EP3110959A1 - Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes - Google Patents

Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzymes

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Publication number
EP3110959A1
EP3110959A1 EP15713257.2A EP15713257A EP3110959A1 EP 3110959 A1 EP3110959 A1 EP 3110959A1 EP 15713257 A EP15713257 A EP 15713257A EP 3110959 A1 EP3110959 A1 EP 3110959A1
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European Patent Office
Prior art keywords
alpha
glucan
saccharide
fraction
synthesis reaction
Prior art date
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EP15713257.2A
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German (de)
French (fr)
Inventor
Kevin D. Nagy
Erwin Columbus HAGO
Jayarama K. Shetty
Susan Marie Hennessey
Robert Dicosimo
Ling Hua
Rodrigo Ramirez
Zhongmei TANG
Zheyong YU
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Nutrition and Biosciences USA 4 Inc
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EI Du Pont de Nemours and Co
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Publication of EP3110959A1 publication Critical patent/EP3110959A1/en
Pending legal-status Critical Current

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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C12P19/08—Dextran
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01005—Dextransucrase (2.4.1.5)
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    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01024—1,4-Alpha-glucan 6-alpha-glucosyltransferase (2.4.1.24)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0102—Alpha-glucosidase (3.2.1.20)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/0107—Glucan 1,6-alpha-glucosidase (3.2.1.70)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01084—Glucan 1,3-alpha-glucosidase (3.2.1.84), i.e. mutanase
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01115—Branched-dextran exo-1,2-alpha-glucosidase (3.2.1.115)

Definitions

  • 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 glucosyi-glucose linkages with an alpha-glucossdase enzyme.
  • Transglucosidases (EC.2.4.1 .24, 1 ,4-alpha-glucan 6-alpha- glucosyltransferase) are D-giucosyltransferase enzymes that catalyze both hydrolytic and transfer reactions on incubation with alpha-D-gluco- oligosaccharides (1951 , Pazur and 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 D- glucose, or panose (6-O-alpha-glucosyl maltose) from maltose.
  • Transglucosidase can also transfer a glucosyl residue to the HO-2 or HO-3 of another D-g!ucosyl unit to form Kojibiose or Nigerose, This enzyme can further transfer a D-glucosy! unit back to HO-4 to reform maltose.
  • Xanthan gum comprises a cellulosic backbone in which alternate glucoses are 1 ,3-iinked 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.
  • transglucosidase to degrade sucrose, melezitose and trehalulose.
  • sugars comprise glucose linked to fructose via 1 ,2- (sucrose), 1 ,3- (melezitose), or 1.1- (trehalulose) linkages.
  • transglucosidase Although various hydrolytic activities of transglucosidase have been disclosed, this type of enzyme is generally considered to be an alpha- glucosidase, given its ability to hydrolyze alpha-linkages between two glucosyl residues.
  • 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.
  • 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.
  • 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
  • the method comprises: contacting the saccharide with an alpha-giucosidase 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.
  • the alpha-glucosidase enzyme of the hydrolysis method is immobilized.
  • the saccharide of the hydrolysis method 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.
  • 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.
  • the fraction is a filtrate of the glucan synthesis reaction.
  • the glucan synthesis reaction produces at least one soluble alpha-glucan product that is (i) a product of a glucosy!transferase, or (ii) a product of the concerted action of both a glucosyitransferase and an alpha-glucanohydrolase capable of hydrolyzing glucan polymers having one or more alpha-1.3-glycosidic linkages or one or more alpha-1 , 8-glycosidic 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.
  • the alpha-glucosidase enzyme is a transglucosidase.
  • the transglucosidase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1
  • 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 glucosyi-glucose linkage, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,8 glucosy!-g!ucose 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.
  • 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.
  • 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.
  • 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 alpha- glucosidase enzyme under suitable conditions, wherein the alpha-g!ucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,6 glucosyi-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).
  • the invention concerns a fermentation method comprising: (a) contacting a fraction obtained from a glucan synthesis reaction with an a!pha-glucosidase enzyme under suitable conditions, wherein the alpha- g!ucosidase enzyme hydro!yzes at least one a!pha-1 ,3 or alpha-1 ,6 glucosyl- glucose 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.
  • Figure 1 H N R spectra of glucan reaction filtrate material before (starting materia!) and after (treated material) hydrolysis treatment with NOVO 188 enzyme (see Examples 2-3).
  • Figure 2 1 H 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).
  • alpha-glucosidase full-length precursor 13 14 form including signal peptide. (3293 bases) (1035 aa)
  • alpha-glucosidase full-length precursor 16 17 form including signal peptide. (3162 bases) (990 aa)
  • 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.
  • saccharide saccharide molecule
  • carbohydrate 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 “monomelic units”, for example.
  • Preferred monosaccharides herein are fructose and glucose.
  • glycosidic linkage and “glycosidic bond” are used
  • alpha-1 ,3 glucosyl-glucose linkage refers to an alpha-1 ,3- glycosidic linkage between two alpha-D-glucose molecules.
  • alpha- 1 ,6 glucosyl-glucose linkage refers to an alpha-1 ,6-glycosidic linkage between two alpha-D-glucose molecules.
  • Alpha-1 ,3 glucosyl-glucose linkage(s) and/or alpha- 1 ,6 glucosyl-glucose linkage(s) herein are comprised within a disaccharide or oligosaccharide in certain embodiments.
  • alpha-1 ,5 g!ucosyl-fructose linkage refers to an alpha-1 ,5- glycosidic 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.
  • A!pha-D ⁇ glucose herein can also be referred to as "glucose”.
  • leucrose A disaccharide containing an a!pha-1 ,5 glucosyl-fructose linkage is referred to herein as leucrose.
  • leucrose and "D-glucopyranosyl- a!pha(1 -5)-D-fructopyranose” are used interchangeably herein.
  • Leucrose has the following structure:
  • alpha-glucosidase "aipha-1 ,4-gIucosidase”, and "alpha ⁇ D ⁇ glycoside 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 alpha-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 alpha- glucosidases with such activity.
  • Transglucosidase (TG), “transglucosidase enzyme”, and “1 ,4- alpha-g!ucan 8 ⁇ alpha-glucosyltransferase” are used interchangeably herein.
  • Transglucosidases (EC 2.4.1 .24) have previously been recognized as D- giucosyltransferase 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.
  • glucoamylase (GA), "glucoamylase enzyme”, and "alpha-1 ,4- glucan glucohydrolase” are used interchangeably herein.
  • Glucoamylases (EC 3.2.1 .3) have previously been recognized as exo-acting enzymes that catalyze hydrolysis of both a!pha-1 ,4 and a!pha-1 ,6 glycosidic linkages from non-reducing ends of glucose-containing di ⁇ , oligo- and poly-saccharides.
  • Glucoamy!ases are now disclosed herein to also have hydrolytic activity toward alpha-1 , 5 glucosyl- fructose 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.
  • Hydrolysis "hydrolysis of, or “hydrolytic activity toward" an alpha-1 ,3 or alpha 1 ,6 glucosyl-glucose linkage herein refers to enzymatic hydrolysis of the alpha- 1 ,3 or alpha-1 ,6 glycosidic linkage between two glucose molecules by an alpha- glucosidase such as a transgiucosidase. Such hydrolysis occurs when contacting a disaccharide or oligosaccharide containing an alpha-1 ,3 and/or alpha-1 ,6 glucosyl-glucose linkage with an alpha-giucosidase herein under suitable conditions.
  • 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.
  • saccharification refers to a process of breaking a saccharide (disaccharide or oligosaccharide) into its monosaccharide
  • 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 ,6 glucosyl- glucose linkage with an alpha-glucosidase refer to those conditions (e.g., temperature, pH, time) that support the hydrolysis of one or more alpha-1 ,3 and/or alpha-1 ,6 glucosyl-glucose linkages in the saccharide by the alpha- glucosidase.
  • Suitable conditions can comprise "aqueous conditions", for example, comprising at least 20 wt% water. Aqueous conditions may
  • 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 transgiucosidase or glucoamylase reaction).
  • immobilized enzyme herein refers to an enzyme that is attached to an inert, insoluble material. Methods for preparing immobilized enzymes are disclosed, for example, in U.S. Patent No. 5541097, which is incorporated herein by reference.
  • glucan and "glucan polymer” are used interchangeably herein and refer to a polysaccharide of glucose monomers linked by glycosidic bonds.
  • An "alpha-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%, 98%, 97%, 98%, 99%, or 100% alpha-glycosidic linkages.
  • insoluble glucan refers to a glucan polymer that is not soluble in aqueous conditions.
  • An example of insoluble glucan herein is poly alpha-1 ,3- glucan with a DP of at least 8 or 9.
  • a giucosyltransferase reaction in certain embodiments as presently disclosed produces at least one insoluble glucan product.
  • soluble glucan soluble alpha-glucan
  • soluble fiber soluble glucan fiber
  • soluble dietary fiber a glucan polymer that is soluble in aqueous conditions.
  • soluble glucan examples are certain oligosaccharides, such as poly alpha-1 ,3- glucan with a DP less than 8, and certain oligosaccharides disclosed in the Examples provided below.
  • soluble alpha-glucan compounds in certain embodiments herein are (i) water-soluble glucose oligomers having a degree of polymerization of 3 or more, (ii) digestion- resistant (i.e., exhibit very slow or no digestibility) with little or no absorption in the human small intestine, and (iii) 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.
  • poly alpha-1 ,3-g!ucan and "alpha-1 ,3-glucan polymer” are used interchangeably herein.
  • Poly aipha-1 ,3-glucan is a polymer comprising glucose monomenc units linked together by glycosidic linkages, wherein at least about 50% of the glycosidic linkages are alpha- 1 ,3-glycosidic linkages.
  • alpha-1 ,3-glycosidic linkage refers to the type of covalent bond 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 giucan herein can be represented as number- average molecular weight (M n ) or as weight-average molecular weight (M w ). Alternatively, molecular weight can be represented as Daltons, grams/mole, DP W (weight average degree of polymerization), or DP n (number average degree of polymerization). Various means are known in the art for calculating these molecular weight measurements such as with high-pressure liquid
  • HPLC high resolution chromatography
  • SEC size exclusion chromatography
  • GPC gel permeation chromatography
  • glucose transferase enzyme gtf enzyme
  • gtf enzyme catalyst gtf, glucansucrase
  • the activity of a gtf enzyme herein catalyzes the reaction of sucrose substrate to make the products giucan 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-termma! to C-terminal direction) a signal peptide, a variable domain, a catalytic domain, and a glucan- binding domain.
  • a glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active
  • sucrose 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.
  • giucan 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 components.
  • Other components that can be in a glucan synthesis reaction herein include fructose, glucose, leucrose, soluble oligosaccharides (e.g., DP2- DP7), and soluble glucan produc (s), for example.
  • one or more alpha- g!ucanohydro!ase 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.
  • DP degree of polymerization
  • alpha-glucanohydrolase and "giucanohydroiase” are used interchangeably herein and refer to an enzyme capable of hydroiyzing an alpha- glucan oligomer.
  • An alpha-giucanohydroiase can be defined by its
  • alpha-glucanohydrolase enzymes herein include dextranases (EC 3.2.1 ,11 ;
  • 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.
  • a fraction can contain dissolved sugars such as 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.
  • glucan reaction filtrate glucan filtrate
  • glucan filtrate 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 alpha- glucosidase herein to hydrolyze leucrose and/or oligosaccharides present in the filtrate.
  • percent by volume percent by volume
  • volume percent percent of solute in a solution
  • volume percent percent of solute in a solution
  • 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.
  • 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.
  • these terms may refer to, for example, a quantity or activity that is at least 1 %, 2%, 3%, 4%, 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.
  • sequence identity refers 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.
  • percentage of sequence 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.
  • BLAST Basic Local Alignment Search Tool
  • NCB National Center for Biotechnology Information
  • BLASTN algorithm polynucleotide sequences
  • BASTP algorithm polypeptide sequences
  • percent identity between sequences may be performed using a Clustal algorithm (e.g., ClustalW or ClustalV).
  • ClustalW ClustalW or ClustalV
  • 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%, 84%, 85%, 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.
  • Isolated refers to any cellular component that is completely separated from its native source (e.g., an isolated polynucleotide or polypeptide molecule).
  • an isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix.
  • 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, transg!ucosidase), or glucosyltransferase enzyme.
  • the enzyme reactions e.g., alpha-glucosidase reaction, glucosyltransferase reaction disclosed herein are synthetic, non-naturally 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 contacting step, the alpha-glucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,6 glucosyl-glucose linkage of the saccharide.
  • this hydrolysis method can alternatively be referred to as a method of reducing the amount of a saccharide in a composition.
  • alpha- glucosidase enzymes can hydrolyze alpha-1 ,3 and alpha-1 , 6 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.
  • 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 , 6 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, non- reducing (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 a!pha-1 ,8 glucosyl- glucose 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.
  • an alpha-g!ucosidase can be a fungal alpha-glucosidase.
  • suitable alpha-g!ucosidases herein include those disclosed in U.S. Patent Nos. 6355487, 5922580, 5795766, 5783252, and 8633008, which are all incorporated herein by reference.
  • alpha-glucosldase enzyme in certain embodiments herein may comprise the amino acid sequence of SEQ ID NO:5, 6, 8, 9, 1 1 , 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or that of D!AZYME RDF ULTRA (DuPont Industrial Biosciences).
  • 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 NO: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 DIAZY E RDF ULTRA, and have hydrolytic activity toward alpha- 1 ,3 and/or alpha-1 ,6 giucosyl-glucose linkages in saccharides.
  • Several of the foregoing sequences are mature a!pha-glucosidases that lack an N-terniinal signal peptide.
  • 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 intraceliularly and obtained from a ceil lysate).
  • a transglucosidase (EC 2.4.1 .24; 1 ,4-aipha-glucan 8-alpha- g!ucosyltransferase) can be used in certain embodiments herein as an alpha- glucosidase to hydrolyze alpha-1 ,3 and/or alpha-1 ,6 giucosyl-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-gluco- oligosaccharides.
  • Transglucosidases as now disclosed herein also have hydrolytic activity toward alpha-1 ,3 and alpha-1 ,6 giucosyl-glucose linkages.
  • a transglucosidase enzyme herein may be derived from any microbial source, such as a bacteria or fungus.
  • fungal transglucosidases include, but are not limited to, those of Trichoderma species (e.g., T. reesei), Aspergillus species and Neosariorya species (e.g., N. fischeri).
  • Aspergillus species from which a transglucosidase may be derived include, but are not limited to, A. niger, A. awamori, A. oryzae, A, terreus, A. c!avatus, A. fumigatus and A. nidulans.
  • transglucosidase enzymes usefu! herein are described in Barker et al (1953, J. Chem. Soc. 3588-3593); Pazur et a!. (1988, Carbohydr, Res. 149:137-147), Nakamura et al. (1997, J. Biotechnoi. 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.
  • thermostable transglucosidase discloses a process for producing thermostable transglucosidase. Yet more examples of
  • transglucosidase enzymes useful herein may be any of those in the GENBANK database (NCBI), such as accession numbers: D45356 (GID:2845159, A. niger), BAD06006.1 (GID:4031328, A. awamori), BAA08125.1 (GID:1054565, A oryzae), XPJ301210809.1 (G!D:1 15492363, A. terreus), XP_001216899.1 (GID:1 15433524, A terreus), XPJ3Q1271891 .1 (GID:121707620, A. davatus), XP ..
  • NCBI GENBANK database
  • a transglucosidase 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 transglucosidase sequences, and have hydro!ytic 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 transglucosidase enzyme in certain embodiments herein may comprise the amino acid sequence of SEQ ID NO:1 (TG L-2000), which is an A niger transglucosidase (U.S. Patent Appl. Publ. No. 2008/0229514).
  • a transglucosidase 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 glucosyl- glucose linkages in saccharides. Any of SEQ ID NO:1 or variants thereof can be produced following the disclosure of U.S. Patent Appl.
  • SEQ ID NO: 1 is a mature transglucosidase that lacks an N-terminal signal peptide. Since SEQ !D 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 !D 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 intracelluiar!y and obtained from a cell lysate).
  • a giucoamyiase (EC 3.2.1 .3; aipha-1 ,4-glucan glucohydrolase) can be used in certain embodiments herein as an alpha-glucosidase.
  • a giucoamyiase can be included with a transglucosidase in each of the hydrolysis reaction settings/conditions disclosed herein.
  • a giucoamyiase can be used to hydroiyze (i) an alpha-1 ,5 glucosyl-fructose linkage, and/or (ii) an alpha-1 ,3 and/or aipha-1 ,8 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 alpha- 1 ,4 and aipha-1 ,6 glycosidic linkages from non-reducing ends of glucose- containing di-, oligo- and poly-saccharides.
  • Glucoamylases as now disclosed herein also have hydrolytic activity toward alpha-1 ,5 glucosyl-fructose linkages.
  • an alpha-glucosidase is not a giucoamyiase.
  • a giucoamyiase enzyme herein may be derived from any microbial source, such as a bacteria or fungus.
  • bacterial glucoamylases include, but are not limited to, those of Bacillus species (e.g., B. alkalophilus, B. amyioiiquefaciens, B. lentus, B. iicheniformis, B. stearothermophiius, B. subWis, B. thuringiensis) and Streptomyces species (e.g., S. lividans).
  • Examples of fungal glucoamylases include, but are not limited to, those of Trichoderma species (e.g., T. reesei, T.
  • thermophsius, T. duponti Mucor species
  • Hypocrea species e.g., H. geiatsnosa, H. orientaiis, H, vinosa, H. citrina
  • Fusarium species e.g., F. oxysporum, F. roseum, F. venenatum
  • Neurospora species e.g., N. crassa
  • Humicoia species e.g., H. grisea, H. insoiens, H, ianuginose
  • Peniciilium species e.g., P.
  • a glucoamyiase 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 glucoamyiase sequences, and have hydrolytic activity toward (i) alpha- 1 ,5 glucosyl-fructose linkages and/or (ii) a!pha-1 ,3 and/or alpha-1.6 g!ucosyl- glucose linkages. All of the foregoing glucoamyiases, when used in a hydrolysis reaction herein, are preferably in a mature form lacking an N-terminal signal peptide.
  • glucoamyiases useful herein include OPTIDEX L-400, GC 147, GC 321 , G ZYME G990 4X, OPT!MAX 7525, DEXTROZY E, DISTILLASE and GLUCZY E, for example.
  • a glucoamyiase enzyme in certain embodiments herein may comprise the amino acid sequence of SEQ ID NO:2 (GC 321 ), which is a T. reesei
  • a glucoamyiase may comprise an amino acid sequence that is at least about 90%, 91 %, 92%, 93%, 94%, 95%, 98%, 97%, 98%, or 99% identical to SEQ ID NO:2 and have hydrolytic activity toward (i) alpha-1 ,5 glucosyl-fructose linkages and/or (ii) alpha-1 ,3 and/or a!pha-1 ,6 glucosyl-glucose linkages.
  • Any of SEQ ID NO:2 or variants thereof can be produced following the disclosures of U.S. Pat. No. 7413887 or U.S. Pat. Appl. PubL No.
  • SEQ !D NQ:2 is a mature g!ucoamylase 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 an epitope) if expressing it without using a signal peptide (such as with an expression system where the enzyme is expressed
  • alpha-glucosidase enzyme herein such as a transglucossdase or glucoamylase may be from a commercial source (e.g., DuPont Industrial
  • 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.
  • an alpha-glucosidase may be produced recombinantly in a heterologous expression system, such as a microbial or fungal heterologous expression system.
  • heterologous expression systems include bacterial (e.g., E. coll, Bacillus sp.) and eukaryotic systems.
  • Eukaryotic systems can employ yeast (e.g., Pichia sp.,
  • Saccha myces sp. or fungal (e.g., Trichoderma sp. such as T, reesei;
  • 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.
  • an alpha-glucosidase enzyme in a mature form can be provided by expressing it without using a signal peptide, such as with an expression system where the enzyme is expressed intrace!lular!y and obtained from a cell iysate.
  • 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).
  • a cell that expresses the enzyme(s) 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 alpha- glucosidase 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.
  • 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.
  • a saccharide can have one or more alpha-1 , 5 glucosyl-fructose linkages.
  • a saccharide herein comprises at least one alpha-1 ,3 or alpha-1 ,8 glucosyl-glucose linkage, the saccharide comprises at least two glucose units.
  • a saccharide herein comprises only glucose units, or both glucose and fructose units. Such a composition may characterize the
  • a saccharide herein may contain other monosaccharides in addition to glucose and fructose, such as galactose, ribose and xylose.
  • 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.
  • DP degree of polymerization
  • a DPS oligosaccharide has 3 monomeric units, for example.
  • the oligosaccharide can be a DPS, DP4, DP5, DP6, DP7, DPS, 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 can comprise other iinkages in addition to at least one alpha-1 ,3 or alpha-1 ,6 glucosyi-glucose linkage.
  • An oligosaccharide in certain embodiments comprises only glucose monomers linked by alpha-1 ,3 and/or alpha-1 ,8 glycosidic Iinkages.
  • such oligosaccharides comprise only alpha-1 ,3 glucosyi-glucose and/or alpha-1 , 6 giucosyl-glucose Iinkages.
  • Examples of such an oligosaccharide contain only alpha-1 ,3 Iinkages or alpha-1 , 6 Iinkages.
  • An oligosaccharide can comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% glucosyi-glucose Iinkages in certain embodiments. In other embodiments, there can be about 75-85% alpha-1 ,3 giucosyl-glucose Iinkages and about 15-25% alpha-1 , 6 giucosyl-glucose Iinkages in oligosaccharides herein.
  • oligosaccharides herein can comprise any percentage (any integer value between 1 % and 99%) of alpha-1 ,3 glucosyi-glucose Iinkages and any percentage (any integer value between 1 % and 99%) of alpha-1 , 6 giucosyl- glucose Iinkages, 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 alpha- 1 ,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 oligosaccharides (i.e., average linkage content).
  • Oligosaccharides comprising only glucose monomers linked by alpha-1 ,3 and/or a!pha-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.
  • alpha-glucosidases e.g., transglucosidase and glucoamylase enzymes
  • leucrose which comprises an alpha-1 ,5 glucosyl-fructose linkage
  • oligosaccharides comprising only alpha-1 ,3 glucosyl-glucose and/or alpha-1 ,6 glucosyl-glucose linkages.
  • an alpha-glucosidase can be used, for example, in a reaction for hydrolyzing alpha-1 ,5 glucosyl-fructose linkages, alpha-1 ,3 glucosyl- glucose 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.
  • 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%, 80%, 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).
  • 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.
  • the saccharide concentration can be measured both before and after performing the hydrolysis method.
  • 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 aipha-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.
  • one or more enzymes can be immobilized by contacting the enzyme(s) with a solution of an amine- reactive material (e.g., glutaraldehyde) to form an adduct (e.g., enzyme- glutaraldehyde adduct), after which the adduct is bonded to a solid carrier that has been treated with a polyamine (e.g., a polyethy!enimine such as EPO !N P- 1050).
  • an amine- reactive material e.g., glutaraldehyde
  • an adduct e.g., enzyme- glutaraldehyde adduct
  • a solid carrier (solid support) to which an alpha-giucosidase 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 amine- reactive material leads to attachment of the enzyme to the solid carrier.
  • polyamines useful herein include po!yethylenediamine, a
  • poiyethylenimine e.g., polydiethylenetriamine, polytriethyienetetramine, po!ypentaethylenehexamine, polyhexamethylenediamine
  • Preferred polyamines are water-soluble and/or have a molecular weight of about from 500 to 100,000 Daltons.
  • polyethylenimine such as EPO IN 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.
  • these amine-reactive materials include glutaraldehyde, succindialdehyde, terephthaldehyde, bis- diazobenzidine-2,2'-disulfon!c acid, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone, diphenyl ⁇ '-dithiocyanate ⁇ '-disuifonic acid, 3-methoxydiphenylmethane-4,4'- diisocyanate, toluene-2-isocyanate-4-isothiocyanate, toluene-2,-4- diisothiocyanate, diazobenzidine, diazobenzidine-3,3'-dianisidirie, ⁇ , ⁇ '-
  • 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 alpha- glucosidase are those conditions that support the hydrolysis of one or more alpha-1 ,3 or alpha-1 ,6 glucosy!-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.
  • an alpha- glucosidase e.g., transglucosidase
  • the disaccharides and oligosaccharides in the disclosed hydrolysis method are typically soluble in water or an aqueous solution.
  • contacting a saccharide herein with an alpha-glucossdase 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.
  • 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%).
  • Aqueous conditions can further comprise a buffer, for example, such as an acidic, neutral, or alkaline buffer, at a suitable
  • buffers/buffering agents include citrate, acetate (e.g., sodium acetate), KH 2 PQ 4 , 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.
  • 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).
  • 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.
  • One or more of a!pha-glucosidase enzymes herein may be used in a hydrolysis reaction in certain embodiments. Both a transg!ucosidase and giucoamylase can be used in a reaction, for example.
  • the amount of an alpha- glucosidase 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.
  • about 0.1 -0.5 vol% or 0.1 -1 .0 vol% of alpha- glucosidase can be used in a hydrolysis reaction.
  • an alpha- g!ucosidase herein can be used at about, or at least about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 ppm in a hydrolysis reaction.
  • a transg!ucosidase unit 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 80 °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.
  • the concentration of leucrose can be about 5, 10, 15, 20, 25, 30, 35, or 40 wt% (or any integer value between 5 and 40 wt%).
  • 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, 8, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 wt%.
  • concentration of total sugars which includes disaccharides and oligosaccharides
  • preferred concentrations of total sugars in a hydrolysis reaction to 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.
  • 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.
  • 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).
  • one or more aipha-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 alpha- glucosidase 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.
  • a glucan synthesis reaction herein can be for producing poly alpha-1 ,3- 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 g!ucosyltransferase enzyme that produces a poly alpha-glucan such as poly alpha-1 ,3-giucan.
  • a g!ucosyltransferase enzyme that produces a poly alpha-glucan such as poly alpha-1 ,3-giucan. Examples of such glucosyltransferase enzymes useful herein are disclosed in U.S. Pat. No. 7000000, and U.S. Pat. Appl. Publ. Nos,
  • a glucosyltransferase enzyme herein may be derived from any microbial source, such as a bacteria or fungus.
  • bacterial glucosyltransferase enzymes are those derived from a Streptococcus species, Leuconostoc species or Lactobacillus species.
  • Streptococcus species include S.
  • Leuconostoc species include L. mesenteroides, L. amelibiosum, L, argentinum, L. camosum, L. citreum, L.
  • Lactobacillus species include L, acidophilus, L, deibrueckii, L, heiveticus, L. saiivarius, L, cases, L, curvatus, L. plantarurn, L. sakei, L. brevis, L, buchneri, L. fermentum and L. reuteh.
  • a glucosyltransferase enzyme herein can be primer-independent or primer-dependent. Primer-independent glucosyltransferase enzymes do not require the presence of a primer to perform glucan synthesis. A primer- dependent glucosyltransferase 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 glucosyltransferase enzyme. Primers that can be used in certain embodiments include dextran and other carbohydrate-based primers, such as hydrolyzed glucan, for example.
  • 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.
  • a glucosyltransferase enzyme may be produced recombinantly in a heterologous expression system, such as a microbial heterologous expression system.
  • heterologous expression systems include bacterial (e.g., E. co/ 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).
  • 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. coil) used to heterologously express the enzyme.
  • the bacteria may be subjected to disruption using a French pressure cell.
  • 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 0.15- 0.3% (v/v), for example, in a reaction solution for producing a poly alpha-glucan such as poly alpha-1 ,3-giucan 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.
  • 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.
  • 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 sucrose” refers to the sucrose concentration in a gtf reaction solution just after a!i the reaction soiution 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.
  • sucrose can have a purity of at least 99.0%, or can be reagent grade sucrose.
  • 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. Appi. No. 61/969,958.
  • ICUMSA International Commission for Uniform Methods of Sugar Analysis
  • ICUMSA International Commission for Uniform Methods of Sugar Analysis
  • ICUMSA can be measured, for example, by ICUMSA Method GS1/3-7 as described by R.J. McCowage, R.M. Urquhart and M.L. Burge (Determination of the Solution Colour of Raw Sugars, Brown Sugars and Coloured Syru s at pH 7.0 - Official , Verlag Dr Albert
  • 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, 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:
  • Buffer concentration in a g!ucan 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 glucan 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.
  • 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
  • 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.
  • branch points include alpha-1 ,6 branch points.
  • the molecular weight of poly alpha-1 ,3-glucan produced in a glucan synthesis reaction herein can be measured as number-average molecular weight (M n ) or weight-average molecular weight (M w ). Alternatively, molecular weight can be measured in Daltons or grams/mole. It may also be useful to refer to the DP W (weight average degree of polymerization) or DP n (number average degree of polymerization) of the poly alpha-1 ,3-glucan polymer.
  • the M n or w of poly alpha-1 ,3-glucan herein may be at least about 1000.
  • the M n or M w can be at least about 1000 to about 600000 (or any integer value between 1000 and 600000), for example.
  • poly alpha-1 ,3-glucan in can have a molecular weight in DP n or DP W of at least about 100, or of at least about 100 to 1000 (or any integer value between 100 and 1000).
  • 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.
  • a fraction has been separated from soluble or insoluble glucan product(s) synthesized in the reaction.
  • 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 alpha- 1 ,3-glucan synthesis reaction.
  • the volume of a fraction in certain embodiments can be at least about 10%, 20%, 30%, 40%, 50%, 80%, 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.
  • 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.
  • 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 equipment known in the art suitable for concentrating a solution.
  • 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
  • a fraction in some aspects can comprise water that replaces the water that was present in the composition from which the fraction was obtained.
  • 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.
  • a fraction can be modified to have a pH of about 4 to 5 before an alpha-glucosidase is added to the fraction.
  • 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 alpha- 1 ,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 ef fraction include sucrose (i.e., residual sucrose not consumed in the gtf reaction), one or more gtf enzymes, glucose, buffer, salts, FermaSure ® , borates, sodium
  • the components of a fraction from a poly alpha- 1 ,3-glucan synthesis reaction include water, fructose, glucose, one or more types of saccharide (!eucrose 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.
  • deactivated e.g., heat-deactivated
  • 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 leucrose and
  • oligosaccharides will increase. Conversely, as the temperature increases, the selectivity of the reaction towards leucrose tends to decrease, while the selectivity towards oligosaccharides is largely unaffected.
  • DS dry solids
  • a glucan synthesis reaction herein can produce one or more soluble alpha-glucan products.
  • a soluble aipha-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 alpha-g!ucanohydro!ase 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:
  • PDI polydispersity index
  • Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl.
  • 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.
  • 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.
  • a soluble a!pha-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.
  • a soluble alpha-glucan fiber composition can comprise 93 to 97% alpha-(1 ,3) glycosidic linkages and less than 3% alpha-(1 ,6) glycosidic linkages and has a weight-average molecular weight corresponding to a DP of 3 to 7 mixture.
  • a soluble alpha-glucan fiber composition can comprise about 95% alpha-(1 ,3) glycosidic linkages and about 1 % alpha-(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 % alpha-(1 ,3.6) linkages.
  • 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.
  • a soluble alpha-glucan fiber composition can comprise a weight average molecular weight (M w ) 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.
  • M w weight average molecular weight
  • 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
  • the relative level of digestibility may alternatively be determined using AOAC 201 1 .25 (Integrated Total Dietary Fiber Assay) (McCleary et a!., 2012, J. AOAC Int., 95 (3), 824-844).
  • 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.
  • 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.
  • 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.
  • a soluble alpha-glucan herein can comprise:
  • DE dextrose equivalence
  • PDI polydispersity index
  • Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl.
  • a soluble alpha-glucan herein can comprise:
  • Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl. No. 62/004,312.
  • a soluble alpha-glucan herein can comprise:
  • Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl. No. 82/004,314.
  • a soluble alpha-glucan herein can comprise:
  • Such a soluble alpha-glucan can be produced as disclosed in U.S. Appl.
  • a soluble alpha-glucan is a direct product of a glucosyltransferase.
  • a glucosyltransferase and conditions for use thereof in a suitable giucan 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-glucan can alternatively be a product, for example, of the concerted action of both a glucosyltransferase and an alpha-glucanohydrolase that is capable of hydrolyzing giucan polymers having one or more alpha-1 ,3- glycosidic linkages or one or more alpha-1 ,6-glycosidic linkages
  • a giucan synthesis reaction for producing a soluble alpha-glucan product can comprise both at least one glucosyltransferase and at least one alpha-glucanohydrolase.
  • a giucan synthesis reaction can initially comprise one or more glucosyltransferases as the only enzyme component(s).
  • Such a reaction produces a first alpha-glucan 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-glucan product.
  • alpha-glucanohydrolase is added to the reaction for a suitable period of time to allow modification of the first product to a soluble alpha-glucan product.
  • Conditions for performing a giucan synthesis reaction in which one or more alpha-glucanohydrolase enzymes are included during giucan synthesis reaction and/or after giucan 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.
  • An a!pha-g!ucanohydro!ase herein can be, for example, a dextranase (capable of hydrolyzing a!pha-1 ,6-linked glycosidic bonds; E.G. 3.2,1.1 1 ), a mutanase (capable of hydrolyzing aipha-1 ,3-Iinked glycosidic bonds; E.G.
  • a mutanase comprising SEQ ID NO:47 can be used in certain aspects.
  • 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 alpha-glucan products can serve directly as suitable conditions in which to perform a hydrolysis reaction herein in which an alpha-giucosidase 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 ,3- glucan, for example.
  • a fraction (e.g., chromatographic fraction) of a glucan synthesis reaction for producing one or more soluble alpha-glucan products can be used as suitable conditions in which to perform alpha- glucosidase-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.
  • a fraction can be a chromatographic fraction of a glucan synthesis reaction that produces one or more soluble alpha-glucan products as disclosed herein.
  • Such a reaction can optionally include one or more alpha-glucanohydrolases 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 60%, 70%, 80%, 90%, 95%), a soluble alpha-glucan product from a reaction composition from which it was produced.
  • a fraction can be subjected to any of the alpha-1 ,5 glucosy!-fructose hydrolysis processes disclosed herein using one or more alpha-glucanases.
  • a chromatographic fraction herein can typica!iy be obtained using a suitable type of liquid chromatography.
  • Liquid chromatography can be performed using size-exclusion chromatography (SEC), column chromatography, high- performance 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 alpha- glucosidase 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.
  • compositions include any of those disclosed herein, such as a hydro!yzed filtrate from a g!ucan synthesis reaction, or a hydrolyzed fraction of a glucan synthesis reaction used to produce soluble aipha-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 can comprise an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 98%, 97%, 98%, or 99% identical to SEQ ID NO:5, 6, 8, 9, 1 1 , 12, 14, 15, 17, 18, 20, 22, 24, 28, 28, 30, 32, 34, 38, 38, or that of DIAZY E RDF ULTRA (DuPont Industrial Biosciences).
  • compositions can comprise an amino acid sequence that is at least 90% identical to SEQ ID NO:1 .
  • any of the alpha-glucosidases disclosed herein can be used to produce the disclosed composition.
  • 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
  • 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 alpha- glucosidase.
  • 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
  • This feature can be taken advantage of to perform enhanced methods of (i) fructose enrichment and (ii) fermentation, for example.
  • chromatography is enhanced when using a glucan filtrate hydrolyzed by an alpha-glucosidase (transglucosidase), as compared to using a filtrate that was not hydrolyzed.
  • transglucosidase alpha-glucosidase
  • 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 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).
  • an alpha-glucosidase e.g., transglucosidase
  • the features of the disclosed fructose enrichment method regarding a!pha-glucosidase (e.g., transg!ucosidase) enzymes, and fractions of a g!ucan synthesis reaction, for example, can be according to any of the disclosures provided herein concerning each of these features.
  • a!pha-glucosidase e.g., transg!ucosidase
  • fractions of a g!ucan 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.
  • 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
  • step (b) 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 aipha-1 ,5 glucosyi-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).
  • 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.
  • alpha- g!ucosidase e.g., transglucosidase or glucoamylase
  • 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.
  • bacteria useful herein include
  • Lactobacillus species Streptococcus species, Bifidobacterium species,
  • yeast useful herein include Saccharomyces species such as S. cerevisiae and S. bayanus,
  • 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- ansi et al. (2006, Fermentation Microbiology and
  • 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 non- hydrolyzed fraction of a glucan synthesis reaction.
  • 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.
  • rate of product formation by a fermentation herein can be increased.
  • Example 7 below demonstrates that leucrose can be fermented to ethanol by yeast provided a feed comprising giucan filtrate that had not been hydrolyzed.
  • 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.
  • a method for fermenting leucrose can comprise adapting a microbe (e.g., yeast such as S. cerevisiae) for utilizing leucrose.
  • a microbe e.g., yeast such as S. cerevisiae
  • 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.
  • an enhanced leucrose fermentation method herein can comprise hydrolysis of leucrose with an alpha-glucosidase (e.g.,
  • transglucosidase or glucoamyiase while simultaneously fermenting the leucrose with a microbe.
  • compositions and methods disclosed herein include:
  • a saccharide comprising at least one alpha-1 ,3 or alpha-1 , 6 giucosyl-glucose linkage, wherein the saccharide is a disaccharide or oligosaccharide
  • the method comprises: contacting the saccharide with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-g!ucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,6 glucosyl-glucose linkage of the
  • saccharide is a byproduct of the glucan synthesis reaction.
  • a composition produced by contacting a saccharide with an alpha- glucosidase enzyme is produced by contacting a saccharide with an alpha- glucosidase enzyme
  • the saccharide is a disaccharide or oligosaccharide and comprises at least one alpha-1 ,3 or aipha-1 ,6 glucosyl-glucose linkage, wherein the enzyme hydro!yzes at least one aipha-1 ,3 or alpha-1 ,6 glucosyl-glucose linkage of the saccharide,
  • composition comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to the contacting.
  • composition of embodiment 1 1 wherein the degree of polymerization of the saccharide before hydrolysis is 3 to 7.
  • a method of enriching fructose present in a fraction of a glucan synthesis reaction comprising:
  • step (b) separating fructose from the hydrolyzed fraction of step (a) to
  • a fermentation method comprising:
  • step (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);
  • the Streptococcus salivahus gtfj enzyme (SEQ ID NO:3) was expressed in E. cols ' strain DH10B using an isopropyl beta-D-1 -thiogaiactopyranoside
  • SEQ ID NO:3 has an N-terminal 42-residue deletion compared to the S. salivahus gtfj amino acid sequence in GENBANK Identification No. 47527, but includes a start methionine. Briefly, E. coli DH10B cells were transformed to express SEQ ID NO:3 from a DNA sequence codon- optimized 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 800 n m) 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 OD 6 oo of 0.8-1 .0. Induced cultures were left on the shaker and harvested 3 hours post induction.
  • Gtfj enzyme (SEQ !D NO:3) was harvested by centrifuging cultured cells
  • the Streptococcus sp. C150 gtf-S enzyme (SEQ ID NO:40) was prepared as follows.
  • SG1 184 is a Bacillus subtiiis expression strain that expresses a truncated version of the glycosyltransferase Gtf-S ("GTF0459") from GTF0459.
  • Streptococcus sp.C150 (GENBANK ® G!:S21278321 ).
  • the gene (SEQ ID NO:41 ) encoding an N-terminal truncated protein GTF0459 (SEQ ID NO:42) from E. coli expression p!asmid pMP79 was cloned into the Nhel and Hindi!! sites of the Bacillus subtiiis integrative expression plasmid p4JH under the aprE promoter and fused with the B. subtiiis AprE signal peptide on the vector.
  • the construct was first transformed into E. coli DH10B and selected on LB with ampici!lin (100 plates.
  • the confirmed construct pDCQ984 expressing GTF0459 was then transformed into ⁇ . subtilis BG6006 containing nine protease deletions (amyE :xylRPxylAcomK-ermC, degUHy32, oppA, AspollE3501 , AaprE, AnprE, Aepr, AispA, Abpr, Avpr, AwprA, Ampr-ybfJ, An rB) and selected on LB plates with chloramphenicol (5 p.g/mL). The colonies grown on LB plates wi
  • chloramphenicol were streaked several times onto LB plates with 25
  • the resulting B. subtilis expression strain, SG1 184 was first grown in LB medium with 25 ug/mL chloramphenicol and then subcultured into Grants! I medium containing 25 ⁇ 9 ⁇ 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- ⁇ filters. The filtered supernatant was aliquoted and frozen at -80 °C.
  • 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.
  • 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 (SEQ ID NO:42) was then stored frozen at -80 °C.
  • the S, mutans MT-4239 gtf-C enzyme (SEQ ID NO:43) was prepared as follows. A gene encoding a truncated version of a glucosyitransferase (gtf) enzyme identified in GENBANK ® as Gl:3130088 (SEQ ID NO:43; gtf-C from S. mutans MT-4239) was synthesized using codons optimized for expression in Bacillus subtilis and synthesized by GenScript.
  • gtf glucosyitransferase
  • the gene (SEQ !D NO:44) encoding GTF0088BsT1 with an N-terminal truncation and a C-terminal T1 truncation (SEQ ID NO:45) was amplified from the GENSCRIPT plasmid and cloned into the Nhel and Hindi!! sites of the Bacillus subtilis integrative
  • the construct was first transformed into E. coil DH10B and selected on LB with ampicillin (100 plates.
  • the confirmed construct pDCQ1021 expressing GTF0088BsT1 was then transformed into B. subtilis BG6006 containing nine protease deletions
  • chloramphenicol were streaked several times onto LB plates with 25
  • the resulting B. subtilis expression strain SG1221 was first grown in LB medium with 25 ug/mL chloramphenicol and then subcultured into Grantsl! medium containing 25 ⁇ 9 ⁇ 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- ⁇ filters. The filtered supernatant was aliquoted and frozen at -80 °C.
  • B. subtilis SG1221 strain expressing GTF0088BsT1 (SEQ ID NG: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, FOAMBLAST 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, 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.
  • 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 m sodium acetate buffer at pH 5.5 in the presence or absence of 25 g/L dextran (MW -1500, Sigma-Aldrich, 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 AM IN EX HPX-87C columns series at 85 °C (Bio- Rad, Hercules, CA) to quantify sucrose concentration.
  • the sucrose was analyzed by HPLC using two AM IN EX HPX-87C columns series at 85 °C (Bio- Rad, Hercules, CA) to quantify sucrose concentration.
  • 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.
  • the nucleotide sequence (SEQ ID NO:46) encoding protein sequence (MUT3325; SEQ ID NO:47) was subcloned into p!asmid pTrex3 at Sacii and AscI restriction sites, a vector designed to express the gene of interest in Trichoderma reesei, under control of CBHI promoter and terminator, with Aspergillus niger acetamidase for selection.
  • the resulting p!asmid was transformed into T. reesei by biolistic injection.
  • the detailed method of biolistic transformation is described in International PCT Patent Application Publication WO2009/126773 A1 , which is incorporated herein by reference.
  • the culture was grown in shake flasks for 4-5 days at 28 °C and 220 rpm.
  • the eel! mass was first removed by centrifugation at 4000g for 10 min and the supernatant was filtered through 0.2- ⁇ sterile filters.
  • the expression of mutanase MUT3325 (SEQ ID NO:47) was confirmed by SDS-PAGE.
  • the production media component is listed below.
  • 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, 0.008 g/L cupric sulfate pentahydrate, 0.0035 g/L manganese sulfate
  • the culture was grown at 32 °C and 170 rpm for 48 hours before being transferred to 8 L of the
  • 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
  • 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 tol OOO 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 giucose-sophorose solids / g biomass / hr.
  • Insoluble mutan polymers required for determining mutanase activity were prepared using secreted enzymes produced by Streptococcus sobrinus ATCC ® 33478TM. Specifically, one loop of glycerol stock of S. sobrinus ATCC ® 33478TM 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.
  • BHI agar plate Brain Heart Infusion agar, Teknova, Hollister, CA
  • the resulting cells were removed by centrifugation and the resulting supernatant was filtered through a 0.2- ⁇ 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
  • 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 p-hydroxybenzoic acid hydrazide solution (PAH BAH) assay (Lever M.. Anal.
  • PAH BAH p-hydroxybenzoic acid hydrazide solution
  • the PAHBAH assay was performed by adding 10 ⁇ of reaction sample supernatant to 100 pL of PAHBAH working solution and heated at 95 °C for 5 min.
  • the working solution was prepared by mixing one part of reagent A (0.05 g/mL p- hydroxy 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).
  • reagent A 0.05 g/mL p- hydroxy benzoic acid hydrazide and 5% by volume of concentrated hydrochloric acid
  • reagent B 0.05 g/mL NaOH, 0.2 g/mL sodium potassium tartrate
  • NMR data were acquired on an Agilent DD2 spectrometer operating at 500 MHz for 1 H using a 5-mm cryogenic triple-resonance pulsed-field gradient (PFG) probe.
  • PPG pulsed-field gradient
  • 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 ⁇ 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 ⁇ 3. Sample temperature was maintained at 25 °C.
  • Samples were prepared by adding 50 ⁇ to a 5-mm NMR tube along with 450 ⁇ of D 2 O and 60 ⁇ of D 2 O containing 12.4 mM DSS (4,4-dimethyi-4 ⁇ siiapentane-1 -sulfonic acid sodium salt) interna! 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 4.85 for beta RE.
  • Sucrose (3000 g) was added to a clean 5-gallon polyethylene bucket.
  • Water (18.1 L) and Fermasure ! M (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 Genera! 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.
  • Table 2 indicates that the concentrated filtrate of the glucan synthesis reaction contains sucrose, fructose, glucose, leucrose and oligosaccharides of DP2-DP7.
  • This example measures the activity of various g!ucoamylase (EC 3.2.1.3), transglucosidase (EC 2.4.1.24), beta-glucossdase (EC 3.2.1 .21 ), alpha-amyiase (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.
  • various g!ucoamylase EC 2.4.1.24
  • beta-glucossdase EC 3.2.1 .21
  • alpha-amyiase EC 3.2.1 .1
  • glucosidase EC 3.2.1
  • Example 2 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 5 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 concentration used in each hydrolysis reaction. Table 5 further provides the percent hydrolysis of the leucrose and DP3+ (at least DP3-DP7)
  • Percent DP3+ hydrolysis was calculated as (1 - (wt% DP3+ oligosaccharides in the final syrup) / (wt% DP3+
  • b DP3+ contains DP3-DP7, but may also contain larger soluble oligosaccharides that have a high ratio of alpha-1 ,6 linkages to a!pha-1 ,3 linkages, when produced using certain gtf enzymes.
  • Table 5 indicates that 1 ,4-aipha-glucosidase and 1 ,6-aIpha-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.
  • use of a memelulanase Example 2.5 showed very little activity.
  • beta-glucosidase enzymes 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 2.1 1 ). 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
  • Giucoamylases (Examples 2.16-2.18) showed a range of activity against ieucrose and the oligosaccharides. Only one tested glucoamyiase (Example 2.18) gave less than 30% hydrolysis of both the Ieucrose and oligosaccharides.
  • alpha-glucosidases such as DIAZY E RDF ULTRA, glucoamyiase and transglucosidase can hydrolyze Ieucrose byproduct present in a glucan reaction filtrate.
  • the ability of alpha-glucosidases to hydrolyze Ieucrose indicates that these enzymes can hydrolyze alpha-1 ,5 glucosyl-fructose linkages. While this activity was shown above using Ieucrose as a substrate, it is believed that this activity can also be extended to
  • oligosaccharides comprising alpha-1 ,5 glucosyl-fructose linkages.
  • alpha-glucosidases such as glucoamyiase 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 aipha-1 ,8 glucosyl- glucose linkages.
  • alpha-glucosidase enzymes were generally effective at hydrolyzing the Ieucrose 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 Ieucrose hydrolysis, and a
  • glucoamyiase e.g., GC 321
  • GC 321 glucoamyiase
  • alpha-glucosidase enzymes can individually hydrolyze (i) alpha-1 ,5 glucosyl-fructose linkages and (ii) alpha-1 , 3 and alpha-1 , 6 glucosyl-glucose linkages in certain saccharides.
  • transg!ucosidase EC 2.4.1 .24
  • beta-glucosidase EC 3.2.1 .21
  • 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 glucan synthesis reaction comprise >90% glucose-glucose linkages, as determined by NMR (General Methods). Of the giucose-glucose linkages, -78% represent alpha-1 ,3 linkages and -22% represent alpha-1 ,6 linkages.
  • NMR was similarly used to determine the linkage profile of material generated using TG L-2000 (SEQ ID NO:1 ) transglucosidase ( Figure 2).
  • TG L-2000 SEQ ID NO:1
  • 210 ⁇ L ⁇ of concentrated filtrate from the material in Table 3, 300 iiL of D2O, and 90 ⁇ » of D 2 0 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 60 °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 re- equilibrated in the probe at 60 °C and shimmed, and measurements were taken within a few minutes of analysis.
  • transglucosidase can hydrolyze (i) alpha-1 ,5 glucosyl-fructose linkages and (ii) alpha-1 ,3 and aipha-1 ,6 glucosyl-giucose linkages in certain saccharides.
  • 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.
  • 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 6-7 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 polyethy!enimine (PEL EPO N P-1050).
  • PEL EPO N P-1050 polyethy!enimine
  • glucoamylase GC-147 having activity defined in Table 4 was added to 315 ml of 0.02 M acetate buffer (pH 4.5).
  • Each of the treated enzyme-giutaraldehyde 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
  • 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 ) immobilized glucoamyiase, 2) immobilized transglucosidase, and 3) immobilized g!ucoamylase followed by immobilized transglucosidase.
  • 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 glucoamyiase and transglucosidase gave the highest hydrolysis of oligosaccharides.
  • immobilized glucoamyiase or transglucosidase represents an effective technique to hydrolyze oligosaccharides containing aipha-1 ,3 and alpha-1 ,6 glucosy!-glucose linkages, as well as leucrose.
  • oligosaccharides elute first, followed by disaccharides, and then
  • 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.
  • 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.
  • a syrup was prepared by concentrating (vacuum at 50 °C) a glucan filtrate that had been treated with 1 vol% of transglucosidase TG L-2000 (SEQ ID NO: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 monosaccharides.
  • the syrup had the final product distribution described in Table A.
  • Hyd rolyzed G I u can Filtrate
  • Example 5 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 g!ucan filtrate that had not been hydrolyzed by a transglucosidase.
  • 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.
  • 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.
  • 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
  • Periodic samples were taken during the fermentation and analyzed for optical density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL- 3 refractometer (Atago), and sugar and ethanol concentrations by HPLC
  • 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 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 are provided in Tables 1 1 and 12, respectively.
  • giucan filtrate can be used in a fermentation process to produce ethanol.
  • Example 7 but using a giucan filtrate that was previously treated with a transglucosidase (TG L-2000, SEQ ID NO:1 ). Hydrolyzed giucan filtrate was prepared as follows. Giucan 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 (IKA KS4000) at 60 °C and 100 rpm for 72 hours. Hydrolyzed giucan filtrate was thus prepared.
  • Yeast (S. cerevisiae) cream (Bom Retiro mill, Brazil) was washed by suspending the cream in tap water (2.4 L, optical density of 65 at 600 rim) and then centrifuging the yeast cream for 5 minutes using a LEGEND XTR centrifuge 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 BIOFL0310 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 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
  • 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.
  • oligosaccharide byproduct components have been saccharified results in increased ethanol yields at faster rates.
  • This saccharification can be done using a transg!ucosidase, for example.
  • Yeast (S. cerevisiae) cream (Bom Retiro mill, Brazil) was washed by suspending the cream in tap water (2.4 L, optical density of 85 at 600 nm) and 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 centnfugation 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.
  • the fermentation was nominally complete in 6 hours, similar to the fermentations where the filtrate was hydrolyzed prior to the fermentation step (Example 8), and gave a slightly superior titer of ethanol (82 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).
  • a saccharifying enzyme such as TG L-2000
  • 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.
  • glucan filtrate components e.g., leucrose
  • This example discloses preparing various alpha-glucosidases in addition to those alpha-glucosidases (transglucosidase, glucoamylase, DIAZY E RDF ULTRA) used in some of the foregoing Examples. These additional alpha- glucosidases were tested for hydroiytic activity against oligosaccharides comprising alpha-1 ,5 glucosyl-fructose linkages or alpha-1 ,3 and/or alpha-1 ,8 glucosyl-glucose linkages in Examples 1 1 , 12, 15 and 16 provided below. Discovery of : an Aspergillus ciavatus alpha- lucQsidase (Aclgiul)
  • a strain of Aspergillus ciavatus was selected as a potential source of other enzymes that may be useful in various industrial applications.
  • One of the genes identified in Aspergillus ciavatus encodes an alpha-glucosidase and the sequence of this gene, called "AcigluT', is provided in SEQ ID NO:4.
  • the corresponding protein encoded by SEQ ID NQ:4 is provided in SEQ ID NO:5.
  • Aclgiul belongs to Glycosyl hydrolase family 31 based on a PFA search (pfam.sanger.ac.uk web link).
  • 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 et al., 201 1 , Nature Methods, 8:785-786).
  • SignalP version 4.0 Nadahl Petersen et al., 201 1 , Nature Methods, 8:785-786.
  • the presence of a signal sequence suggests that Aclgiul is a secreted enzyme.
  • the amino acid sequence of the predicted mature form of Aclgiul is set forth as SEQ ID NO:8.
  • Plasmid pJG294 was transformed into a quad deleted Trichoderma reesei strain (described in WO05/001038) 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 NO:8, 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.
  • Neosariorya fischeri A strain of Neosariorya fischeri was selected as a potential source of other enzymes that may be useful in various industrial applications.
  • One of the genes identified in Neosariorya 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.
  • Nfiglul belongs to G!ycosy! hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link).
  • the protein SEO ID NO:8 has a signal peptide with a length of 19 amino acids as predicted by SignalP version 4.0 (Nordah!
  • Nfiglul is a secreted enzyme.
  • the amino acid sequence of the predicted mature form of Nfiglul is set forth as SEQ ID NO: 9.
  • a synthetic Nfiglul gene was cloned into pTrexSg expression vector (described in U.S. Patent Appl. Publ. No. 201 1/0136197) and the resulting piasmid was designated as pJG295.
  • the sequence of the Nfiglul gene was confirmed by DNA sequencing.
  • Piasmid pJG295 was transformed into a quad deleted Trichoderrna reesei strain (described in WO05/001036) 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 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.
  • 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 "Ncrglui", is provided in SEQ ID NO:10.
  • the corresponding protein encoded by SEQ ID NO: 10 is provided in SEQ ID NO:1 1 .
  • Ncrglui belongs to Glycosyl hydrolase family 31 based on a PFAM search
  • 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 et al., 201 1 , Nature Methods, 8:785-786). The presence of a signal sequence suggests that Ncrglui is a secreted enzyme.
  • the amino acid sequence of the predicted mature form of Ncrglul is set forth as SEQ ID NO:12.
  • a synthetic Ncrglul gene was cloned into pTrexSgM expression vector (described in U.S. Patent Appl. Publ. No. 201 1/0136197) and the resulting plasmid was designated as pJG296. The sequence of the Ncrglul gene was confirmed by DNA sequencing.
  • Plasmid pJG298 was transformed into a quad deleted Trichoderma reesei strain (described in WO05/001036) using a biolistic method (Te'o VS et 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.
  • Rasamsonia cornposticola A strain of Rasamsonia cornposticola was selected as a potential source of other enzymes that may be useful in various industrial applications.
  • One of the genes identified in Rasamsonia cornposticola encodes an alpha-glucosidase and the sequence of this gene, called a TauSec098", is provided in SEQ ID NO: 13.
  • the corresponding protein encoded by SEQ ID NO: 13 is provided in SEQ ID NO: 14.
  • TauSec098 belongs to Glycosyl hydrolase family 31 and contains an N-terminal CBM 20 domain based on a PFA search
  • 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 et ai, 201 1 , Nature Methods, 8:785-786).
  • SignalP version 4.0 Naphl Petersen et ai, 201 1 , Nature Methods, 8:785-786.
  • the presence of a signal sequence suggests that TauSec098 is a secreted enzyme.
  • the amino acid sequence of the predicted mature form of TauSec098 is set forth as SEQ ID NO:15.
  • a synthetic TauSec098 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 pGX256- TauSec098.
  • the sequence of the TauSec098 gene was confirmed by DNA sequencing.
  • Plasmid pGX258-TauSec098 was transformed into a quad-deleted
  • Trichoderma reesei strain (described in WO05/001036) 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 ( ⁇ ) sulfate 5 mg/L; zinc sulfate 1 .4 mg/L; cobalt (I!) chloride 1 mg/L; manganese (N) sulfate 1 .6 mg/L; agar 20 g/L; pH 4.25). Transformed colonies (about 50-100) appeared in about 1 week.
  • Rasamsonia composticola 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 “TauSec099", is provided in SEQ ID NO: 16.
  • the corresponding protein encoded by SEQ ID NO: 16 is provided in
  • SEQ ID NO: 17 TauSec099 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 a!., 201 1 , Nature Methods, 8:785-786). The presence of a signal sequence suggests that TauSec099 is a secreted enzyme. The amino acid sequence of the predicted mature form of TauSec099 is set forth as SEQ ID NO: 18.
  • a synthetic TauSecQQQ gene was cloned into the Trichoderma reesei expression vector pGXT (a pTTT-derived plasmid) by Generay Biotech Co.
  • Plasmid pGX256-TauSec099 was transformed into a quad-deleted
  • Trichoderma reesei strain (described in WO05/001036) using protoplast transformation (Te'o et a!., 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 (li) 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.
  • BoGiul An aipha-glucosidase gene, "BloGiul”, was identified from Bifidobacterium longum subsp. longum JDIV1301 .
  • the nucleic acid sequence for the BloGiul gene (SEQ ID NO:19, GENBANK Acc. No. NC014169.1 , complement sequence from positions 140600 to 142414) and the amino acid sequence of the
  • the DNA sequence encoding the entire BloG!ul protein (SEQ ID NO:20) was optimized for expression in B. subtiiis, then synthesized (yielding SEQ ID NO:21 ) and inserted into the p3JM plasmid by Generay Biotech Co. (Shanghai, China), resulting in p3JM-BloGlu1 .
  • the p3JM-BioGlu1 plasmid contains an aprE promoter to drive expression of the optimized BioGiul sequence (SEQ ID NO:20) was optimized for expression in B. subtiiis, then synthesized (yielding SEQ ID NO:21 ) and inserted into the p3JM plasmid by Generay Biotech Co. (Shanghai, China), resulting in p3JM-BloGlu1 .
  • the p3JM-BioGlu1 plasmid contains an aprE promoter to drive expression of the optimized BioGiul sequence (SEQ
  • Plasmid p3JM-BloGlu1 was used to transform ⁇ .
  • subtiiis cells degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr
  • the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol.
  • NCBI database (GENBANK Acc. No. WP_007054665.1 ).
  • 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-BioGlu2.
  • the p3JM-BioGlu2 plasmid contains an aprE promoter to drive expression of the optimized BioGiu2 sequence (SEQ ID NO:23).
  • Plasmid p3JM-BloG!u2 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 CaC ) to express BloG!u2 protein (SEQ ID NO:24).
  • the DNA sequence encoding the entire BioGluS protein (SEQ ID NO:28) was optimized for expression in B. subtilis, then synthesized (yielding SEQ ID NO:27) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in p3JM-BioGlu3.
  • the p3JM-B!oGlu3 plasmid contains an aprE promoter to drive expression of the optimized BioGluS sequence (SEQ ID NO:27).
  • Plasmid p3JM-BloGlu3 was used to transform ⁇ .
  • subtiiis cells degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Arnpr, AispA, Abpr
  • the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol.
  • MBD medium a MOPS-based defined medium supplemented with an additional 5 mM CaCfe
  • BpsGlul 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 ).
  • a DNA sequence encoding BpsGlul protein was optimized for expression in B, subtiiis, then synthesized (yielding SEQ ID NO:29) and inserted into the 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 p3J -BpsGlu1 plasmid contains an aprE promoter to drive expression of the optimized BpsGlul sequence (SEQ ID NO:29)
  • Plasmid p3JM-BpsGIu1 was used to transform B. subtilis cells
  • BthGlul An alpha-glucosidase gene, "BthGlul”, was identified from Bifidobacterium thermophiium RBL67.
  • the nucleic acid sequence of the BthGlul gene (SEQ D 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 NQ:31 were found in GENBANK Acc. No. YP_007592840.1.
  • the DNA sequence encoding the entire BthGlul protein (SEQ ID NO:32) was optimized for expression in B. subtilis, then synthesized (yielding SEQ ID NQ:33) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in p3JM-BthGiu1.
  • the p3JM-BthGlu1 plasmid contains an aprE promoter to drive expression of the optimized BthGlul sequence (SEQ ID NO:33).
  • Plasmid p3JM-BthGlu1 was used to transform B. subtilis cells (degUHy32,
  • 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. WPJ303827971 .1 ).
  • a DNA sequence encoding BbrGlu2 protein was optimized for expression in B, subtiiis, then synthesized (yielding SEQ D NO:35) and inserted into the p3J plasmid by Generay Biotech Co., resulting in p3JM-BbrGlu2.
  • SEQ ID NG:3S encodes the amino acid sequence of SEQ ID NQ:36.
  • the p3JM-BbrGlu2 plasmid contains an aprE promoter to drive expression of the optimized BbrGlu2 sequence (SEQ ID NO:35)
  • Plasmid p3JM-BbrGlu2 was used to transform B. subtiiis ceils (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 MQPS-based defined medium supplemented with an additional 5 mM
  • BbrGluS alpha-glucosidase gene
  • BbrGluS 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. YP_005583701 .1 .
  • the p3JM-BbrGlu5 plasmid contains an aprE promoter to drive expression of the optimized BbrGluS sequence (SEQ ID NO:39).
  • Plasmid p3J -BbrGlu5 was used to transform B. suhtilis ceils (degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Ampr, AispA, Abpr), and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm chloramphenicol.
  • 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 (SEQ 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 pre- equilibrated 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 alpha-giucosidase (SEQ 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 pre-equi!ibraied with 20 m 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 eiuted from the column with 1 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.
  • TauSec098 SEQ ID NO:15
  • TauSec099 SEQ ID NO:18 alpha- glucosidases were purified via hydrophobic interaction chromatography. For each purification, ammonium sulphate was added to about 180 mL of
  • the target protein (SEQ ID NO: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 AMICON ULTRA-15 devices.
  • the final product which was greater than 90% pure, was stored in 40% glycerol at -80 °C until usage.
  • BloGlul (SEQ ID NO:20), BloGlu2 (SEQ ID NO:24) and B!oGluS (SEQ ID NO:
  • alpha-glucosidases were all purified in three steps. For each purification, the crude broth from a 1 -L DASGIP ferrnenter 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 loaded onto a 40-mL HiPrep l M Phenyl FF column pre-equi!ibrated 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 H 2 0 in three column volumes each. All fractions were analyzed using SDS-PAGE and activity assays.
  • the fractions containing target protein (SEQ ID NO:20, SEQ ID NQ:24, or SEQ ID NO:28) were pooled, concentrated and subsequently loaded onto a HiLoadTM 28/60 SuperdexTM 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 AMICQN ULTRA-15 devices, and stored in 40% glycerol at -20 °C until usage.
  • BpsGlui SEQ ID NO:30
  • BthGlul SEQ ID NG:32 alpha- glucosidases alpha- glucosidases were purified in two 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 centrifuged at 8000 X g for 30 min. The resulting pellet was re-suspended in 20 mM Tris pH 8.0 (buffer A).
  • BbrGlu2 SEQ ID NO:36
  • BbrGluS SEQ ID NQ:38 alpha- g!ucosidases alpha- g!ucosidases were purified in four steps. For each purification, the crude broth from a 1-L DASGIP fermente 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 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 ⁇ ' 1 Phenyl FF column pre-equilibrated with 20 mM HEPES pH 7.0, 1 M ammonium sulfate.
  • the target protein (SEQ ID NO:36 or SEQ ID NO: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 VIVAFLOW 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to a HiPrep l M Q 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.
  • alpha-glucosidases were expressed and purified. These alpha-glucosidases were tested for hydro!ytic activity against alpha-1 ,5 glucosyl-fructose linkages and alpha-1 ,3 and/or alpha-1 ,6 g!ucosyl-glucose linkages in Examples 1 1 , 12, 15 and 16 provided below.
  • alpha-glucosidases have hydro!ytic 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 glucosyl-fructose linkages and alpha-1 ,3 and alpha-1 ,6 glucosy!-glucose linkages.
  • each aipha-glucosidase disclosed in Example 10 was assayed based on the release of glucose from a particular substrate (isomaltose, maltose, panose, leucrose, or nigerose) when the substrate was 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-ethylbenzothiazo!lne-8-su!fonic acid (ABTS) by peroxide that was generated from coupled GOX/HRP enzymes reacted with glucose.
  • ABTS 2,2'-azino-bis 3-ethylbenzothiazo!lne-8-su!fonic acid
  • 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-glucosidase samples and glucose standard were prepared in MILLIQ water.
  • alpha-glucosidase samples were tested with only one dosage at 10 ppm due to a limited stock of substrate solutions.
  • Each alpha-glucosidase sample (10 pL) was transferred into a new microtiter plate (Corning 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).
  • Each enzyme was used at one dosage (10 ppm) against nigerose.
  • alpha-giucosidases besides exhibiting hydrolytic activity against alpha-1 ,4 giucosyl-glucose linkage (maltose), also exhibit hydrolytic activity against alpha-1 ,6 giucosyl-glucose linkage (isomaltose), alpha-1 ,3 glucosyl-glucose linkage (nigerose), and aipha-1 ,5 glucosyl-fructose linkage (leucrose) (Table 17).
  • 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 ,6 glucosyl-glucose linkages.
  • 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, DP3 and HS (higher sugars, DP4+) in a filtrate of an insoluble glucan (poly alpha-1 , 3-glucan) synthesis reaction was studied.
  • 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 CS11 GC, #227 resin; Na ⁇ ion form; 5% divinyl benzene (crosslinking); 0.34 mm particle size: 1 .64 m bed length; 0.093 m column diameter.
  • the concentrated sugar solution i.e., concentrated filtrate
  • Table 3 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.
  • the resin 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 convert the resin to the sodium form.
  • the sugar solution (0.6 L) was then fed to the column, after which the column was eluted using water at a flow rate of 50 mL/min.
  • the run conditions of the chromatographic separation are summarized as follows: 0.8 L feed size, 25 g dry solids/100 g solution, 65 °C column temperature, 50 mL/min flow rate. An oligosaccharide solution was eluted between 11 and 21 minutes. A small amount of salts - indicated by an increase in conductivity - was eluted at the same time.
  • the oligosaccharide fraction thus prepared was analyzed by HPLC to determine its product distribution. In total, the fraction contained >89% of oligosaccharides containing three or more hexose units and less than 1.5% of identifiable mono- and di-saccharides.
  • This fraction was concentrated to a total dry weight of 317 g/L using a thin film evaporator (LCI Corporation, Charlotte, NC) followed by rotary evaporation with a ROTAVAPOR (R-151 ; Buchi, New Castle, DE).
  • the product distribution of the concentrated fraction as measured by HPLC appears in Table 18.
  • 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 series HPLC system with an AMINEX HPX-42A column (300 mm x 7.8 mm) at 85 °C. The sample (10 ⁇ ) was applied to the HPLC column and separated with an isocratic gradient of MILL!-Q water as the mobile phase at a flow rate of 0.6 mL/min. Oligosaccharide products were detected using a refractive index detector. The numbers provided in Table 19 below reflect the average of peak area percentages (from duplication of each sample) of each DP n as a fraction of the total from DP1 to DP7.
  • alpha-glucosidase can be used to break down oligosaccharides with alpha-1 ,5 glucosyl-fructose linkages and/or alpha-1 ,3 and a!pha-1 ,6 glucosyl-g!ucose linkages.
  • the results provided in Table 19 also suggest that fungal alpha-glucosidases have better hydrolytic activity towards soluble oligosaccharides compared with the bacterial alpha-glucosidases.
  • 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 ⁇ . 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 AM!NEX HPX-42A column (300 mm x 7.8 mm) at 85 °C. The sample (10 ⁇ ) was applied to the HPLC column and separated with an isocratic gradient of MILLI-Q water as the mobile phase at a flow rate of 0.6 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 DP n as a fraction of the total.
  • a!pha-g!ucosidase can be used to hydro!yze 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.
  • 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.
  • a giucan synthesis reaction was used to produce at least one soluble alpha-g!ucan product, This soluble product resulted from the concerted action of both a glucosyltransferase (GTF0459, SEQ ID NO:42) and an a!pha-g!ucanohydrolase (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 giucan synthesis reaction. This fraction was used in Examples 15 and 16 below to test the activity of alpha- glucosidases thereupon.
  • 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 I EX/SEC column
  • a glucan synthesis reaction was used to produce at least one soluble alpha-glucan product.
  • This Example also demonstrated the preparation of a chromatographic fraction from a glucan synthesis reaction that produced a soluble alpha-glucan product. This fraction was used in Examples 15 and 16 below to test the activity of alpha-glucosidases thereupon.
  • This Example describes using alpha-glucosidase to hydrolyze Ieucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that produced soluble alpha-glucan product.
  • 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 ⁇ . of 0.5 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-foid in water for HPLC analysis (General Methods). The percentages reported in Table 23 reflect the average of peak area percentages (from duplicate analyses of each sample) of each DP n as a fraction of the total. The results indicate that the fungal alpha-giucosidases had better hydrolytic activity towards glucan oligomers when compared to the bacterial alpha-glucosidases.
  • 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 alpha- g!ucan product.
  • a fraction e.g., chromatographic fraction
  • Example 15 is further to Example 15, describing the use of alpha- glucosidase to hydrolyze leucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that produced soluble alpha-glucan product.
  • alpha-glucosidases that were most active for hydrolysis of oiigomer/leucrose fractions from qtf-S/MUT3325 and qtf-C reactions (Example 15) was performed by analyzing sugar compositions resulting in reactions containing enzymes dosed at equal protein concentrations. Incubations of alpha- glucosidases (dosed at 4 ppm; for blends, the ratio of the two enzymes was 1 :1 and total dosage was 4 ppm) and oiigomer/leucrose substrate (10% ds) were performed at pH 5.5 in the presence of 2 mM calcium chloride at 80 °C and 65 °C, respectively. The reactions were quenched by adding 50 ⁇ !_ of 0.5 M NaOH after 23 hours of incubation.
  • 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 Methods). The percentages reported in Table 24 (below) reflect the average of peak area percentages (from duplicate analyses of each sample) of each DP n as a fraction of the total. The results indicate that TauSec098 was efficacious for hydrolysis of DP2 to DP7 oligomers and TauSec099 outperformed TG L-2000 for leucrose hydrolysis when the incubation was performed at 65 °C. The blends of TauSec098 with TauSec099 (or TG L-2000) were effective for hydrolysis of oligomers and leucrose for DP1 production.
  • alpha-glucosidase can be used to hydrolyze leucrose and other oligosaccharides present in a fraction (e.g., chromatographic fraction) obtained from a g!ucan synthesis reaction, such as one that synthesizes a soluble alpha- glucan product.
  • a fraction e.g., chromatographic fraction

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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

TITLE
ENZYMATIC HYDROLYSIS OF D!SACCHARIDES AND OLIGOSACCHARIDES
USING ALPHA-GLUCOSIDASE ENZYMES
This application claims the benefit of U.S. Provisional Application Nos. 61/945,233 (filed February 27, 2014), 61/945,241 (filed February 27, 2014), 62/004,290 (filed May 29, 2014), 62/004,308 (filed May 29, 2014), 62/004,312 (filed May 29, 2014), 62/004,300 (filed May 29, 2014), 62/004,314 (filed May 29, 2014), and 62/004,305 (filed May 29, 2014), all of which are incorporated herein by reference in their entireties.
FIELD OF INVENTION
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 glucosyi-glucose linkages with an alpha-glucossdase enzyme.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
The official copy of the sequence listing is submitted electronically via EFS-VVeb as an ASCII formatted sequence listing with a file named
CL6220USNP__SequenceListing__ST25.txt created on February 1 1 , 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-alpha- glucosyltransferase) are D-giucosyltransferase enzymes that catalyze both hydrolytic and transfer reactions on incubation with alpha-D-gluco- oligosaccharides (1951 , Pazur and 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 D- glucose, or panose (6-O-alpha-glucosyl maltose) from maltose.
Transglucosidase can also transfer a glucosyl residue to the HO-2 or HO-3 of another D-g!ucosyl unit to form Kojibiose or Nigerose, This enzyme can further transfer a D-glucosy! unit back to HO-4 to reform maltose.
As a result of transglucosylation reactions with transglucosidase, malto- oligosaccharide residues are converted to isoma!to-oligosaccharides (!MO) 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-iinked 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 alpha- glucosidase, 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. 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-giucosidase 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 glucosy!transferase, or (ii) a product of the concerted action of both a glucosyitransferase and an alpha-glucanohydrolase capable of hydrolyzing glucan polymers having one or more alpha-1.3-glycosidic linkages or one or more alpha-1 , 8-glycosidic 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. 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 glucosyi-glucose linkage, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,8 glucosy!-g!ucose 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 alpha- glucosidase enzyme under suitable conditions, wherein the alpha-g!ucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,6 glucosyi-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 with an a!pha-glucosidase enzyme under suitable conditions, wherein the alpha- g!ucosidase enzyme hydro!yzes at least one a!pha-1 ,3 or alpha-1 ,6 glucosyl- glucose 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.
BRIEF DESCRIPTION OF THE DRAWI GS AND SEQUENCES
Figure 1 : H N R spectra of glucan reaction filtrate material before (starting materia!) and after (treated material) hydrolysis treatment with NOVO 188 enzyme (see Examples 2-3).
Figure 2: 1 H 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
glucosidase, fu!I-!ength precursor form
including signal peptide.
"NfigiuT. Neosartorya fischeri alpha- glucosidase, mature form lacking signal 9 peptide. (969 aa)
"Ncrg!uT, Neurospora crassa alpha- glucosidase, full-length precursor form 10 1 1 including signal peptide. (3385 bases) (1044 aa)
"Ncrg!ul ", Neurospora crassa alpha- g!ucosidase, mature form lacking signal 12 peptide. (1022 aa)
"TauSecQ98", Rasamsonia composticoia
alpha-glucosidase, full-length precursor 13 14 form including signal peptide. (3293 bases) (1035 aa)
"TauSec098", Rasamsonia composticoia
a!pha-g!ucosidase, mature form lacking 15 signal peptide. (1013 aaj
"TauSec099", Rasamsonia composticoia
alpha-glucosidase, full-length precursor 16 17 form including signal peptide. (3162 bases) (990 aa)
"TauSec099", Rasamsonia composticoia
alpha-glucosidase, mature form lacking 18 signal peptide. (973 aa)
"BloGlul ", Bifidobacterium long urn
(subsp. iongum JDM301 ) alpha- 19 20 glucosidase (wild type). (1815 bases) (604 aa)
"BloGlul ", Bifidobacterium iongum
(subsp. Iongum JDM301 ) alpha- 21
glucosidase, codon-optimized sequence. ( 812 bases)
"BloGlu2", Bifidobacterium iongum 22 alpha-glucosidase (wild type). (604 aa)
"BloGlu2", Bifidobacterium Iongum
alpha-glucosidase, codon-optimized
sequence encoding amino acid 23 24 sequence. ( 812 bases) (604 aa)
"BloGlu3", Bifidobacterium Iongum
(subsp. F8) alpha-glucosidase (wiid 25 26 type) (1815 bases) (604 aa)
"BloGlu3", Bifidobacterium Iongum
(subsp. F8) alpha-glucosidase, codon- optimized sequence encoding amino 27
acid sequence. (1812 bases)
28
"BpsGlul ", Bifidobacterium (585 aa)
DETAILED DESCRIPTION OF THE INVENTION The disclosures of all 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 "monomelic 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 glucose- glucose linkage" and "glucose-alpha 1 ,3-glucose" herein refers to an alpha-1 ,3- glycosidic linkage between two alpha-D-glucose molecules. The terms "alpha- 1 ,6 glucosyl-glucose linkage", "alpha-1 ,6 glucose-glucose linkage" and "glucose- alpha 1 ,6-glucose" herein refers to an alpha-1 ,6-glycosidic linkage between two alpha-D-glucose molecules. Alpha-1 ,3 glucosyl-glucose linkage(s) and/or alpha- 1 ,6 glucosyl-glucose linkage(s) herein are comprised within a disaccharide or oligosaccharide in certain embodiments.
The terms "alpha-1 ,5 g!ucosyl-fructose linkage", "alpha-1 ,5 glucose- fructose linkage" and "glucose-alpha-1 ,5-fructose" herein refers to an alpha-1 ,5- glycosidic 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. "A!pha-D~glucose" herein can also be referred to as "glucose".
A disaccharide containing an a!pha-1 ,5 glucosyl-fructose linkage is referred to herein as leucrose. The terms "leucrose" and "D-glucopyranosyl- a!pha(1 -5)-D-fructopyranose" are used interchangeably herein. Leucrose has the following structure:
The terms "alpha-glucosidase", "aipha-1 ,4-gIucosidase", and "alpha~D~ glycoside 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 alpha-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 alpha- glucosidases with such activity.
The terms "transglucosidase" (TG), "transglucosidase enzyme", and "1 ,4- alpha-g!ucan 8~alpha-glucosyltransferase" are used interchangeably herein. Transglucosidases (EC 2.4.1 .24) have previously been recognized as D- giucosyltransferase 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 ,4- glucan glucohydrolase" are used interchangeably herein. Glucoamylases (EC 3.2.1 .3) have previously been recognized as exo-acting enzymes that catalyze hydrolysis of both a!pha-1 ,4 and a!pha-1 ,6 glycosidic linkages from non-reducing ends of glucose-containing di~, oligo- and poly-saccharides. Glucoamy!ases are now disclosed herein to also have hydrolytic activity toward alpha-1 , 5 glucosyl- fructose 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 alpha- 1 ,3 or alpha-1 ,6 glycosidic linkage between two glucose molecules by an alpha- glucosidase such as a transgiucosidase. Such hydrolysis occurs when contacting a disaccharide or oligosaccharide containing an alpha-1 ,3 and/or alpha-1 ,6 glucosyl-glucose linkage with an alpha-giucosidase 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) into 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 ,6 glucosyl- glucose 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 ,6 glucosyl-glucose linkages in the saccharide by the alpha- glucosidase. Suitable conditions can comprise "aqueous conditions", for example, comprising at least 20 wt% water. 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 transgiucosidase 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 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 "alpha-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%, 98%, 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 ,3- glucan with a DP of at least 8 or 9. A giucosyltransferase reaction in certain embodiments as presently disclosed produces at least one insoluble glucan product.
The terms "soluble glucan", "soluble alpha-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 ,3- glucan with a DP less than 8, and certain oligosaccharides disclosed in the Examples provided below. A giucosyltransferase reaction in certain
embodiments as presently disclosed produces at least one soluble glucan product. Another set of features that characterizes soluble alpha-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) digestion- resistant (i.e., exhibit very slow or no digestibility) with little or no absorption in the human small intestine, and (iii) 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-g!ucan" and "alpha-1 ,3-glucan polymer" are used interchangeably herein. Poly aipha-1 ,3-glucan is a polymer comprising glucose monomenc 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 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 giucan herein can be represented as number- average 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, "glucansucrase" and the like are used interchangeably herein. The activity of a gtf enzyme herein catalyzes the reaction of sucrose substrate to make the products giucan 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-termma! to C-terminal direction) a signal peptide, a variable domain, a catalytic domain, and a glucan- binding domain. A glucosyltransferase herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active
EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238, 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 "giucan synthesis reaction", "giucan reaction" "gtf reaction" and the like are used interchangeably herein and refer to a reaction that is performed by a glucosyltransferase enzyme. A giucan 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 components. Other components that can be in a glucan synthesis reaction herein include fructose, glucose, leucrose, soluble oligosaccharides (e.g., DP2- DP7), and soluble glucan produc (s), for example. Also, one or more alpha- g!ucanohydro!ase 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 "giucanohydroiase" are used interchangeably herein and refer to an enzyme capable of hydroiyzing an alpha- glucan oligomer. An alpha-giucanohydroiase 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 g!ycosidic bonds), mutanases (EC 3.2.1 .59; capable of endohydrolyzing alpha-1 ,3-linked glycosidic bonds), and alternanases (EC 3.2.1 .-; capable of endohydrolyticaliy cleaving alternan).
Various factors including, but not limited to, level of branching, the type of branching, and the relative branch length within certain alpha-g!ucans 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 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 alpha- glucosidase 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 (PDI= 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 quantity or activity that is at least 1 %, 2%, 3%, 4%, 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 (NCB!) 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., ClustalW or ClustalV). 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, WINDOWS and DIAGONALS
SAVED=5. For nucleic acids, these parameters may be ΚΤυΡίΕ=2, GAP PENALTY^, WINDOWS and DIAGONALS SAVED=4. Alternatively still, percent identity between sequences may be performed using an EMBOSS algorithm (e.g., needle) with parameters such as GAP OPEN=10, GAP
EXTEND^O.S, END GAP PENALTY=false, END GAP QPEN=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%, 84%, 85%, 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, transg!ucosidase), or glucosyltransferase enzyme. The enzyme reactions (e.g., alpha-glucosidase reaction, glucosyltransferase reaction) disclosed herein are synthetic, non-naturally 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 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 alpha- glucosidase enzymes can hydrolyze alpha-1 ,3 and alpha-1 , 6 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 , 6 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, non- reducing (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 a!pha-1 ,8 glucosyl- glucose 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-g!ucosidase can be a fungal alpha-glucosidase. Other examples of suitable alpha-g!ucosidases herein include those disclosed in U.S. Patent Nos. 6355487, 5922580, 5795766, 5783252, and 8633008, which are all incorporated herein by reference.
An alpha-glucosldase enzyme in certain embodiments herein may comprise the amino acid sequence of SEQ ID NO:5, 6, 8, 9, 1 1 , 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or that of D!AZYME 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 NO: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 DIAZY E RDF ULTRA, and have hydrolytic activity toward alpha- 1 ,3 and/or alpha-1 ,6 giucosyl-glucose linkages in saccharides. Several of the foregoing sequences, for example, are mature a!pha-glucosidases that lack an N-terniinal 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 intraceliularly and obtained from a ceil lysate).
A transglucosidase (EC 2.4.1 .24; 1 ,4-aipha-glucan 8-alpha- g!ucosyltransferase) can be used in certain embodiments herein as an alpha- glucosidase to hydrolyze alpha-1 ,3 and/or alpha-1 ,6 giucosyl-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-gluco- oligosaccharides. Transglucosidases as now disclosed herein also have hydrolytic activity toward alpha-1 ,3 and alpha-1 ,6 giucosyl-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 Neosariorya species (e.g., N. fischeri). Examples of Aspergillus species from which a transglucosidase may be derived include, but are not limited to, A. niger, A. awamori, A. oryzae, A, terreus, A. c!avatus, A. fumigatus and A. nidulans. Other examples of transglucosidase enzymes usefu! herein are described in Barker et al (1953, J. Chem. Soc. 3588-3593); Pazur et a!. (1988, Carbohydr, Res. 149:137-147), Nakamura et al. (1997, J. Biotechnoi. 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.
4889296, which is incorporated herein by reference, discloses a process for producing thermostable transglucosidase. Yet more examples of
transglucosidase enzymes useful herein may be any of those in the GENBANK database (NCBI), such as accession numbers: D45356 (GID:2845159, A. niger), BAD06006.1 (GID:4031328, A. awamori), BAA08125.1 (GID:1054565, A oryzae), XPJ301210809.1 (G!D:1 15492363, A. terreus), XP_001216899.1 (GID:1 15433524, A terreus), XPJ3Q1271891 .1 (GID:121707620, A. davatus), XP..751811 .1 (GiD:70993928, A fumigatus), XP...659621 .1 (G!D:67523121. A nidulans), XPJ3Q 1266999.1 (GID:1 19500484, N. fischeri) and XP_001258585.1 (GID:1 19473371 , N. fischeri), which are al! incorporated herein by reference. Alternatively, a transglucosidase 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 transglucosidase sequences, and have hydro!ytic 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 transglucosidase enzyme in certain embodiments herein may comprise the amino acid sequence of SEQ ID NO:1 (TG L-2000), which is an A niger transglucosidase (U.S. Patent Appl. Publ. No. 2008/0229514). Alternatively, a transglucosidase 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 glucosyl- glucose linkages in saccharides. Any of SEQ ID NO:1 or variants thereof can be produced following the disclosure of U.S. Patent Appl. Publ. No. 2008/0229514, 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 !D 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 !D 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 intracelluiar!y and obtained from a cell lysate).
A giucoamyiase (EC 3.2.1 .3; aipha-1 ,4-glucan glucohydrolase) can be used in certain embodiments herein as an alpha-glucosidase. For example, a giucoamyiase can be included with a transglucosidase in each of the hydrolysis reaction settings/conditions disclosed herein. In this context, a giucoamyiase can be used to hydroiyze (i) an alpha-1 ,5 glucosyl-fructose linkage, and/or (ii) an alpha-1 ,3 and/or aipha-1 ,8 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 alpha- 1 ,4 and aipha-1 ,6 glycosidic linkages from non-reducing ends of glucose- containing di-, oligo- and poly-saccharides. Glucoamylases as now disclosed herein also have hydrolytic activity toward alpha-1 ,5 glucosyl-fructose linkages. In certain embodiments, an alpha-glucosidase is not a giucoamyiase.
A giucoamyiase 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. alkalophilus, B. amyioiiquefaciens, B. lentus, B. iicheniformis, B. stearothermophiius, B. subWis, B. thuringiensis) and Streptomyces 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. sfrictipilis, T. asperel!um, T, koni!angbra, T. hazianum), Aspergillus species (e.g., A. niger, A. oryzae, A.
terreus, A. clavatus, A. nidulans, A. kawachi, A. awamori), Rhszopus species (e.g., R. oryzae, R. niveus), Taiaromyces species (e.g., T. emersonii, T.
thermophsius, T. duponti), Mucor species, Hypocrea species (e.g., H. geiatsnosa, H. orientaiis, H, vinosa, H. citrina), Fusarium species (e.g., F. oxysporum, F. roseum, F. venenatum), Neurospora species (e.g., N. crassa), Humicoia species (e.g., H. grisea, H. insoiens, H, ianuginose), Peniciilium species (e.g., P.
notatum, P. chrysogenum) and Saccharomycopsis species (e.g., S. fibulsgera). Examples of these bacteria! and fungal glucoamy!ases for use herein are disclosed in U.S. Pat. Appl. Publ. No. 2013/0102035, which is incorporated herein by reference. Other examples of giucoamy!ase enzymes useful herein are described in Svensson et a!. (1983, Carisberg Res. Commun. 48:529-544), Boe! et al. (1984, EMBO J. 3:1097-1102); Hayashida et a!. (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. (1988, Agric. Bio!. Chem. 50:957-964), Ashikari et al. (1989, App!. Microbiol. Biotechnol. 32:129-133), U.S. Pat. No. 4883864; U.S. Pat. No. 4618579,
Houghton-Larsen et al. (2003, Appi. Microbiol. Biotechnol. 82:210-217) and U.S. Pat. No. 7413887, all of which are incorporated herein by reference.
Alternatively, a glucoamyiase 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 glucoamyiase sequences, and have hydrolytic activity toward (i) alpha- 1 ,5 glucosyl-fructose linkages and/or (ii) a!pha-1 ,3 and/or alpha-1.6 g!ucosyl- glucose linkages. All of the foregoing glucoamyiases, when used in a hydrolysis reaction herein, are preferably in a mature form lacking an N-terminal signal peptide. Commercially available glucoamyiases useful herein include OPTIDEX L-400, GC 147, GC 321 , G ZYME G990 4X, OPT!MAX 7525, DEXTROZY E, DISTILLASE and GLUCZY E, for example.
A glucoamyiase enzyme in certain embodiments herein may comprise the amino acid sequence of SEQ ID NO:2 (GC 321 ), which is a T. reesei
glucoamyiase. Alternatively, a glucoamyiase may comprise an amino acid sequence that is at least about 90%, 91 %, 92%, 93%, 94%, 95%, 98%, 97%, 98%, or 99% identical to SEQ ID NO:2 and have hydrolytic activity toward (i) alpha-1 ,5 glucosyl-fructose linkages and/or (ii) alpha-1 ,3 and/or a!pha-1 ,6 glucosyl-glucose linkages. Any of SEQ ID NO:2 or variants thereof can be produced following the disclosures of U.S. Pat. No. 7413887 or U.S. Pat. Appl. PubL No. 2013/0102035, for example, which are incorporated herein by reference. SEQ !D NQ:2 is a mature g!ucoamylase 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 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).
An alpha-glucosidase enzyme herein such as a transglucossdase 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. coll, Bacillus sp.) and eukaryotic systems. Eukaryotic systems can employ yeast (e.g., Pichia sp.,
Saccha myces 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 can be provided by expressing it without using a signal peptide, such as with an expression system where the enzyme is expressed intrace!lular!y and obtained from a cell iysate. 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 alpha- glucosidase 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. cerevisiae), Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kioeckera, and Schvsanniomyces. 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 ,8 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. 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, DPS, 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 iinkages in addition to at least one alpha-1 ,3 or alpha-1 ,6 glucosyi-glucose linkage. For example, there may also be one or more alpha-1 ,5 glucosyl-fructose Iinkages 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 Iinkages. Thus, such oligosaccharides comprise only alpha-1 ,3 glucosyi-glucose and/or alpha-1 , 6 giucosyl-glucose Iinkages. Examples of such an oligosaccharide contain only alpha-1 ,3 Iinkages or alpha-1 , 6 Iinkages. An oligosaccharide can comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% glucosyi-glucose Iinkages in certain embodiments. In other embodiments, there can be about 75-85% alpha-1 ,3 giucosyl-glucose Iinkages and about 15-25% alpha-1 , 6 giucosyl-glucose Iinkages in oligosaccharides herein. Alternatively, oligosaccharides herein can comprise any percentage (any integer value between 1 % and 99%) of alpha-1 ,3 glucosyi-glucose Iinkages and any percentage (any integer value between 1 % and 99%) of alpha-1 , 6 giucosyl- glucose Iinkages, 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 alpha- 1 ,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 oligosaccharides (i.e., average linkage content). Oligosaccharides comprising only glucose monomers linked by alpha-1 ,3 and/or a!pha-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 glucosyl- glucose 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%, 80%, 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). 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 aipha-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 amine- reactive material (e.g., glutaraldehyde) to form an adduct (e.g., enzyme- glutaraldehyde adduct), after which the adduct is bonded to a solid carrier that has been treated with a polyamine (e.g., a polyethy!enimine such as EPO !N P- 1050).
A solid carrier (solid support) to which an alpha-giucosidase 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 amine- reactive material leads to attachment of the enzyme to the solid carrier.
Examples of polyamines useful herein include po!yethylenediamine, a
poiyethylenimine (e.g., polydiethylenetriamine, polytriethyienetetramine, po!ypentaethylenehexamine, polyhexamethylenediamine),
polymethylenedicyclohexylamine, poiymethyienedianiiine,
poiytetraethylenepentamine, polyphenylenediamine and blends of two or more of these polyamine compounds. Preferred polyamines are water-soluble and/or have a molecular weight of about from 500 to 100,000 Daltons. A
polyethylenimine such as EPO IN 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, terephthaldehyde, bis- diazobenzidine-2,2'-disulfon!c acid, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone, diphenyl^^'-dithiocyanate^^'-disuifonic acid, 3-methoxydiphenylmethane-4,4'- diisocyanate, toluene-2-isocyanate-4-isothiocyanate, toluene-2,-4- diisothiocyanate, diazobenzidine, diazobenzidine-3,3'-dianisidirie, Ν,Ν'- hexamethyiene 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 alpha- glucosidase (e.g., transglucosidase) are those conditions that support the hydrolysis of one or more alpha-1 ,3 or alpha-1 ,6 glucosy!-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 saccharide herein with an alpha-glucossdase 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), KH2PQ4, 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. One or more of a!pha-glucosidase enzymes herein may be used in a hydrolysis reaction in certain embodiments. Both a transg!ucosidase and giucoamylase can be used in a reaction, for example. The amount of an alpha- glucosidase 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 alpha- glucosidase can be used in a hydrolysis reaction. Alternatively still, an alpha- g!ucosidase herein can be used at about, or at least about, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 ppm in a hydrolysis reaction. A transg!ucosidase 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 80 °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 wt% (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, 8, 7, 8, 9, 10, 1 1 , 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 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 aipha-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 alpha- glucosidase 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 ,3- 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 g!ucosyltransferase enzyme that produces a poly alpha-glucan such as poly alpha-1 ,3-giucan. Examples of such glucosyltransferase 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 glucosyltransferase enzyme herein may be derived from any microbial source, such as a bacteria or fungus. Examples of bacterial glucosyltransferase enzymes are those derived from a Streptococcus species, Leuconostoc species or Lactobacillus species. Examples of Streptococcus species include S.
sallvarlus, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S.
gailoiyticus and S. sanguinis. Examples of Leuconostoc species include L. mesenteroides, L. amelibiosum, L, argentinum, L. camosum, L. citreum, L.
cremoris, L dextranicum and L. fructosum. Examples of Lactobacillus species include L, acidophilus, L, deibrueckii, L, heiveticus, L. saiivarius, L, cases, L, curvatus, L. plantarurn, L. sakei, L. brevis, L, buchneri, L. fermentum and L. reuteh.
A glucosyltransferase enzyme herein can be primer-independent or primer-dependent. Primer-independent glucosyltransferase enzymes do not require the presence of a primer to perform glucan synthesis. A primer- dependent glucosyltransferase 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 glucosyltransferase 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. 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. co/ 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. coil) 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 0.15- 0.3% (v/v), for example, in a reaction solution for producing a poly alpha-glucan such as poly alpha-1 ,3-giucan 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 sucrose" refers to the sucrose concentration in a gtf reaction solution just after a!i the reaction soiution 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. Appi. 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 Analysis in ICUMSA Methods of Sugar Analysis: Official and Tentative Methods Recommended by the International Commission for Uniform Methods of Sugar Analysis (ICUMSA) (Ed. H.C.S. de Whalley, Elsevier Pub. Co., 1984), 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. Urquhart and M.L. Burge (Determination of the Solution Colour of Raw Sugars, Brown Sugars and Coloured Syru s 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, 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 g!ucan 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 glucan 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
!inear/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 glucan 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 Mn or w 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 DPn or DPW of at least about 100, or of at least about 100 to 1000 (or any integer value between 100 and 1000). 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 alpha- 1 ,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%, 80%, 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 aipha-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 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 alpha- 1 ,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 ef 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 alpha- 1 ,3-glucan synthesis reaction include water, fructose, glucose, one or more types of saccharide (!eucrose 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 leucrose and
oligosaccharides will increase. Conversely, as the temperature increases, the selectivity of the reaction towards leucrose tends to decrease, while the selectivity towards oligosaccharides is largely unaffected. It 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 aipha-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 alpha-g!ucanohydro!ase 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-giycosidic 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 (Pa¾s) 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. 82/004,290.
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 alpha-(1 ,3) and aipha-{1 ,6) glycosidic linkage content embodiments described above, a soluble a!pha-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% alpha-(1 ,6) glycosidic linkages and has a weight-average molecular weight corresponding to 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 % alpha-(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 % alpha-(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 digestibility may alternatively be determined using AOAC 201 1 .25 (Integrated Total Dietary Fiber Assay) (McCleary et a!., 2012, J. AOAC Int., 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. 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% aIpha-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 ; h) a solubility of at least 20% (w/w) in water at 25 °C; and
i) a po!ydispersity 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 alpha-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. 82/004,314.
As another example, a soluble alpha-glucan herein can comprise:
a) a range of:
i) 1 % to 50% of alpha-1 ,3-giycosldic 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; 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-glucan can be produced as disclosed in U.S. Appl.
No. 62/004,305.
In certain embodiments, a soluble alpha-glucan is a direct product of a glucosyltransferase. Such a glucosyltransferase, and conditions for use thereof in a suitable giucan 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-glucan can alternatively be a product, for example, of the concerted action of both a glucosyltransferase and an alpha-glucanohydrolase that is capable of hydrolyzing giucan polymers having one or more alpha-1 ,3- glycosidic linkages or one or more alpha-1 ,6-glycosidic linkages, in some aspects, a giucan synthesis reaction for producing a soluble alpha-glucan product can comprise both at least one glucosyltransferase and at least one alpha-glucanohydrolase. In other aspects, a giucan synthesis reaction can initially comprise one or more glucosyltransferases as the only enzyme component(s). Such a reaction produces a first alpha-glucan 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-glucan product. Thus, there are different ways by which to synthesize a soluble alpha-glucan product through the concerted action of both a glucosyltransferase and an alpha- glucanohydrolase. Conditions for performing a giucan synthesis reaction in which one or more alpha-glucanohydrolase enzymes are included during giucan synthesis reaction and/or after giucan 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. An a!pha-g!ucanohydro!ase herein can be, for example, a dextranase (capable of hydrolyzing a!pha-1 ,6-linked glycosidic bonds; E.G. 3.2,1.1 1 ), a mutanase (capable of hydrolyzing aipha-1 ,3-Iinked glycosidic bonds; E.G.
3.2.1 .59), a mycodextranase (capable of endohydrolysis of (1 -4)-alpha-D- glucosldlc 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 endohydrolytically cleaving alternan; E.G. 3.2.1 .-; see U.S. Patent No. 5786196).
A mutanase comprising SEQ ID NO: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 alpha-glucan products can serve directly as suitable conditions in which to perform a hydrolysis reaction herein in which an alpha-giucosidase 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 ,3- glucan, for example. Alternatively, a fraction (e.g., chromatographic fraction) of a glucan synthesis reaction for producing one or more soluble alpha-glucan products can be used as suitable conditions in which to perform alpha- glucosidase-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 alpha-glucan products as disclosed herein. Such a reaction can optionally include one or more alpha-glucanohydrolases 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 60%, 70%, 80%, 90%, 95%), a soluble alpha-glucan product from a reaction composition from which it was produced. Once separated from all or most of a soluble alpha-g!ucan product, a fraction can be subjected to any of the alpha-1 ,5 glucosy!-fructose hydrolysis processes disclosed herein using one or more alpha-glucanases.
A chromatographic fraction herein can typica!iy be obtained using a suitable type of liquid chromatography. Liquid chromatography can be performed using size-exclusion chromatography (SEC), column chromatography, high- performance 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 alpha- glucosidase 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 hydro!yzed filtrate from a g!ucan synthesis reaction, or a hydrolyzed fraction of a glucan synthesis reaction used to produce soluble aipha-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 can comprise an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 98%, 97%, 98%, or 99% identical to SEQ ID NO:5, 6, 8, 9, 1 1 , 12, 14, 15, 17, 18, 20, 22, 24, 28, 28, 30, 32, 34, 38, 38, or that of DIAZY E 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. 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 alpha- glucosidase. 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 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).
The features of the disclosed fructose enrichment method regarding a!pha-glucosidase (e.g., transg!ucosidase) enzymes, and fractions of a g!ucan 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 aipha-1 ,5 glucosyi-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. The features of the disclosed fermentation method regarding alpha- g!ucosidase (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. coii) and Bacillus species. Examples of yeast useful herein include Saccharomyces species such as S. cerevisiae and S. bayanus,
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- ansi 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 non- hydrolyzed 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. 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. cerevisiae) 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 glucoamyiase) while simultaneously fermenting the leucrose with a microbe.
Non-limiting examples of compositions and methods disclosed herein include:
1 . A method of hydrolyzing an aipha-1 ,3 or alpha-1 ,6 giucosyl-glucose
linkage in a saccharide comprising at least one alpha-1 ,3 or alpha-1 , 6 giucosyl-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-g!ucosidase 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.
The method of embodiment 1 , wherein the alpha-glucosidase enzyme is immobilized,
The method of embodiment 1 or 2, wherein the degree of polymerization of the saccharide before hydrolysis is 3 to 7.
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,
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. The method of embodiment 5, wherein the glucan synthesis reaction produces at least one insoluble alpha-glucan product.
The method of embodiment 6, wherein the fraction is a filtrate of the glucan synthesis reaction.
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 aipha-1 ,3-glycosidic linkages or one or more alpha-1 ,6- glycosidic linkages.
The method of embodiment 8, wherein the fraction is a chromatographic fraction of the glucan synthesis reaction. The method of any one of embodiments 1-9, wherein the alpha- glucosidase enzyme is a transg!ucosidase.
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 aipha-1 ,6 glucosyl-glucose linkage, wherein the enzyme hydro!yzes at least one aipha-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.
The composition of embodiment 1 1 , wherein the degree of polymerization of the saccharide before hydrolysis is 3 to 7.
The composition of embodiment 1 1 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. 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). 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 alpha-1 ,6 glucosyl-glucose 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 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 it 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 milliliters), "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 millimeters), "mL/min" means milliliters per minute, "m" means meter(s), "μί_" means microliter(s), "mmol" means millimoie(s), "min" means minute(s), "mol%" means mole percent, "M" means molar, "mg/g" means milligram per gram, "rpm" means revolutions per minute, " Pa" means megaPascais.
GENERAL METHODS
All reagents were obtained from Sigma-Aidrich (St. Louis, MO) unless stated otherwise. Sucrose was obtained from VWR (Radnor, PA). Preparation of Crude Extracts of G lucosyltransf erase (qtf) Enzymes
The Streptococcus salivahus gtfj enzyme (SEQ ID NO:3) was expressed in E. cols' strain DH10B using an isopropyl beta-D-1 -thiogaiactopyranoside
(!PTG)-induced expression system. SEQ ID NO:3 has an N-terminal 42-residue deletion compared to the S. salivahus gtfj amino acid sequence in GENBANK Identification No. 47527, but includes a start methionine. Briefly, E. coli DH10B cells were transformed to express SEQ ID NO:3 from a DNA sequence codon- optimized 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 800nm) 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 OD6oo of 0.8-1 .0. Induced cultures were left on the shaker and harvested 3 hours post induction.
Gtfj enzyme (SEQ !D NO:3) 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 cent ifuged at 16000 rpm at 4 °C to pellet the unbroken cells and cell debris. The crude extract (containing soluble Gtfj enzyme, SEQ ID NO:3) was separated from the pellet and analyzed by Bradford protein assay to determine protein concentration (mg/rrsL).
The Streptococcus sp. C150 gtf-S enzyme (SEQ ID NO:40) was prepared as follows. SG1 184 is a Bacillus subtiiis expression strain that expresses a truncated version of the glycosyltransferase Gtf-S ("GTF0459") from
Streptococcus sp.C150 (GENBANK® G!:S21278321 ). The gene (SEQ ID NO:41 ) encoding an N-terminal truncated protein GTF0459 (SEQ ID NO:42) from E. coli expression p!asmid pMP79 was cloned into the Nhel and Hindi!! sites of the Bacillus subtiiis integrative expression plasmid p4JH under the aprE promoter and fused with the B. subtiiis AprE signal peptide on the vector. The construct was first transformed into E. coli DH10B and selected on LB with ampici!lin (100 plates. The confirmed construct pDCQ984 expressing GTF0459 was then transformed into β. subtilis BG6006 containing nine protease deletions (amyE :xylRPxylAcomK-ermC, degUHy32, oppA, AspollE3501 , AaprE, AnprE, Aepr, AispA, Abpr, Avpr, AwprA, Ampr-ybfJ, An rB) and selected on LB plates with chloramphenicol (5 p.g/mL). The colonies grown on LB plates wi
chloramphenicol were streaked several times onto LB plates with 25
chloramphenicol. The resulting B. subtilis expression strain, SG1 184, was first grown in LB medium with 25 ug/mL chloramphenicol and then subcultured into Grants! I medium containing 25 μ9 Γηί 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-μηι filters. The filtered supernatant was aliquoted and frozen at -80 °C.
B. subtilis SG1 184 strain, expressing GTF0459 (SEQ 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 (SEQ ID NO:42) was then stored frozen at -80 °C.
The S, mutans MT-4239 gtf-C enzyme (SEQ ID NO:43) was prepared as follows. A gene encoding a truncated version of a glucosyitransferase (gtf) enzyme identified in GENBANK® as Gl:3130088 (SEQ ID NO:43; gtf-C from S. mutans MT-4239) was synthesized using codons optimized for expression in Bacillus subtilis and synthesized by GenScript. The gene (SEQ !D NO:44) encoding GTF0088BsT1 with an N-terminal truncation and a C-terminal T1 truncation (SEQ ID NO:45) was amplified from the GENSCRIPT plasmid and cloned into the Nhel and Hindi!! 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 plates. The confirmed construct pDCQ1021 expressing GTF0088BsT1 was 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 the LB plates with chloramphenicol (5 p.g/mL). The colonies grown on LB plates wi
chloramphenicol were streaked several times onto LB plates with 25
chloramphenicol. The resulting B. subtilis expression strain SG1221 was first grown in LB medium with 25 ug/mL chloramphenicol and then subcultured into Grantsl! medium containing 25 μ9 ίηί 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-μηι filters. The filtered supernatant was aliquoted and frozen at -80 °C.
B. subtilis SG1221 strain, expressing GTF0088BsT1 (SEQ ID NG: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, FOAMBLAST 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, 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 GTFQ088BsT1 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 m sodium acetate buffer at pH 5.5 in the presence or absence of 25 g/L dextran (MW -1500, Sigma-Aldrich, 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 AM IN EX HPX-87C columns series at 85 °C (Bio- Rad, 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)-Glucanohydroiase (rnutanase) A gene encoding the Penicillium marneffei ATCC^ 18224™ rnutanase identified in GEN BANK® as G!:212533325 was synthesized by GenScript (Piscataway, NJ). The nucleotide sequence (SEQ ID NO:46) encoding protein sequence (MUT3325; SEQ ID NO:47) was subcloned into p!asmid pTrex3 at Sacii and AscI restriction sites, a vector designed to express the gene of interest in Trichoderma reesei, under control of CBHI promoter and terminator, with Aspergillus niger acetamidase for selection. The resulting p!asmid was transformed into T. reesei by biolistic injection. The detailed method of biolistic transformation is described in International PCT Patent Application Publication WO2009/126773 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 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 eel! mass was first removed by centrifugation at 4000g for 10 min and the supernatant was filtered through 0.2-μιη sterile filters. The expression of mutanase MUT3325 (SEQ ID NO:47) was confirmed by SDS-PAGE.
The production media component is listed below.
NREL-Trich Lactose Defined
MnS04.H20 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, 0.008 g/L cupric sulfate pentahydrate, 0.0035 g/L manganese sulfate
monohydraie 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 tol OOO 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 giucose-sophorose solids / g biomass / hr. At the end of run, the biomass was removed by centrifugation and the supernatant containing the MUT3325 mutanase (SEQ 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 Chaifont, Buckinghamshire, UK). The concentrated protein was stored at -80 °C. Determination of Aipha-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-μΓη 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, -10 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 p-hydroxybenzoic acid hydrazide solution (PAH BAH) 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 μί of reaction sample supernatant to 100 pL of PAHBAH working solution and heated at 95 °C for 5 min. The working solution was prepared by mixing one part of reagent A (0.05 g/mL p- hydroxy 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® HP- 87C 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 Aminex¾' 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® UltiMate1 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 (PFG) 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 Ή 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 μ3. Sample temperature was maintained at 25 °C. Samples were prepared by adding 50 μί to a 5-mm NMR tube along with 450 μΐ of D2O and 60 μΐ of D2O containing 12.4 mM DSS (4,4-dimethyi-4~siiapentane-1 -sulfonic acid sodium salt) interna! 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 4.85 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 Fermasure ! M (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 Genera! 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 poiy- alpha-1 ,3-giucan 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 40- 50 °C) to a total sugar concentration of about 320 g/L sugars. The composition of the concentrated filtrate is provided in Table 2. Table 2
Composition of a Concentrated Filtrate of a G!ucan Synthesis Reaction
Table 2 indicates that the concentrated filtrate of the glucan synthesis 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 g!ucoamylase (EC 3.2.1.3), transglucosidase (EC 2.4.1.24), beta-glucossdase (EC 3.2.1 .21 ), alpha-amyiase (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 DIAZY E RDF ULTRA, transglucosidase (EC 2.4.1 .24) and glucoamy!ase (EC 3.2.1.3), which are all alpha-giucosidase, 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
Com position of a Concentrated Filtrate of a G I u ca n S nth es is Reaction 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 [aipha(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
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 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 Fsitrate by
Various Enzymes
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 a!pha-1 ,3 linkages, when produced using certain gtf enzymes.
Table 5 indicates that 1 ,4-aipha-glucosidase and 1 ,6-aIpha-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 puilulanase (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 2.1 1 ). 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 beta- glucosidase suggests that the presence of other enzymes in the tested beta- glucosidase formulations, such as glucoamylase or another type of alpha- glucosidase, could be responsible for the observed activity.
Conversely, the results in Table 5 indicate that transglucosidase (TG L- 2000, Example 2.8) 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 activity (Example 2.8), indicating that the observed hydrolysis is due to the transglucosidase enzyme and not background activity.
Giucoamylases (Examples 2.16-2.18) showed a range of activity against ieucrose and the oligosaccharides. Only one tested glucoamyiase (Example 2.18) gave less than 30% hydrolysis of both the Ieucrose and oligosaccharides.
The results in Table 5 indicate that alpha-glucosidases such as DIAZY E RDF ULTRA, glucoamyiase and transglucosidase can hydrolyze Ieucrose byproduct present in a glucan reaction filtrate. The ability of alpha-glucosidases to hydrolyze Ieucrose indicates that these enzymes can hydrolyze alpha-1 ,5 glucosyl-fructose linkages. While this activity was shown above using Ieucrose 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 glucoamyiase 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 aipha-1 ,8 glucosyl- glucose linkages.
Since alpha-glucosidase enzymes were generally effective at hydrolyzing the Ieucrose 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 Ieucrose hydrolysis, and a
glucoamyiase (e.g., GC 321 ) enzyme that efficiently hydrolyzes oligosaccharide byproducts.
Thus, alpha-glucosidase enzymes can individually hydrolyze (i) alpha-1 ,5 glucosyl-fructose linkages and (ii) alpha-1 , 3 and alpha-1 , 6 glucosyl-glucose linkages in certain saccharides. EXAMPLE 3
Comparison of Linkage Distributions of Glucan Reaction Fi!trate Components before an d after Enzyme H yd rolysis
This example measures the hydrolytic activity of transg!ucosidase (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 glucan synthesis reaction comprise >90% glucose-glucose linkages, as determined by NMR (General Methods). Of the giucose-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 2.1 1 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 NO:1 ) transglucosidase (Figure 2). 210 μL· of concentrated filtrate from the material in Table 3, 300 iiL of D2O, and 90 μί» of D20 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 60 °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 re- equilibrated 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 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 a!pha-1 ,3 and a!pha-1 ,8 linkages into glucose and can convert leucrose into fructose and glucose. Thus, transglucosidase can hydrolyze (i) alpha-1 ,5 glucosyl-fructose linkages and (ii) alpha-1 ,3 and aipha-1 ,6 glucosyl-giucose 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 GC- 147 (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 6-7 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 polyethy!enimine (PEL EPO N P-1050). 3500 rrsL 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-giutaraldehyde 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-g!utaraldehyde adduct containing treated transglucosidase.
Each of the treated enzyme-giutaraldehyde 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 ) immobilized glucoamyiase, 2) immobilized transglucosidase, and 3) immobilized g!ucoamylase followed by immobilized transglucosidase.
Table 6
Application of Immobilized Glucoamylase and Transglucosidase Enzymes to
Hydrolyze Oligosaccharides and Leucrose
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 glucoamyiase and transglucosidase gave the highest hydrolysis of oligosaccharides.
Thus, use of an immobilized glucoamyiase or transglucosidase, or both types of immobilized enzymes, represents an effective technique to hydrolyze oligosaccharides containing aipha-1 ,3 and alpha-1 ,6 glucosy!-glucose linkages, as well as leucrose. These results are consistent with those of Example 2. Immobilization of other aipha-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. 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
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
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 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, afterwhsch the column was eluted using water at a flow rate of 30 L/hr.
Table 8
Physical Parameters of the Column
In this separation, leucrose remained in the column longer than sucrose, perhaps due to complexation of leucrose with the calcium cation, and in fact, co- 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
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 leucrose 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 vol% of transglucosidase TG L-2000 (SEQ ID NO: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 monosaccharides. The syrup had the final product distribution described in Table A.
Table A
Composition of a Concentrated Giucan Filtrate that Was Hydrolyzed before
Concentration
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
Two fractions containing fructose were isolated. Fraction 6.1 eluted between 73 and 103 minutes, and fraction 8.2 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 8.2 in >90% purity. The product distribution in each fraction (6.1 and 6.2) as measured by HPLC appears in Table C. Table C
Product Distribution of Chromatographic Fractions Containing Fructose from a
Hyd rolyzed G I u can Filtrate
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 g!ucan 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 (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 884 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 PAL- 3 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
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 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 are provided in Tables 1 1 and 12, respectively.
Table 11
Feed and Time Course Fermentation Profiles Using the First Recycle of Yeast
Cells
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
Colls
6 <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 leucrose was consumed in the first fermentation, although the yeast cells started to acclimate and consume leucrose 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 leucrose were present in the medium, even after the last cycle.
Thus, giucan filtrate can be used in a fermentation process to produce ethanol.
EXAMPLE 8
Production of Ethanol by Fermenting Hydrolyzed Giucan Filtrate
This example demonstrates that fermenting a giucan filtrate in which the leucrose 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 giucan filtrate that was previously treated with a transglucosidase (TG L-2000, SEQ ID NO:1 ). Hydrolyzed giucan filtrate was prepared as follows. Giucan 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 (IKA KS4000) at 60 °C and 100 rpm for 72 hours. Hydrolyzed giucan filtrate was thus prepared.
Yeast (S. cerevisiae) cream (Bom Retiro mill, Brazil) was washed by suspending the cream in tap water (2.4 L, optical density of 65 at 600 rim) and then centrifuging the yeast cream for 5 minutes using a LEGEND XTR centrifuge 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 BIOFL0310 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 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 PAL- 3 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
Concentrations (g/L) of ethanoi (EtOH) and sugar compounds in the feed and at various fermentation time points (0-22 hours) are iisted.
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 Glucan Filtrate
Concentrations (g/L) of ethanol (EtOH) and sugar compounds in the feed and at various fermentation time points (0-21 hours) are Iisted. Table 15
Feed and Time Course Fermentation Profiles Using the Second Recycle of Yeast
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 hydroiyzing 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 g!ucan 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 transg!ucosidase, 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. cerevisiae) cream (Bom Retiro mill, Brazil) was washed by suspending the cream in tap water (2.4 L, optical density of 85 at 600 nm) and 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 centnfugation 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 g!ucan 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 884 mL/hr, and the fermentation was allowed to proceed for 48 hours.
Periodic samples were taken during the fermentation and analyzed for optica! density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL- 3 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
5 <1 14 <1 2 - - - 45
6 <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 ethano! (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 fermentations where the filtrate was hydrolyzed prior to the fermentation step (Example 8), and gave a slightly superior titer of ethanol (82 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
ration
This example discloses preparing various alpha-glucosidases in addition to those alpha-glucosidases (transglucosidase, glucoamylase, DIAZY E RDF ULTRA) used in some of the foregoing Examples. These additional alpha- glucosidases were tested for hydroiytic activity against oligosaccharides comprising alpha-1 ,5 glucosyl-fructose linkages or alpha-1 ,3 and/or alpha-1 ,8 glucosyl-glucose linkages in Examples 1 1 , 12, 15 and 16 provided below. Discovery of : an Aspergillus ciavatus alpha- lucQsidase (Aclgiul)
A strain of Aspergillus ciavatus was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Aspergillus ciavatus encodes an alpha-glucosidase and the sequence of this gene, called "AcigluT', is provided in SEQ ID NO:4. The corresponding protein encoded by SEQ ID NQ:4 is provided in SEQ ID NO:5. Aclgiul belongs to Glycosyl hydrolase family 31 based on a PFA 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 et al., 201 1 , Nature Methods, 8:785-786). The presence of a signal sequence suggests that Aclgiul is a secreted enzyme.__The amino acid sequence of the predicted mature form of Aclgiul is set forth as SEQ ID NO:8.
Expression of Aspergillus ciavatus alpha-glucosidase Aclgiul
A synthetic Aclgiul gene was cloned into pTrexSgM expression vector
(described in U.S. Patent Appl. Publ. No. 201 1/0136197, incorporated herein by reference) and the resulting plasmid was designated as pJG294. The sequence of the Aclgiul gene was confirmed by DNA sequencing.
Plasmid pJG294 was transformed into a quad deleted Trichoderma reesei strain (described in WO05/001038) 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 NO:8, 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 Neosariorya fischeri alpha-glucosidase Nfiqiul
A strain of Neosariorya fischeri was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Neosariorya 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. Nfiglul belongs to G!ycosy! hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEO ID NO:8) has a signal peptide with a length of 19 amino acids as predicted by SignalP version 4.0 (Nordah! Petersen et al., 201 1 , Nature Methods, 8:785-788). The presence of a signal sequence suggests that Nfiglul is a secreted enzyme. The amino acid sequence of the predicted mature form of Nfiglul is set forth as SEQ ID NO: 9.
Expression of Neosartorya fischeri alpha-glucosidase Nfiglul
A synthetic Nfiglul gene was cloned into pTrexSg expression vector (described in U.S. Patent Appl. Publ. No. 201 1/0136197) and the resulting piasmid was designated as pJG295. The sequence of the Nfiglul gene was confirmed by DNA sequencing.
Piasmid pJG295 was transformed into a quad deleted Trichoderrna reesei strain (described in WO05/001036) 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 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 Ncrglui
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 "Ncrglui", is provided in SEQ ID NO:10. The corresponding protein encoded by SEQ ID NO: 10 is provided in SEQ ID NO:1 1 . Ncrglui 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 et al., 201 1 , Nature Methods, 8:785-786). The presence of a signal sequence suggests that Ncrglui is a secreted enzyme. The amino acid sequence of the predicted mature form of Ncrglul is set forth as SEQ ID NO:12.
Exp ress i o n of Neurospora crassa a I p h a -q i u cos i d ase N erg I u 1
A synthetic Ncrglul gene was cloned into pTrexSgM expression vector (described in U.S. Patent Appl. Publ. No. 201 1/0136197) and the resulting plasmid was designated as pJG296. The sequence of the Ncrglul gene was confirmed by DNA sequencing.
Plasmid pJG298 was transformed into a quad deleted Trichoderma reesei strain (described in WO05/001036) using a biolistic method (Te'o VS et 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 cornposticola alpha-glucosidase Tau8ec098
A strain of Rasamsonia cornposticola was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Rasamsonia cornposticola encodes an alpha-glucosidase and the sequence of this gene, called aTauSec098", is provided in SEQ ID NO: 13. The corresponding protein encoded by SEQ ID NO: 13 is provided in SEQ ID NO: 14. TauSec098 belongs to Glycosyl hydrolase family 31 and contains an N-terminal CBM 20 domain based on a PFA 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 et ai, 201 1 , Nature Methods, 8:785-786). The presence of a signal sequence suggests that TauSec098 is a secreted enzyme. The amino acid sequence of the predicted mature form of TauSec098 is set forth as SEQ ID NO:15.
Expression of Rasamsonia cornposticola alpha-glucosidase TauSec098
A synthetic TauSec098 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 pGX256- TauSec098. The sequence of the TauSec098 gene was confirmed by DNA sequencing.
Plasmid pGX258-TauSec098 was transformed into a quad-deleted
Trichoderma reesei strain (described in WO05/001036) 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 (Π) sulfate 5 mg/L; zinc sulfate 1 .4 mg/L; cobalt (I!) chloride 1 mg/L; manganese (N) 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 *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 TauSec098 enzyme (SEQ ID NO: 15). Discovery of Rasamsonia composticola alpha-giucosidase TauSec099
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 "TauSec099", is provided in SEQ ID NO: 16. The corresponding protein encoded by SEQ ID NO: 16 is provided in
SEQ ID NO: 17. TauSec099 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 a!., 201 1 , Nature Methods, 8:785-786). The presence of a signal sequence suggests that TauSec099 is a secreted enzyme. The amino acid sequence of the predicted mature form of TauSec099 is set forth as SEQ ID NO: 18.
Expression of Rasamsoma composticoia alpha -qlucosidase TauSec099
A synthetic TauSecQQQ 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 pGX256- TauSec099. The sequence of the TauSec0998 gene was confirmed by DNA sequencing.
Plasmid pGX256-TauSec099 was transformed into a quad-deleted
Trichoderma reesei strain (described in WO05/001036) using protoplast transformation (Te'o et a!., 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 (li) 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 ><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 TauSec099 enzyme (SEQ ID NO: 18). Sequences of Bifidobacterium longum aipha-glucosidase BloGiul
An aipha-glucosidase gene, "BloGiul", was identified from Bifidobacterium longum subsp. longum JDIV1301 . The nucleic acid sequence for the BloGiul gene (SEQ ID NO:19, GENBANK Acc. 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 . Expression of Bifidobacterium ionqum alpha-glucosidase BioGiul
The DNA sequence encoding the entire BloG!ul protein (SEQ ID NO:20) was optimized for expression in B. subtiiis, then synthesized (yielding SEQ ID NO:21 ) and inserted into the p3JM plasmid by Generay Biotech Co. (Shanghai, China), resulting in p3JM-BloGlu1 . The p3JM-BioGlu1 plasmid contains an aprE promoter to drive expression of the optimized BioGiul sequence (SEQ ID
NO:21 ).
Plasmid p3JM-BloGlu1 was used to transform β. 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 CaC ) to express BioGiul protein (SEQ ID NO:20).
Sequences of Bifidobacterium ionqum alpha-qlucosidase BloGiu2
An alpha-glucosidase, B!oGlu2, was identified from Bifidobacterium longum. The amino acid sequence (SEQ ID NO:22) of BloGlu2 was found in the
NCBI database (GENBANK Acc. No. WP_007054665.1 ).
Expression of Bifidobacterium Ionqum 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-BioGlu2. 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 BioGiu2 sequence (SEQ ID NO:23).
Plasmid p3JM-BloG!u2 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 CaC ) to express BloG!u2 protein (SEQ ID NO:24).
Sequences of Bifidobacterium lonqum alpha-q!ucosidase BioGluS
An aipha-glucosidase gene, "BloGiu3", was identified from Bifidobacterium iongum subsp. !ongum F8. The nucleic acid sequence for the BioGluS gene (SEQ ID NO:25, GENBANK Acc. No. NC_021008.1 , positions 2130827 to 2132441 ), and the amino acid sequence of the hypothetical protein (SEQ ID NO:26) encoded by SEQ ID NO:25 were found in GENBANK Acc. No.
YPJ307768249.1 .
Expression of Bifidobacterium lonqum alpha-giucosidase BioGluS
The DNA sequence encoding the entire BioGluS protein (SEQ ID NO:28) was optimized for expression in B. subtilis, then synthesized (yielding SEQ ID NO:27) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in p3JM-BioGlu3. The p3JM-B!oGlu3 plasmid contains an aprE promoter to drive expression of the optimized BioGluS sequence (SEQ ID NO:27).
Plasmid p3JM-BloGlu3 was used to transform β. subtiiis cells (degUHy32, AnprB, Avpr, Aepr, AscoC, AwprA, Arnpr, 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 BloGlu3 protein (SEQ ID NO:28).
Sequences of Bifidobacterium pseudoion um alpha-glucosidase BpsGlul
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 pseudoionqum a!pha-q!ucosidase BpsGlul
A DNA sequence encoding BpsGlul protein was optimized for expression in B, subtiiis, then synthesized (yielding SEQ ID NO:29) and inserted into the 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 p3J -BpsGlu1 plasmid contains an aprE promoter to drive expression of the optimized BpsGlul sequence (SEQ ID NO:29)
Plasmid p3JM-BpsGIu1 was used to transform B. subtilis cells
(degUHy32, ΔηρπΒ, 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 PGR 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 NO:30).
Sequences of Bifidobacterium thermophiium alpha-qlucosidase BthGlul
An alpha-glucosidase gene, "BthGlul", was identified from Bifidobacterium thermophiium RBL67. The nucleic acid sequence of the BthGlul gene (SEQ D 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 NQ:31 were found in GENBANK Acc. No. YP_007592840.1.
Expression of Bifidobacterium■ thermophiium alpha-q lucosidase 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 NQ:33) and inserted into the p3JM plasmid by Generay Biotech Co., resulting in p3JM-BthGiu1. The p3JM-BthGlu1 plasmid contains an aprE promoter to drive expression of the optimized BthGlul sequence (SEQ ID NO:33).
Plasmid p3JM-BthGlu1 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 PGR 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 BthGlul protein (SEQ ID NO:32). Sequences of Bifidobacterium hreve 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. WPJ303827971 .1 ).
Expression of Bifidobacterium breve alpha-glucosidase BbrGlu2
A DNA sequence encoding BbrGlu2 protein was optimized for expression in B, subtiiis, then synthesized (yielding SEQ D NO:35) and inserted into the p3J plasmid by Generay Biotech Co., resulting in p3JM-BbrGlu2. SEQ ID NG:3S encodes the amino acid sequence of SEQ ID NQ:36. The p3JM-BbrGlu2 plasmid contains an aprE promoter to drive expression of the optimized BbrGlu2 sequence (SEQ ID NO:35)
Plasmid p3JM-BbrGlu2 was used to transform B. subtiiis ceils (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 MQPS-based defined medium supplemented with an additional 5 mM
CaCI2) to express SEQ ID NO:36.
Sequences of Bifidobacterium breve alpha-glucosidase BbrGluS
An alpha-glucosidase gene, "BbrGluS", was identified from
Bifidobacterium breve ACS-G71 -V-Sch8b. The nucleic acid sequence of the
BbrGluS 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. YP_005583701 .1 .
Expression of Bifidobacterium breve alpha-glucosidase BbrGluS
The DNA sequence encoding the entire BbrGluS protein (SEQ ID NQ:38) was optimized for expression in B. subtiiis, then synthesized (yielding SEQ ID
NO:39) and inserted into the p3J plasmid by Generay Biotech Co., resulting in p3JM-BbrGIu5. The p3JM-BbrGlu5 plasmid contains an aprE promoter to drive expression of the optimized BbrGluS sequence (SEQ ID NO:39).
Plasmid p3J -BbrGlu5 was used to transform B. suhtilis ceils (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 PGR and sequencing, was selected and subjected to fermentation in a 250-mL shake flask with BD medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCI2) to express BbrGluS protein (SEQ ID NO:38).
Purification of alpha-glucosidases from expression cultures
AclGlui and NcrGlul
Both AclGlui (SEO ID NO:8) and NcrGlul (SEQ ID NO:12) alpha- glucosidases 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 (SEQ 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 pre- equilibrated 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.
NfiGlui
NfiGlul alpha-giucosidase (SEQ 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 pre-equi!ibraied with 20 m 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 eiuted from the column with 1 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.
TauSec098 and TauSec099
Both TauSec098 (SEQ ID NO:15) and TauSec099 (SEQ ID NO:18) alpha- glucosidases 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 ferrnenter to a final concentration of 1 M. This solution was then loaded onto a 50-mL HIPREP phenyi-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 eiuted stepwise with 75%, 50% and 0% buffer A using three CVs each, followed by two CVs of M!LLIQ H2O. All fractions were analyzed by SDS-PAGE. The target protein (SEQ ID NO: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 AMICON 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 NO:20), BloGlu2 (SEQ ID NO:24) and B!oGluS (SEQ ID
NO:26) alpha-glucosidases were all purified in three steps. For each purification, the crude broth from a 1 -L DASGIP ferrnenter 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 loaded onto a 40-mL HiPrep l M Phenyl FF column pre-equi!ibrated 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 H20 in three column volumes each. All fractions were analyzed using SDS-PAGE and activity assays. The fractions containing target protein (SEQ ID NO:20, SEQ ID NQ:24, or SEQ ID NO:28) 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 AMICQN ULTRA-15 devices, and stored in 40% glycerol at -20 °C until usage.
BpsGlui and BthGlul
Both BpsGlui (SEQ ID NO:30) and BthGlul (SEQ ID NG:32) alpha- glucosidases were purified in two 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 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 H20 in three column volumes each. All fractions were analyzed using SDS-PAGE and activity assays. The target protein (SEQ ID NO:30 or SEQ ID NO:32) was present in the eluate from the 0% buffer B elution step; this eluate was pooled and concentrated using 10K
A ICON 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 (SEQ ID NO:36) and BbrGluS (SEQ ID NQ:38) alpha- g!ucosidases were purified in four steps. For each purification, the crude broth from a 1-L DASGIP fermente 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 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 ^'1 Phenyl FF column pre-equilibrated with 20 mM HEPES pH 7.0, 1 M ammonium sulfate. The target protein (SEQ ID NO:36 or SEQ ID NO: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 VIVAFLOW 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to a HiPrep l M Q 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 AMICON 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 hydro!ytic activity against alpha-1 ,5 glucosyl-fructose linkages and alpha-1 ,3 and/or alpha-1 ,6 g!ucosyl-glucose linkages in Examples 1 1 , 12, 15 and 16 provided below.
EXAMPLE 1 1
Testing Alpha-Giucosidases for Hydrolvtic Activity Against Various Glycosidic
This example discloses testing whether alpha-glucosidases have hydro!ytic 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 glucosyl-fructose linkages and alpha-1 ,3 and alpha-1 ,6 glucosy!-glucose linkages.
Substrate Specificity of Alpha-Gliicosidases
The substrate specificity of each aipha-glucosidase disclosed in Example 10 was assayed based on the release of glucose from a particular substrate (isomaltose, maltose, panose, leucrose, or nigerose) when the substrate was 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-ethylbenzothiazo!lne-8-su!fonic 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 μί. of 0.5 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-glucosidase samples and glucose standard were prepared in MILLIQ water. For nigerose, alpha- glucosidase samples were tested with only one dosage at 10 ppm due to a limited stock of substrate solutions. Each alpha-glucosidase sample (10 pL) was transferred into a new microtiter plate (Corning 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 μί of each reaction mix, as well as 10 L 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 1 1 second intervals for 5 min using a SOFTMAX PRO plate reader (Molecular Devices). The output was the reaction rate, V0, for each enzyme concentration. Linear regression was used to determine the slope of the plot V0 vs. enzyme dose. The specific activity of each alpha-glucosidase 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 mol glucose / min. 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 ransglucosidase (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
Each enzyme was used at one dosage (10 ppm) against nigerose.
Interestingly, it was found that alpha-giucosidases, besides exhibiting hydrolytic activity against alpha-1 ,4 giucosyl-glucose linkage (maltose), also exhibit hydrolytic activity against alpha-1 ,6 giucosyl-glucose linkage (isomaltose), alpha-1 ,3 glucosyl-glucose linkage (nigerose), and aipha-1 ,5 glucosyl-fructose linkage (leucrose) (Table 17).
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 ,6 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, DP3 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 Alpha- Glucosidase Activit
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 CS11 GC, #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 convert the resin to the sodium form. The sugar solution (0.6 L) was then fed to the column, after which the column was eluted using water at a flow rate of 50 mL/min. The run conditions of the chromatographic separation are summarized as follows: 0.8 L feed size, 25 g dry solids/100 g solution, 65 °C column temperature, 50 mL/min flow rate. An oligosaccharide solution was eluted between 11 and 21 minutes. A small amount of salts - indicated by an increase in conductivity - was eluted at the same time. The oligosaccharide fraction thus prepared was analyzed by HPLC to determine its product distribution. In total, the fraction contained >89% of oligosaccharides containing three or more hexose units and less than 1.5% of identifiable mono- and di-saccharides. This fraction was concentrated to a total dry weight of 317 g/L using a thin film evaporator (LCI Corporation, Charlotte, NC) followed by rotary evaporation with a ROTAVAPOR (R-151 ; Buchi, New Castle, DE). The product distribution of the concentrated fraction as measured by HPLC appears in Table 18.
Table 18
Product Distribution of Concentrated Oligosaccharide Fraction
Primary Screening of Alpha-Giucosidases on Giucan Oligomer Hydrolysis
The activities of eleven different alpha-glucosidases (Example 10), as well 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 50 ML 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 series HPLC system with an AMINEX HPX-42A column (300 mm x 7.8 mm) at 85 °C. The sample (10 μΐ) was applied to the HPLC column and separated with an isocratic gradient of MILL!-Q water as the mobile phase at a flow rate of 0.6 mL/min. Oligosaccharide products were detected using a refractive index detector. The numbers provided in Table 19 below reflect the average of peak area percentages (from duplication of each sample) of each DPn as a fraction of the total from DP1 to DP7.
Table 19
Analysis Glucan Filtrate Oligosaccharides Following Treatment with Alpha-
Glucosidase
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 aipha-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 alpha- glucosidase against various glycosidic linkages in addition to alpha-1.4 linkages (Example 1 1 ), it is apparent that alpha-glucosidase can be used to break down oligosaccharides with alpha-1 ,5 glucosyl-fructose linkages and/or alpha-1 ,3 and a!pha-1 ,6 glucosyl-g!ucose linkages. The results provided in Table 19 also suggest that fungal alpha-glucosidases have better hydrolytic activity towards soluble oligosaccharides compared with the bacterial alpha-glucosidases.
Confirmation of Alpha-Glucosidase Hydrolytic Activity Toward Oligosaccharide 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 μί. 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 AM!NEX HPX-42A column (300 mm x 7.8 mm) at 85 °C. The sample (10 μί) was applied to the HPLC column and separated with an isocratic gradient of MILLI-Q water as the mobile phase at a flow rate of 0.6 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. Thus, a!pha-g!ucosidase can be used to hydro!yze 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 Oligomer/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 (GTFG459, 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 I EX/SEC column chromatography using the following resin and conditions: FINEX CS 11 GC SAC in Ca + 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 Ql igomer/Leucrose Fraction from g tf-S/M UT3325 Reaction In this Example, a giucan synthesis reaction was used to produce at least one soluble alpha-g!ucan product, This soluble product resulted from the concerted action of both a glucosyltransferase (GTF0459, SEQ ID NO:42) and an a!pha-g!ucanohydrolase (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 giucan synthesis reaction. This fraction was used in Examples 15 and 16 below to test the activity of alpha- glucosidases thereupon.
EXAMPLE 14
Isolation of Olsgomer/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 , SEQ 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 I EX/SEC column
chromatography using the following resin and conditions: FINEX CS 11 GC SAC in Ca + 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 oligosacharide and monosaccharide components of the isolated fraction thus prepared. Analysis of Qligomer/Leucrose Fraction from Gtf-C Reaction
In this Example, a glucan synthesis reaction was used to produce at least one soluble alpha-glucan product. This Example also demonstrated the preparation of a chromatographic fraction from a glucan synthesis reaction that produced a soluble alpha-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 Oligomer/LeucrQse Fractions from Gtf-S/MUT3325 and Gtf-C Reactions
This Example describes using alpha-glucosidase to hydrolyze Ieucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that produced soluble alpha-glucan product.
Specifically, study was made on the effect of alpha-glucosidases disclosed in Example 10 on the hydrolysis of Ieucrose and oligosaccharides in the fractions prepared in Examples 13 and 14.
A total of twelve alpha-glucosidases and two benchmark enzymes (oligo- 1 ,6-glucosidase and TG L-2000 transgiucosidase) were screened using o!igomer/leucrose fractions from gtf-S/MUT3325 (Example 13) and gtf-C
(Example 14) reactions as substrate material. All the enzymes (alpha- glucosidases 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 μί. of 0.5 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-foid in water for HPLC analysis (General 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-giucosidases had better hydrolytic activity towards glucan oligomers when compared to the bacterial alpha-glucosidases.
2015/017644
7644
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-g!ucosidase 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 a!pha-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 aipha- 1.5 glucosy!-fructose linkages and also likely alpha-1.3 and alpha-1.6 glucosyl- glucose 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 alpha- g!ucan product.
EXAMPLE 18
Select Screening of Alpha-Glucosidases using Oligomer/Leucrose Fractions from
Gtf-8/ UT3325 and Gtf-C Reactions
This Example is further to Example 15, describing the use of alpha- glucosidase to hydrolyze leucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that produced soluble alpha-glucan product.
Evaluation of alpha-glucosidases that were most active for hydrolysis of oiigomer/leucrose fractions from qtf-S/MUT3325 and qtf-C reactions (Example 15) was performed by analyzing sugar compositions resulting in reactions containing enzymes dosed at equal protein concentrations. Incubations of alpha- glucosidases (dosed at 4 ppm; for blends, the ratio of the two enzymes was 1 :1 and total dosage was 4 ppm) and oiigomer/leucrose substrate (10% ds) were performed at pH 5.5 in the presence of 2 mM calcium chloride at 80 °C and 65 °C, respectively. The reactions were quenched by adding 50 μ!_ of 0.5 M NaOH after 23 hours of incubation.
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 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 TauSec098 was efficacious for hydrolysis of DP2 to DP7 oligomers and TauSec099 outperformed TG L-2000 for leucrose hydrolysis when the incubation was performed at 65 °C. The blends of TauSec098 with TauSec099 (or TG L-2000) were effective for hydrolysis of oligomers and leucrose for DP1 production.
Thus, alpha-glucosidase can be used to hydrolyze leucrose and other oligosaccharides present in a fraction (e.g., chromatographic fraction) obtained from a g!ucan synthesis reaction, such as one that synthesizes a soluble alpha- glucan product.
Table 24
Sugar Composition Analysis of Select Screening of Ajpha-Glucosidases Using Qjigomer/Leucrose Fractions from
Gtf~S/MUT3325 and Gtf-C reactions
Blank 7.0 0.4 0.7 1 .3 3.2 7.9 0.0
5

Claims

What is claimed is:
1 A method of hydrolyzing an alpha-1 ,3 or alpha-1 ,6 glucosyl-glucose
linkage in a saccharide comprising at least one aipha-1 ,3 or alpha-1 ,8 glucosyl-glucose linkage, wherein the saccharide is a disaccharide or oligosaccharide, and wherein the method comprises:
contacting the saccharide with an a!pha-glucosidase enzyme under suitable conditions, wherein said alpha-glucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,8 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 said contacting.
2. The method of claim 1 , wherein the a!pha-glucosidase enzyme is
immobilized.
The method of claim 1 , wherein the degree of polymerization of the saccharide before hydrolysis is 3 to 7.
The method of claim 1 , 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 said contacting.
The method of claim 1 , 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.
The method of claim 5, wherein the glucan synthesis reaction produces at least one insoluble alpha-giucan product.
7. The method of claim 6, wherein the fraction is a filtrate of the glucan synthesis reaction.
8. The method of claim 5, wherein the glucan synthesis reaction produces at least one soluble alpba-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 aIpha-1 ,3-gIycosidic linkages or one or more alpha-1 ,6- glycosidic linkages.
9. The method of claim 8, wherein the fraction is a chromatographic fraction of the glucan synthesis reaction.
10. The method of claim 1 , wherein the aipha-glucosidase enzyme is a
transglucosidase.
1 1 . A composition produced by contacting a saccharide with an alpha- glucosidase enzyme,
wherein said saccharide is a disaccharide or oligosaccharide and comprises at least one alpha-1 ,3 or alpha-1 ,6 glucosyl-glucose linkage, wherein said enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,6 glucosyl-glucose linkage of the saccharide,
and wherein said composition comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to said contacting.
12. The composition of claim 11 , wherein the degree of polymerization of the saccharide before hydrolysis is 3 to 7. The composition of claim 11. 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.
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 aipha-glucosidase enzyme under suitable conditions, wherein said alpha-g!ucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,6 giucosyl-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).
A fermentation method comprising:
(a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under suitable conditions, wherein said alpha-glucosidase enzyme hydrolyzes at least one alpha-1 ,3 or alpha-1 ,6 g!ucosyl-glucose linkage of a disaccharide or oligosaccharide comprised within said fraction;
(b) fermenting the fraction of step (a) with a microbe to yield a product, wherein said 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.
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