr Thr Ala Asn Thr Ala Asn Phe Asp Val Lys Pro Thr Thr Thr 100 105 110 Ser Glu Gln Ser Lys Thr Asp Asn Ser Asp Lys Ile Ile Ala Thr Ser 115 120 125 Lys Ala Val Asn Arg Leu Thr Ala Thr Gly Lys Phe Val Pro Ala Asn 130 135 140 Asn Asn Thr Ala His Pro Lys Thr Val Thr Asp Lys Ile Val Pro Ile 145 150 155 160 Lys Pro Lys Ile Gly Lys Leu Lys Gln Pro Ser Ser Leu Ser Gln Asp 165 170 175
    Page 108
    CL6220WOPCT_SequenceListing_ST25.txt
    Asp Ile Ala Ala 180 Leu Gly Asn Val Lys 185 Asn Ile Arg Lys Val 190 Asn Gly Lys Tyr Tyr Tyr Tyr Lys Glu Asp Gly Thr Leu Gln Lys Asn Tyr Ala 195 200 205 Leu Asn Ile Asn Gly Lys Thr Phe Phe Phe Asp Glu Thr Gly Ala Leu 210 215 220 Ser Asn Asn Thr Leu Pro Ser Lys Lys Gly Asn Ile Thr Asn Asn Asp 225 230 235 240 Asn Thr Asn Ser Phe Ala Gln Tyr Asn Gln Val Tyr Ser Thr Asp Ala 245 250 255 Ala Asn Phe Glu His Val Asp His Tyr Leu Thr Ala Glu Ser Trp Tyr 260 265 270 Arg Pro Lys Tyr Ile Leu Lys Asp Gly Lys Thr Trp Thr Gln Ser Thr 275 280 285 Glu Lys Asp Phe Arg Pro Leu Leu Met Thr Trp Trp Pro Asp Gln Glu 290 295 300 Thr Gln Arg Gln Tyr Val Asn Tyr Met Asn Ala Gln Leu Gly Ile His 305 310 315 320 Gln Thr Tyr Asn Thr Ala Thr Ser Pro Leu Gln Leu Asn Leu Ala Ala 325 330 335 Gln Thr Ile Gln Thr Lys Ile Glu Glu Lys Ile Thr Ala Glu Lys Asn 340 345 350 Thr Asn Trp Leu Arg Gln Thr Ile Ser Ala Phe Val Lys Thr Gln Ser 355 360 365 Ala Trp Asn Ser Asp Ser Glu Lys Pro Phe Asp Asp His Leu Gln Lys 370 375 380 Gly Ala Leu Leu Tyr Ser Asn Asn Ser Lys Leu Thr Ser Gln Ala Asn 385 390 395 400 Ser Asn Tyr Arg Ile Leu Asn Arg Thr Pro Thr Asn Gln Thr Gly Lys 405 410 415 Lys Asp Pro Arg Tyr Thr Ala Asp Arg Thr Ile Gly Gly Tyr Glu Phe 420 425 430 Leu Leu Ala Asn Asp Val Asp Asn Ser Asn Pro Val Val Gln Ala Glu 435 440 445
    Page 109
    CL6220WOPCT_SequenceListing_ST25.txt
    Gln Leu 450 Asn Trp Leu His Phe 455 Leu Met Asn Phe Gly Asn 460 Ile Tyr Ala Asn Asp Pro Asp Ala Asn Phe Asp Ser Ile Arg Val Asp Ala Val Asp 465 470 475 480 Asn Val Asp Ala Asp Leu Leu Gln Ile Ala Gly Asp Tyr Leu Lys Ala 485 490 495 Ala Lys Gly Ile His Lys Asn Asp Lys Ala Ala Asn Asp His Leu Ser 500 505 510 Ile Leu Glu Ala Trp Ser Tyr Asn Asp Thr Pro Tyr Leu His Asp Asp 515 520 525 Gly Asp Asn Met Ile Asn Met Asp Asn Arg Leu Arg Leu Ser Leu Leu 530 535 540 Tyr Ser Leu Ala Lys Pro Leu Asn Gln Arg Ser Gly Met Asn Pro Leu 545 550 555 560 Ile Thr Asn Ser Leu Val Asn Arg Thr Asp Asp Asn Ala Glu Thr Ala 565 570 575 Ala Val Pro Ser Tyr Ser Phe Ile Arg Ala His Asp Ser Glu Val Gln 580 585 590 Asp Leu Ile Arg Asn Ile Ile Arg Ala Glu Ile Asn Pro Asn Val Val 595 600 605 Gly Tyr Ser Phe Thr Met Glu Glu Ile Lys Lys Ala Phe Glu Ile Tyr 610 615 620 Asn Lys Asp Leu Leu Ala Thr Glu Lys Lys Tyr Thr His Tyr Asn Thr 625 630 635 640 Ala Leu Ser Tyr Ala Leu Leu Leu Thr Asn Lys Ser Ser Val Pro Arg 645 650 655 Val Tyr Tyr Gly Asp Met Phe Thr Asp Asp Gly Gln Tyr Met Ala His 660 665 670 Lys Thr Ile Asn Tyr Glu Ala Ile Glu Thr Leu Leu Lys Ala Arg Ile 675 680 685 Lys Tyr Val Ser Gly Gly Gln Ala Met Arg Asn Gln Gln Val Gly Asn 690 695 700 Ser Glu Ile Ile Thr Ser Val Arg Tyr Gly Lys Gly Ala Leu Lys Ala 705 710 715 720
    Page 110
    CL6220WOPCT_SequenceListing_ST25.txt
    Thr Asp Thr Gly Asp Arg Thr Thr Arg Thr Ser Gly Val Ala Val 735 Ile 725 730 Glu Gly Asn Asn Pro Ser Leu Arg Leu Lys Ala Ser Asp Arg Val Val 740 745 750 Val Asn Met Gly Ala Ala His Lys Asn Gln Ala Tyr Arg Pro Leu Leu 755 760 765 Leu Thr Thr Asp Asn Gly Ile Lys Ala Tyr His Ser Asp Gln Glu Ala 770 775 780 Ala Gly Leu Val Arg Tyr Thr Asn Asp Arg Gly Glu Leu Ile Phe Thr 785 790 795 800 Ala Ala Asp Ile Lys Gly Tyr Ala Asn Pro Gln Val Ser Gly Tyr Leu 805 810 815 Gly Val Trp Val Pro Val Gly Ala Ala Ala Asp Gln Asp Val Arg Val 820 825 830 Ala Ala Ser Thr Ala Pro Ser Thr Asp Gly Lys Ser Val His Gln Asn 835 840 845 Ala Ala Leu Asp Ser Arg Val Met Phe Glu Gly Phe Ser Asn Phe Gln 850 855 860 Ala Phe Ala Thr Lys Lys Glu Glu Tyr Thr Asn Val Val Ile Ala Lys 865 870 875 880 Asn Val Asp Lys Phe Ala Glu Trp Gly Val Thr Asp Phe Glu Met Ala 885 890 895 Pro Gln Tyr Val Ser Ser Thr Asp Gly Ser Phe Leu Asp Ser Val Ile 900 905 910 Gln Asn Gly Tyr Ala Phe Thr Asp Arg Tyr Asp Leu Gly Ile Ser Lys 915 920 925 Pro Asn Lys Tyr Gly Thr Ala Asp Asp Leu Val Lys Ala Ile Lys Ala 930 935 940 Leu His Ser Lys Gly Ile Lys Val Met Ala Asp Trp Val Pro Asp Gln 945 950 955 960 Met Tyr Ala Phe Pro Glu Lys Glu Val Val Thr Ala Thr Arg Val Asp 965 970 975 Lys Tyr Gly Thr Pro Val Ala Gly Ser Gln Ile Lys Asn Thr Leu Tyr 980 985 990
    Page 111
    CL6220WOPCT_SequenceListing_ST25.txt
    Val Val Asp Gly Lys Ser Ser Gly Lys Asp Gln Gln Ala Lys Tyr Gly 995 1000 1005
    Gly Ala 1010 Phe Leu Glu Glu Leu 1015 Gln Ala Lys Tyr Pro 1020 Glu Leu Phe Ala Arg Lys Gln Ile Ser Thr Gly Val Pro Met Asp Pro Ser Val 1025 1030 1035 Lys Ile Lys Gln Trp Ser Ala Lys Tyr Phe Asn Gly Thr Asn Ile 1040 1045 1050 Leu Gly Arg Gly Ala Gly Tyr Val Leu Lys Asp Gln Ala Thr Asn 1055 1060 1065 Thr Tyr Phe Ser Leu Val Ser Asp Asn Thr Phe Leu Pro Lys Ser 1070 1075 1080 Leu Val Asn Pro Asn His Gly Thr Ser Ser Ser Val Thr Gly Leu 1085 1090 1095 Val Phe Asp Gly Lys Gly Tyr Val Tyr Tyr Ser Thr Ser Gly Tyr 1100 1105 1110 Gln Ala Lys Asn Thr Phe Ile Ser Leu Gly Asn Asn Trp Tyr Tyr 1115 1120 1125 Phe Asp Asn Asn Gly Tyr Met Val Thr Gly Ala Gln Ser Ile Asn 1130 1135 1140 Gly Ala Asn Tyr Tyr Phe Leu Ser Asn Gly Ile Gln Leu Arg Asn 1145 1150 1155 Ala Ile Tyr Asp Asn Gly Asn Lys Val Leu Ser Tyr Tyr Gly Asn 1160 1165 1170 Asp Gly Arg Arg Tyr Glu Asn Gly Tyr Tyr Leu Phe Gly Gln Gln 1175 1180 1185 Trp Arg Tyr Phe Gln Asn Gly Ile Met Ala Val Gly Leu Thr Arg 1190 1195 1200 Val His Gly Ala Val Gln Tyr Phe Asp Ala Ser Gly Phe Gln Ala 1205 1210 1215 Lys Gly Gln Phe Ile Thr Thr Ala Asp Gly Lys Leu Arg Tyr Phe 1220 1225 1230 Asp Arg Asp Ser Gly Asn Gln Ile Ser Asn Arg Phe Val Arg Asn 1235 1240 1245
    Page 112
    CL6220WOPCT_SequenceListing_ST25.txt
    Ser Lys 1250 Gly Glu Trp Phe Leu 1255 Phe Asp His Asn Gly 1260 Val Ala Val Thr Gly Thr Val Thr Phe Asn Gly Gln Arg Leu Tyr Phe Lys Pro 1265 1270 1275 Asn Gly Val Gln Ala Lys Gly Glu Phe Ile Arg Asp Ala Asp Gly 1280 1285 1290 His Leu Arg Tyr Tyr Asp Pro Asn Ser Gly Asn Glu Val Arg Asn 1295 1300 1305 Arg Phe Val Arg Asn Ser Lys Gly Glu Trp Phe Leu Phe Asp His 1310 1315 1320 Asn Gly Ile Ala Val Thr Gly Ala Arg Val Val Asn Gly Gln Arg 1325 1330 1335 Leu Tyr Phe Lys Ser Asn Gly Val Gln Ala Lys Gly Glu Leu Ile 1340 1345 1350 Thr Glu Arg Lys Gly Arg Ile Lys Tyr Tyr Asp Pro Asn Ser Gly 1355 1360 1365 Asn Glu Val Arg Asn Arg Tyr Val Arg Thr Ser Ser Gly Asn Trp 1370 1375 1380 Tyr Tyr Phe Gly Asn Asp Gly Tyr Ala Leu Ile Gly Trp His Val 1385 1390 1395 Val Glu Gly Arg Arg Val Tyr Phe Asp Glu Asn Gly Val Tyr Arg 1400 1405 1410 Tyr Ala Ser His Asp Gln Arg Asn His Trp Asn Tyr Asp Tyr Arg 1415 1420 1425 Arg Asp Phe Gly Arg Gly Ser Ser Ser Ala Ile Arg Phe Arg His 1430 1435 1440 Ser Arg Asn Gly Phe Phe Asp Asn Phe Phe Arg Phe 1445 1450 1455
    <210> 44 <211> 2715 <212> DNA <213> Streptococcus mutans <400> 44 gtgaacggca aatactacta ctacaaagaa gatggaacat tacagaaaaa ctacgcactg aatatcaacg gcaaaacatt tttctttgat gaaacgggag cgttatccaa taacacattg
    120
    Page 113
    ccgagcaaaa CL6220WOPCT_SequenceListing_ST25.txt 180 aaggcaacat cacgaacaac gataacacaa actcctttgc tcaatataac caggtgtact caacggatgc agcgaatttt gaacatgtcg accattatct gacagccgaa 240 tcctggtatc gccctaaata catccttaaa gatggaaaaa catggacgca gtctacagaa 300 aaagacttta gaccgctgct tatgacgtgg tggcctgatc aagaaacaca acgccagtat 360 gtcaattaca tgaacgccca actgggcatc catcagacat ataacacagc aacgagcccg 420 ctgcagctta atttagctgc ccaaacgatc cagacaaaaa tcgaagaaaa aatcacggct 480 gagaaaaata caaactggtt gagacaaacg atcagcgcat ttgttaaaac acagtctgcg 540 tggaatagcg attctgaaaa accgtttgat gaccatttgc aaaaaggagc attgttgtat 600 tccaacaact caaaactgac gtcccaggca aattcaaact accgtatctt gaaccggaca 660 ccgacgaatc aaacaggcaa aaaagatcct agatatacgg cggaccgcac aattggcgga 720 tacgaatttc tgcttgctaa cgatgttgac aattctaacc cggttgtgca agccgaacag 780 ttgaactggc tgcattttct tatgaacttt ggaaacatct acgcgaacga tcctgacgct 840 aattttgatt caattagagt cgatgccgta gacaatgttg atgcagactt attgcaaatc 900 gcgggagatt atcttaaagc agcgaaaggt attcataaaa acgataaagc tgccaatgac 960 catttaagca tcttggaagc atggtcttat aatgatacac cgtacttaca tgatgacgga 1020 gataacatga tcaacatgga caaccgtttg cggctgagcc tgctttattc tttagccaaa 1080 ccgttgaacc agcgtagcgg catgaatcct ctgatcacaa actctcttgt aaatcggacg 1140 gatgacaacg ctgaaacagc agcggttccg tcctattcat ttattagagc ccatgattct 1200 gaagtgcaag accttatcag aaatattatc cgcgcagaaa ttaatcctaa cgtcgtaggc 1260 tactcattta cgatggaaga aatcaaaaaa gcgtttgaaa tctacaacaa agatttattg 1320 gctacagaga aaaaatatac gcattacaac acagcgttaa gctatgctct gcttttaacg 1380 aataaatcaa gcgtgccgag agtctattac ggcgatatgt ttacagatga cggacagtat 1440 atggctcata aaacgatcaa ctacgaagct atcgaaacat tgctgaaagc cagaattaaa 1500 tatgtctctg gtggccaagc catgcgcaac caacaagtgg gaaattccga aattatcacg 1560 tcagtccgtt atggcaaagg agcgcttaaa gctacagata cgggcgacag aacaacgcgc 1620 acatcaggag tggcagtcat cgaaggcaat aacccgtccc ttagattaaa agcgtcagat 1680 cgcgttgtgg tcaacatggg agctgcccat aaaaatcagg cttatcggcc tcttttattg 1740 acaacggata atggcattaa agcctatcat tcagaccaag aagcagcggg tctggtccgt 1800 tacacgaacg atcggggcga acttatcttt acagctgccg acattaaagg atatgcaaat 1860 cctcaggttt caggctactt aggagtatgg gttcctgtgg gcgcagcggc tgatcaagac 1920 gtcagagtag ccgcatccac ggcgccgtca acagacggaa aaagcgtaca tcagaacgcg 1980 gctctggata gccgcgttat gtttgaaggc ttttctaact ttcaagcctt tgcaacgaaa 2040 aaagaagaat acacaaacgt agttatcgca aaaaatgtag ataaatttgc ggaatgggga 2100 gttacggact ttgaaatggc gccgcagtat gtatcttcca cagatggcag ctttctggac 2160
    Page 114
    CL6220WOPCT_SequenceListing_ST25.txt
    tctgttatcc aaaacggata tgcatttacg gatagatacg accttggcat ctcaaaacct 2220 aacaaatacg gaacagcgga tgacctggtt aaagccatca aagcacttca tagcaaaggc 2280 attaaagtaa tggcagattg ggttccggac cagatgtatg cgtttcctga aaaagaagtg 2340 gtcacagcta cgcgcgtaga taaatatggt acgccggttg ctggcagcca aatcaaaaac 2400 acactgtacg tagttgatgg caaatcaagc ggaaaagacc aacaggccaa atatggaggt 2460 gcatttctgg aagaacttca agctaaatac cctgaacttt ttgcccgtaa acagatctct 2520 acaggagtgc cgatggaccc gtccgtcaaa atcaaacagt ggtcagcaaa atactttaac 2580 ggcacaaaca tcttaggtcg gggcgcagga tatgtgttga aagaccaagc gacaaacacg 2640 tactttagcc tggtgtctga taatacattt ctgccgaaat cacttgtcaa tcctaaccat 2700 ggaacgtctt cctaa 2715
    <210> 45 <211> 904 <212> PRT <213> Streptococcus mutans <400> 45
    Val 1 Asn Gly Lys Tyr Tyr Tyr 5 Tyr Lys Glu Asp 10 Gly Thr Leu Gln 15 Lys Asn Tyr Ala Leu Asn Ile Asn Gly Lys Thr Phe Phe Phe Asp Glu Thr 20 25 30 Gly Ala Leu Ser Asn Asn Thr Leu Pro Ser Lys Lys Gly Asn Ile Thr 35 40 45 Asn Asn Asp Asn Thr Asn Ser Phe Ala Gln Tyr Asn Gln Val Tyr Ser 50 55 60 Thr Asp Ala Ala Asn Phe Glu His Val Asp His Tyr Leu Thr Ala Glu 65 70 75 80 Ser Trp Tyr Arg Pro Lys Tyr Ile Leu Lys Asp Gly Lys Thr Trp Thr 85 90 95 Gln Ser Thr Glu Lys Asp Phe Arg Pro Leu Leu Met Thr Trp Trp Pro 100 105 110 Asp Gln Glu Thr Gln Arg Gln Tyr Val Asn Tyr Met Asn Ala Gln Leu 115 120 125 Gly Ile His Gln Thr Tyr Asn Thr Ala Thr Ser Pro Leu Gln Leu Asn 130 135 140 Leu Ala Ala Gln Thr Ile Gln Thr Lys Ile Glu Glu Lys Ile Thr Ala 145 150 155 160
    Page 115
    CL6220WOPCT_SequenceListing_ST25.txt
    Glu Lys Asn Thr Asn 165 Trp Leu Arg Gln Thr 170 Ile Ser Ala Phe Val 175 Lys Thr Gln Ser Ala Trp Asn Ser Asp Ser Glu Lys Pro Phe Asp Asp His 180 185 190 Leu Gln Lys Gly Ala Leu Leu Tyr Ser Asn Asn Ser Lys Leu Thr Ser 195 200 205 Gln Ala Asn Ser Asn Tyr Arg Ile Leu Asn Arg Thr Pro Thr Asn Gln 210 215 220 Thr Gly Lys Lys Asp Pro Arg Tyr Thr Ala Asp Arg Thr Ile Gly Gly 225 230 235 240 Tyr Glu Phe Leu Leu Ala Asn Asp Val Asp Asn Ser Asn Pro Val Val 245 250 255 Gln Ala Glu Gln Leu Asn Trp Leu His Phe Leu Met Asn Phe Gly Asn 260 265 270 Ile Tyr Ala Asn Asp Pro Asp Ala Asn Phe Asp Ser Ile Arg Val Asp 275 280 285 Ala Val Asp Asn Val Asp Ala Asp Leu Leu Gln Ile Ala Gly Asp Tyr 290 295 300 Leu Lys Ala Ala Lys Gly Ile His Lys Asn Asp Lys Ala Ala Asn Asp 305 310 315 320 His Leu Ser Ile Leu Glu Ala Trp Ser Tyr Asn Asp Thr Pro Tyr Leu 325 330 335 His Asp Asp Gly Asp Asn Met Ile Asn Met Asp Asn Arg Leu Arg Leu 340 345 350 Ser Leu Leu Tyr Ser Leu Ala Lys Pro Leu Asn Gln Arg Ser Gly Met 355 360 365 Asn Pro Leu Ile Thr Asn Ser Leu Val Asn Arg Thr Asp Asp Asn Ala 370 375 380 Glu Thr Ala Ala Val Pro Ser Tyr Ser Phe Ile Arg Ala His Asp Ser 385 390 395 400 Glu Val Gln Asp Leu Ile Arg Asn Ile Ile Arg Ala Glu Ile Asn Pro 405 410 415 Asn Val Val Gly Tyr Ser Phe Thr Met Glu Glu Ile Lys Lys Ala Phe 420 425 430
    Page 116
    CL6220WOPCT_SequenceListing_ST25.txt
    Glu Ile Tyr Asn Lys Asp Leu Leu 440 Ala Thr Glu Lys Lys 445 Tyr Thr His 435 Tyr Asn Thr Ala Leu Ser Tyr Ala Leu Leu Leu Thr Asn Lys Ser Ser 450 455 460 Val Pro Arg Val Tyr Tyr Gly Asp Met Phe Thr Asp Asp Gly Gln Tyr 465 470 475 480 Met Ala His Lys Thr Ile Asn Tyr Glu Ala Ile Glu Thr Leu Leu Lys 485 490 495 Ala Arg Ile Lys Tyr Val Ser Gly Gly Gln Ala Met Arg Asn Gln Gln 500 505 510 Val Gly Asn Ser Glu Ile Ile Thr Ser Val Arg Tyr Gly Lys Gly Ala 515 520 525 Leu Lys Ala Thr Asp Thr Gly Asp Arg Thr Thr Arg Thr Ser Gly Val 530 535 540 Ala Val Ile Glu Gly Asn Asn Pro Ser Leu Arg Leu Lys Ala Ser Asp 545 550 555 560 Arg Val Val Val Asn Met Gly Ala Ala His Lys Asn Gln Ala Tyr Arg 565 570 575 Pro Leu Leu Leu Thr Thr Asp Asn Gly Ile Lys Ala Tyr His Ser Asp 580 585 590 Gln Glu Ala Ala Gly Leu Val Arg Tyr Thr Asn Asp Arg Gly Glu Leu 595 600 605 Ile Phe Thr Ala Ala Asp Ile Lys Gly Tyr Ala Asn Pro Gln Val Ser 610 615 620 Gly Tyr Leu Gly Val Trp Val Pro Val Gly Ala Ala Ala Asp Gln Asp 625 630 635 640 Val Arg Val Ala Ala Ser Thr Ala Pro Ser Thr Asp Gly Lys Ser Val 645 650 655 His Gln Asn Ala Ala Leu Asp Ser Arg Val Met Phe Glu Gly Phe Ser 660 665 670 Asn Phe Gln Ala Phe Ala Thr Lys Lys Glu Glu Tyr Thr Asn Val Val 675 680 685 Ile Ala Lys Asn Val Asp Lys Phe Ala Glu Trp Gly Val Thr Asp Phe 690 695 700
    Page 117
    Glu 705 Met Ala Pro Gln CL6220WOPCT_SequenceListing_ST25.txt Asp 720 Tyr 710 Val Ser Ser Thr Asp 715 Gly Ser Phe Leu Ser Val Ile Gln Asn Gly Tyr Ala Phe Thr Asp Arg Tyr Asp Leu Gly 725 730 735 Ile Ser Lys Pro Asn Lys Tyr Gly Thr Ala Asp Asp Leu Val Lys Ala 740 745 750 Ile Lys Ala Leu His Ser Lys Gly Ile Lys Val Met Ala Asp Trp Val 755 760 765 Pro Asp Gln Met Tyr Ala Phe Pro Glu Lys Glu Val Val Thr Ala Thr 770 775 780 Arg Val Asp Lys Tyr Gly Thr Pro Val Ala Gly Ser Gln Ile Lys Asn 785 790 795 800 Thr Leu Tyr Val Val Asp Gly Lys Ser Ser Gly Lys Asp Gln Gln Ala 805 810 815 Lys Tyr Gly Gly Ala Phe Leu Glu Glu Leu Gln Ala Lys Tyr Pro Glu 820 825 830 Leu Phe Ala Arg Lys Gln Ile Ser Thr Gly Val Pro Met Asp Pro Ser 835 840 845 Val Lys Ile Lys Gln Trp Ser Ala Lys Tyr Phe Asn Gly Thr Asn Ile 850 855 860 Leu Gly Arg Gly Ala Gly Tyr Val Leu Lys Asp Gln Ala Thr Asn Thr 865 870 875 880 Tyr Phe Ser Leu Val Ser Asp Asn Thr Phe Leu Pro Lys Ser Leu Val 885 890 895 Asn Pro Asn His Gly Thr Ser Ser
    900 <210> 46 <211> 1308 <212> DNA <213> Penicillium marneffei <400> 46 atgaagcaaa ccacttccct cctcctctca gccatcgcgg caaccagcag cttcagcgga ctaacagccg ctcaaaaact cgcctttgcg cacgtcgtcg tcggcaacac tgcagcacac acccaatcca cctgggaaag cgacattact ctcgcccata actccggtct agatgccttt gccttgaacg gtggattccc cgatggcaac atccccgcac aaatcgccaa cgcttttgcg gcttgtgaag ccctttcaaa tggcttcaag ctattcattt cgtttgacta cctcggtggt
    120
    180
    240
    300
    Page 118
    ggtcagccct ggcctgcctc CL6220WOPCT_SequenceListing_ST25 agaggttgtg tctatgctga agcagtatgc .txt cagttccgat 360 tgttatttgg cctatgatgg caagcccttt gtctcaactt ttgagggcac cggaaatatt 420 gcggattggg cgcacggagg tcccattcgg tcggcggtgg atgtttactt tgtgccggat 480 tggacgagtt tggggcctgc tgggattaag tcgtatctcg acaatatcga tggatttttc 540 agctggaaca tgtggcctgt aggtgcggcc gatatgaccg acgagcctga tttcgaatgg 600 ctcgatgcaa ttgggtccga caagacgtac atgatgggcg tttcgccatg gttcttccac 660 agtgcaagcg gaggcaccga ctgggtctgg cgtggtgatg acctctggga tgaccgatgg 720 attcaagtca cctgcgtcga ccctcaattt gtccaggtcg tcacatggaa cgactggggt 780 gaatcctcct acatcggccc cttcgtgacc gctagcgaag tccccgccgg ctcattagcc 840 tacgtcgaca acatgtcaca ccaaagcttc cttgacttct tgcctttcta catcgccacc 900 ttcaaaggcg acacattcaa catctcccgc gaccagatgc aatactggta ccgcctcgca 960 cccgccgcag caggcagcgc gtgcggcgta tacggcaatg atcccgatca aggccagact 1020 accgttgacg tcaactccat cgttcaggac aaggtgtttt tcagtgcttt gttgacggct 1080 gatgctactg taacggtgca gattggtagt aatgctgcgg tttcatatga tggtgttgct 1140 ggtatgaacc actggagtca ggactttaat ggccagaccg gcgcggttac gtttagtgtt 1200 gtcaggggtg gcgctacagt taagagtggt attggagccg agattacggc ttcgacttcg 1260 ttgtcgaatg ggtgcactaa ttacaaccct tgggttggta gtttctaa 1308
    <210> 47 <211> 435 <212> PRT <213> Penicillium marneffei
    <400> 47 Met Lys Gln Thr Thr Ser Leu Leu Leu Ser Ala Ile Ala Ala Thr Ser 1 5 10 15 Ser Phe Ser Gly Leu Thr Ala Ala Gln Lys Leu Ala Phe Ala His Val 20 25 30 Val Val Gly Asn Thr Ala Ala His Thr Gln Ser Thr Trp Glu Ser Asp 35 40 45 Ile Thr Leu Ala His Asn Ser Gly Leu Asp Ala Phe Ala Leu Asn Gly 50 55 60 Gly Phe Pro Asp Gly Asn Ile Pro Ala Gln Ile Ala Asn Ala Phe Ala 65 70 75 80 Ala Cys Glu Ala Leu Ser Asn Gly Phe Lys Leu Phe Ile Ser Phe Asp 85 90 95 Tyr Leu Gly Gly Gly Gln Pro Trp Pro Ala Ser Glu Val Val Ser Met
    100 105 110
    Page 119
    CL6220WOPCT_SequenceListing_ST25.txt
    Leu Lys Gln Tyr 115 Ala Ser Ser Asp Cys Tyr 120 Leu Ala Tyr Asp 125 Gly Lys Pro Phe Val Ser Thr Phe Glu Gly Thr Gly Asn Ile Ala Asp Trp Ala 130 135 140 His Gly Gly Pro Ile Arg Ser Ala Val Asp Val Tyr Phe Val Pro Asp 145 150 155 160 Trp Thr Ser Leu Gly Pro Ala Gly Ile Lys Ser Tyr Leu Asp Asn Ile 165 170 175 Asp Gly Phe Phe Ser Trp Asn Met Trp Pro Val Gly Ala Ala Asp Met 180 185 190 Thr Asp Glu Pro Asp Phe Glu Trp Leu Asp Ala Ile Gly Ser Asp Lys 195 200 205 Thr Tyr Met Met Gly Val Ser Pro Trp Phe Phe His Ser Ala Ser Gly 210 215 220 Gly Thr Asp Trp Val Trp Arg Gly Asp Asp Leu Trp Asp Asp Arg Trp 225 230 235 240 Ile Gln Val Thr Cys Val Asp Pro Gln Phe Val Gln Val Val Thr Trp 245 250 255 Asn Asp Trp Gly Glu Ser Ser Tyr Ile Gly Pro Phe Val Thr Ala Ser 260 265 270 Glu Val Pro Ala Gly Ser Leu Ala Tyr Val Asp Asn Met Ser His Gln 275 280 285 Ser Phe Leu Asp Phe Leu Pro Phe Tyr Ile Ala Thr Phe Lys Gly Asp 290 295 300 Thr Phe Asn Ile Ser Arg Asp Gln Met Gln Tyr Trp Tyr Arg Leu Ala 305 310 315 320 Pro Ala Ala Ala Gly Ser Ala Cys Gly Val Tyr Gly Asn Asp Pro Asp 325 330 335 Gln Gly Gln Thr Thr Val Asp Val Asn Ser Ile Val Gln Asp Lys Val 340 345 350 Phe Phe Ser Ala Leu Leu Thr Ala Asp Ala Thr Val Thr Val Gln Ile 355 360 365 Gly Ser Asn Ala Ala Val Ser Tyr Asp Gly Val Ala Gly Met Asn His 370 375 380
    Page 120
    CL6220WOPCT_SequenceListing_ST25.txt
    Trp 385 Ser Gln Asp Phe Asn 390 Gly Gln Thr Gly Ala 395 Val Thr Phe Ser Val 400 Val Arg Gly Gly Ala Thr Val Lys Ser Gly Ile Gly Ala Glu Ile Thr 405 410 415 Ala Ser Thr Ser Leu Ser Asn Gly Cys Thr Asn Tyr Asn Pro Trp Val
    420 425 430
    Gly Ser Phe 435
    Page 121
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