JP2017518025A - Enzymatic hydrolysis of disaccharides and oligosaccharides using alpha-glucosidase enzyme - Google Patents

Abstract

Disclosed are methods for hydrolyzing alpha-1,3 or alpha-1,6 glucosyl-glucose linkages in sugars (disaccharides or oligosaccharides). This method comprises contacting a sugar with an alpha-glucosidase enzyme, such as transglucosidase, under suitable conditions, during which the enzyme is at least one alpha-1,3 or alpha-1,6 glucosyl of the sugar. -Hydrolyze glucose bonds. This method is useful, for example, in reducing the amount of oligosaccharides in a filtrate isolated from a glucan synthesis reaction.

Description

  No. 61 / 945,233 (filed Feb. 27, 2014), 61 / 945,241, all of which are incorporated herein by reference in their entirety. (Submitted on February 27, 2014), No. 62 / 004,290 (submitted on May 29, 2014), No. 62 / 004,308 (submitted on May 29, 2014) 62 / 004,312 (submitted on May 29, 2014), 62 / 004,300 (submitted on May 29, 2014), 62 / 004,314 (Claimed May 29, 2014) and 62 / 004,305 (submitted May 29, 2014).

  The present invention is in the field of enzymatic hydrolysis of small sugar polymers. Specifically, the present invention relates to hydrolyzing disaccharides and oligosaccharides containing one or more alpha-1,3 or alpha-1,6 glucosyl-glucose bonds with an alpha-glucosidase enzyme.

Reference to Electronic Submitted Sequence List A formal copy of the sequence list was created on February 11, 2015 and has a size of 266 kilobytes CL6220USNP_SequenceListing_ST25. A file named txt is submitted electronically via EFS-Web as a sequence listing in ASCII format and is filed concurrently with this specification. The sequence listing contained in this ASCII format document is part of this specification and is incorporated herein by reference in its entirety.

  Transglucosidase (EC 2.4.1.24, 1,4-alpha-glucan 6-alpha-glucosyltransferase) is both hydrolyzable and transferable in incubation with alpha-D-gluco-oligosaccharides. Is a D-glucosyltransferase enzyme that catalyzes (Non-patent Document 1). Maltose is the most preferred substrate for the transglucosylation reaction with this enzyme. Transfer occurs most frequently for HO-6, which produces isomaltose from D-glucose or panose (6-O-alpha-glucosyl maltose) from maltose. Transglucosidase can also transfer a glucosyl residue to another D-glucosyl unit, HO-2 or HO-3, to form cordobiose or nigerose. This enzyme can further transfer the D-glucosyl unit back to HO-4 to reform maltose.

  As a result of the transglucosylation reaction with transglucosidase, isomalt-oligos containing a higher proportion of glucosyl residues where malto-oligosaccharide residues are linked from the non-reducing end by alpha-D-1,6 glycosidic bonds Converted to sugar (IMO). In Asia, IMO sugar is used in many food and beverage formulations. Brier et al. (US Pat. No. 5,697,097) disclosed the use of transglucosidase to produce IMO from barley wort.

  Poulose et al. (US Pat. No. 5,639,097) disclosed the use of transglucosidases to degrade polysaccharides such as xanthan and guar gum. Xanthan gum includes a cellulose skeleton in which glucose is alternately 1,3-linked to branches containing mannose and glucuronic acid. The guar gum backbone contains beta-1,4-linked mannose residues in which galactose residues are alpha-1,6-linked with every other mannose.

  Lantero et al. (US Pat. No. 6,057,096) disclosed the use of transglucosidase to degrade sucrose, melezitose and trehalose. These sugars include glucose linked to fructose via 1,2- (sucrose), 1,3- (melezitose) or 1,1- (trehalose) linkages.

  Although various hydrolytic activities of transglucosidases have been disclosed, this type of enzyme is generally considered to be an alpha-glucosidase given its ability to hydrolyze an alpha bond between two glucosyl residues. It is regarded. For example, transglucosidase is associated with having maltase activity (hydrolysis of alpha-1,4 glycosidic linkage between two glucosyl residues of maltose), which is a type of alpha-glucosidase activity.

  Despite the foregoing disclosure, surprisingly, alpha-glucosidases such as transglucosidase (EC 2.4.1.24) hydrolyze the alpha-1,3 and alpha-1,6 glycosidic linkages of glucosyl-glucose. This time it was found that it could be decomposed. 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

US Patent Application Publication No. 2003/0167929 US Patent Application Publication No. 2008/0229514 US Pat. No. 5,770,437

1951, Pazur and French, J.A. Amer. Chem. Soc. 73: 3536

  In one embodiment, the present invention hydrolyzes an alpha-1,3 or alpha-1,6 glucosyl-glucose bond at a sugar comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond. A method, wherein the sugar is a disaccharide or an oligosaccharide, the method comprising contacting the sugar with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme is at least one alpha- of the sugar. The method relates to hydrolyzing 1,3 or alpha-1,6 glucosyl-glucose bonds, wherein the amount of sugar is reduced compared to the amount of sugar present prior to the contacting step.

  In another embodiment, the hydrolytic alpha-glucosidase enzyme is immobilized.

  In another embodiment, the hydrolyzed sugar has a degree of polymerization of 3-7 prior to hydrolysis. In another embodiment, the sugar concentration after the contacting step is less than 50% of the sugar concentration present before the contacting step.

  In another embodiment, suitable conditions for the hydrolysis method include (i) a glucan synthesis reaction, or (ii) a fraction obtained from a glucan synthesis reaction, and the sugar is a byproduct of the glucan synthesis reaction. In another embodiment, the glucan synthesis reaction produces at least one insoluble alpha-glucan product. In another embodiment, the fraction is a filtrate of a glucan synthesis reaction. In another embodiment, the glucan synthesis reaction comprises (i) the product of a glucosyltransferase, or (ii) one or more alpha-1,3-glycosidic linkages or one or more alpha-1,6-glycosidic linkages. At least one soluble alpha-glucan product is produced that is the product of the concerted action of both glucosyltransferase and alpha-glucanohydrolase capable of hydrolyzing the glucan polymer it has. In another embodiment, where the glucan synthesis reaction produces at least one soluble alpha-glucan product, this fraction is a chromatographic fraction of the glucan synthesis reaction.

  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 present invention is a composition produced by contacting a sugar with an alpha-glucosidase enzyme, wherein the sugar is a disaccharide or an oligosaccharide and at least one alpha-1,3 or An alpha-1,6 glucosyl-glucose linkage, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the sugar and the composition is Relates to a composition comprising a reduced amount of sugar compared to the amount of sugar present.

  In another embodiment, the sugar of the composition has a degree of polymerization of 3-7 before hydrolysis. The sugar concentration after the contacting step is, for example, less than 50% of the sugar concentration present before the contacting step.

  In another embodiment, the saccharide of the composition is in (i) a glucan synthesis reaction, or (ii) a fraction obtained from a glucan synthesis reaction, and the saccharide is a byproduct of the glucan synthesis reaction. In another embodiment, the fraction is a glucan synthesis reaction filtrate or a glucan synthesis reaction chromatographic fraction.

  In another embodiment, the present invention provides a method for concentrating fructose present in a fraction of a glucan synthesis reaction, wherein (a) the fraction obtained from the glucan synthesis reaction is combined with an alpha-glucosidase enzyme under appropriate conditions. Contacting, wherein the alpha-glucosidase enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond of a disaccharide or oligosaccharide contained within the fraction. And (b) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition having a higher fructose concentration compared to the fructose concentration of the fraction of step (a). Including methods.

  In another embodiment, the present invention provides (a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme is included in the fraction. Hydrolyzing, contacting, at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond of a disaccharide or oligosaccharide to be obtained (b) using a microorganism to obtain a product ( fermenting the fraction of a), wherein the fermentation can be carried out after or simultaneously with step (a), and (c) optionally the product of (b) And wherein the yield of the product of (b) is increased compared to the yield of the product fermenting a fraction of the glucan synthesis reaction that is not in contact with the enzyme. About the method

NOVO188 prehydrolysis treatment with the enzyme is a ¹ H NMR spectrum of glucan reaction filtrate material (starting material) and after (treated substance) (see Examples 2-3). TG L-2000 prehydrolysis treatment with transglucosidase is a ¹ H NMR spectrum of glucan reaction filtrate material (starting material) and after (treated substance) (see Examples 2-3).

  The disclosures of all cited patents and non-patent literature are hereby incorporated by reference in their entirety.

  As used herein, the term “invention” or “disclosed invention” is not intended to be limiting and the invention as defined in the claims or described herein. Is generally applied to any of the above. These terms are used interchangeably herein.

  The terms “sugar”, “sugar molecule” and “carbohydrate” are used interchangeably herein and refer to a disaccharide or oligosaccharide unless otherwise indicated. As used herein, “disaccharide” refers to a carbohydrate having two monosaccharides linked by a glycosidic bond. As used herein, “oligosaccharide” refers to a carbohydrate composed of 2 to 9 monosaccharides linked by, for example, glycosidic bonds. Oligosaccharides may also be referred to herein as “oligomers”. A monosaccharide contained within a disaccharide or oligosaccharide may be referred to as, for example, a “monosaccharide unit” or “monomer unit”. Preferred monosaccharides herein are fructose and glucose.

  The terms “glycosidic link” and “glycosidic bond” are used interchangeably herein and refer to a type of covalent bond that links a carbohydrate molecule to another carbohydrate molecule.

  As used herein, the terms “alpha-1,3 glucosyl-glucose bond”, “alpha-1,3 glucose-glucose bond” and “glucose-alpha1,3-glucose” refer to two alpha-D- Refers to the alpha-1,3-glycosidic bond between glucose molecules. As used herein, the terms “alpha-1,6 glucosyl-glucose bond”, “alpha-1,6 glucose-glucose bond” and “glucose-alpha1,6-glucose” refer to two alpha-D- Refers to the alpha-1,6-glycosidic bond between glucose molecules. The alpha-1,3 glucosyl-glucose linkages and / or alpha-1,6 glucosyl-glucose linkages herein are included within a disaccharide or oligosaccharide in certain embodiments.

  As used herein, the terms “alpha-1,5 glucosyl-fructose linkage”, “alpha-1,5 glucose-fructose linkage” and “glucose-alpha-1,5-fructose” refer to alpha-D-glucose. Refers to the alpha-1,5-glycosidic bond between the molecule and the fructose molecule. The alpha-1,5 glucosyl-fructose linkages herein are included in a disaccharide or oligosaccharide in certain embodiments.

  As used herein, “alpha-D-glucose” can also refer to “glucose”.

A disaccharide containing an alpha-1,5 glucosyl-fructose linkage is referred to herein as leucus. The terms “leucrose” and “D-glucopyranosyl-alpha (1-5) -D-fructopyranose” are used interchangeably herein. Leukrose has the following structure:
Have

  The terms “alpha-glucosidase”, “alpha-1,4-glucosidase” and “alpha-D-glucoside glucohydrolase” are used interchangeably herein. Alpha-glucosidase (EC 3.2.1.20) (where “EC” refers to Enzyme Commission number) has so far been non-reduced (1,1) from oligosaccharides (eg, disaccharides) and polysaccharide substrates. 4)-has been recognized as an enzyme that catalyzes the hydrolytic release of bound alpha-D-glucose residues. It is now disclosed herein that alpha-glucosidase also has hydrolytic activity on alpha-1,5 glucosyl-fructose bonds and hydrolytic activity on alpha-1,3 and alpha-1,6 glucosyl-glucose bonds. Is done. Transglucosidase and glucoamylase enzymes are examples of alpha-glucosidases with such activity.

  The terms “transglucosidase” (TG), “transglucosidase enzyme” and “1,4-alpha-glucan 6-alpha-glucosyltransferase” are used interchangeably herein. Transglucosidase (EC 2.4.1.24) has heretofore been a D-glucosyltransferase enzyme that catalyzes both hydrolyzable and transfer reactions in incubation with certain alpha-D-gluco-oligosaccharides. Has been recognized. It is now disclosed herein that transglucosidase also has hydrolytic activity on alpha-1,5 glucosyl-fructose bonds and hydrolytic activity on alpha-1,3 and alpha-1,6 glucosyl-glucose bonds. The

  The terms “glucoamylase” (GA), “glucoamylase enzyme” and “alpha-1,4-glucan glucohydrolase” are used interchangeably herein. Glucoamylase (EC 3.2.1.3) has so far hydrolyzed both alpha-1,4 and alpha-1,6 glycosidic linkages from the non-reducing ends of glucose-containing disaccharides, oligosaccharides and polysaccharides. It has been recognized as an exo-type enzyme that catalyzes. It is now disclosed herein that glucoamylase also has hydrolytic activity on alpha-1,5 glucosyl-fructose linkages.

  Enzymatic hydrolysis is a process in which an enzyme promotes the breaking of bonds in a molecule by the addition of water components. As used herein, an alpha-1,3 or alpha-1,6 glucosyl-glucose bond “hydrolyzes”, “hydrolyzes” or “hydrolyzing activity” is an alpha-glucosidase, such as transglucosidase Refers to the enzymatic hydrolysis of an alpha-1,3 or alpha-1,6 glycosyl bond between two glucose molecules. Such hydrolysis involves contacting a disaccharide or oligosaccharide containing an alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkage with the alpha-glucosidase herein under suitable conditions. When it happens. Accordingly, a “hydrolysis reaction” herein includes at least (i) a disaccharide or oligosaccharide containing alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkages, and (ii) alpha. -Containing glucosidase.

  As used herein, the term “saccharification” refers to the process of breaking a sugar (disaccharide or oligosaccharide) into monosaccharide components. Sugars can be saccharified herein in hydrolysis reactions.

  “Suitable conditions” for contacting a sugar (disaccharide or oligosaccharide) comprising at least one alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkage with an alpha-glucosidase herein. Refers to conditions (eg, temperature, pH, time) that support the hydrolysis of one or more alpha-1,3 and / or alpha-1,6 glucosyl-glucose bonds in the sugar by alpha-glucosidase. Suitable conditions may include “aqueous conditions” including, for example, at least 20 wt% water. Aqueous conditions can characterize solutions or mixtures. A solution or mixture in which a sugar containing at least one alpha-1,3 and / or alpha-1,6 glucosyl-glucose bond is contacted with an alpha-glucosidase is for example an alpha-glucosidase reaction (eg a transglucosidase or glucoamylase reaction). Can be called.

  As used herein, an “immobilized” enzyme refers to an enzyme that is attached to an inert, insoluble material. A method for preparing an immobilized enzyme is disclosed, for example, in US Pat. No. 5,541,097, 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. As used herein, “alpha-glucan” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% glucan polymers containing alpha-glycoside linkages.

  As used herein, “insoluble glucan” refers to a glucan polymer that is not soluble in aqueous conditions. An example of an insoluble glucan herein is a polyalpha-1,3-glucan with a DP of at least 8 or 9. The glucosyltransferase reaction in certain embodiments as disclosed herein produces at least one insoluble glucan product.

  Terms such as “soluble glucan”, “soluble alpha-glucan”, “soluble fiber”, “soluble glucan fiber”, “soluble dietary fiber” are used interchangeably herein and are soluble in aqueous conditions Refers to glucan polymer. Examples of soluble glucans herein include certain oligosaccharides, such as polyalpha-1,3-glucans with a DP of less than 8, and certain oligos disclosed in the examples provided below. It is sugar. The glucosyltransferase reaction in certain embodiments as disclosed herein produces at least one soluble glucan product. Another set of properties that characterize the soluble alpha-glucan compounds in certain embodiments herein are that they are (i) water-soluble glucose oligomers having a degree of polymerization of 3 or greater, (ii) almost all in the human small intestine. Or digestion resistance that is not absorbed at all (ie very slow or no digestibility) and (iii) at least partially fermentable in the lower gastrointestinal tract. The digestibility of the soluble glucan fiber composition can be measured using, for example, AOAC method 2009.01.

  The terms “polyalpha-1,3-glucan” and “alpha-1,3-glucan polymer” are used interchangeably herein. A polyalpha-1,3-glucan is a polymer comprising glucose monomer units linked together by glycosidic bonds, wherein at least about 50% of the glycosidic bonds are alpha-1,3-glycosidic bonds. The term “alpha-1,3-glycosidic bond” as used herein is a covalent bond that links alpha-D-glucose molecules together through carbons 1 and 3 in adjacent alpha-D-glucose rings. Refers to the type.

The “molecular weight” of glucan in the present specification can be expressed as a number average molecular weight (M _n ) or a weight average molecular weight (M _w ). Alternatively, molecular weight can be expressed 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 by high pressure liquid chromatography (HPLC), size exclusion chromatography (SEC) or gel permeation chromatography (GPC).

  Terms such as “glucosyltransferase enzyme”, “gtf enzyme”, “gtf enzyme catalyst”, “gtf”, “glucan sucrase” are used interchangeably herein. The activity of the gtf enzyme herein catalyzes the reaction of the sucrose substrate to make the products glucan and fructose. Other products (by-products) of the gtf reaction include glucose (resulting from when glucose is hydrolyzed from the glucosyl-gtf enzyme intermediate complex), various soluble oligosaccharides (eg DP2-DP7) and leucus (glucosyl). -A result from the case where glucose of the gtf enzyme intermediate complex is linked to fructose). Wild-type forms of glucosyltransferase enzymes generally contain a signal peptide (in the N-terminal to C-terminal direction), a variable domain, a catalytic domain and a glucan binding domain. Glucosyltransferases herein are glycosyl hydrolase family 70 (GH70) (Cantarel et al., Nucleic Acids Res. 37: D233-238, 2009) according to the CAZy (Carbohydrate-Active EnZymes) database. Classified below.

  As used herein, the term “sucrose” refers to a non-reducing disaccharide composed of alpha-D-glucose and beta-D-fructose molecules linked by alpha-1,2-glycosidic bonds. Sucrose is commonly known as sugar.

  The terms “glucan synthesis reaction”, “glucan reaction”, “gtf reaction” and the like are used interchangeably herein and refer to a reaction performed by a glucosyltransferase enzyme. A glucan synthesis reaction, as used herein, generally refers to a solution containing at least one active glucosyltransferase enzyme in a solution containing sucrose and water, and optionally other components. Other components that may be present during the glucan synthesis reaction herein include, for example, fructose, glucose, leucorose, soluble oligosaccharides (eg DP2-DP7) and soluble glucan products. Also, one or more alpha-glucanohydrolase enzymes can be included in the glucan synthesis reaction in some embodiments. Certain glucan products, such as polyalpha-1,3-glucan having a degree of polymerization (DP) of at least 8 or 9, are insoluble in water and are therefore not dissolved in the glucan synthesis reaction, but rather emerge from solution. It is understood that you get.

  The terms “alpha-glucanohydrolase” and “glucanohydrolase” are used interchangeably herein and refer to an enzyme capable of hydrolyzing an alpha-glucan oligomer. An alpha-glucanohydrolase can be defined by its endo-type hydrolytic activity on certain alpha-D-glycoside bonds. Examples of alpha-glucanohydrolase enzymes herein include dextranase (EC 3.2.1.11; capable of endo-hydrolyzing alpha-1,6-linked glycosidic bonds), mutanase (EC 3. 2.1.59; alpha-1,3-linked glycosidic bonds can be endo-hydrolyzed) and alternanase (EC 3.2.1.-; alternan can be cleaved to endo-hydrolyzable) Can be mentioned. Various factors within certain alpha-glucans, including but not limited to branching level, branching type and relative branching length, are alpha-glucanohydrolases that endo-hydrolyze some glycosidic bonds. Can adversely affect your ability.

  The “percent dry solid” of the glucan synthesis reaction refers to the wt% of all sugars in the glucan synthesis reaction. The percent dry solid of the gtf reaction can be calculated, for example, based on the amount of sucrose used to prepare the reaction.

  As used herein, “fraction” of a glucan synthesis reaction refers to the solution portion of the glucan synthesis reaction. The fraction can be part or all of the solution from the glucan synthesis reaction and is separated from the soluble or insoluble glucan product synthesized in the reaction. The fraction may optionally also be referred to as “mother liquor” in embodiments where the product is an insoluble (solid) glucan product. An example of fractionation is a filtrate of a glucan synthesis reaction. The fraction can also contain dissolved sugars, such as sucrose, fructose, glucose, leucus, soluble oligosaccharides (eg DP2-DP7), so the fraction is also referred to as a “mixed sugar solution” derived from the glucan synthesis reaction. obtain. As used herein, “hydrolyzed fraction” refers to a fraction that has been treated with alpha-glucosidase herein to hydrolyze leucos and / or oligosaccharides present in the fraction. .

  Terms such as “filtrate”, “glucan reaction filtrate”, “glucan filtrate” are used interchangeably herein and are separated from the solid glucan product synthesized in the glucan synthesis reaction by filtration. Refers to the fraction that is. As used herein, “hydrolyzed filtrate” refers to a filtrate that has been treated with alpha-glucosidase herein to hydrolyze leucorose and / or oligosaccharides present in the filtrate. .

  Terms such as “percent by volume”, “volume percent”, “vol%”, “v / v%” are used interchangeably herein. The percentage by volume of solute in solution can be determined using the formula: [(solute volume) / (solution volume)] × 100%.

  Terms such as “percent by weight”, “weight percent (wt%)”, “weight-weight percent (% w / w)” are used interchangeably herein. Percent by weight refers to the percentage of a substance on a mass basis when it is included in a composition, mixture or solution. All percentages herein are weight percentages unless otherwise indicated.

As used herein, “polydispersity index”, “PDI”, “heterogeneity index”, “dispersion degree”, etc. are used in a sample of a polymer (such as a glucose oligomer, such as a soluble alpha-glucan). It can be calculated by dividing the weight average molecular weight by the number average molecular weight (PDI = M _w / M _n ).

  The terms “increased”, “enhanced” and “improved” are used interchangeably herein. These terms refer to a greater amount or activity, such as an amount or activity that is slightly greater than the original amount or activity, or an amount or activity that is much greater than the original amount or activity, all in between. Amount or activity. Alternatively, these terms include, for example, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% of the increased amount or activity compared to the amount or activity being compared. %, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% greater amount or activity.

  The terms “sequence identity” or “identity” as used herein with respect to polynucleotide or polypeptide sequences are the same when aligned for maximum match over a specified comparison frame. Refers to a nucleobase or amino acid residue in two sequences. Thus, “percent sequence identity” or “percent identity” refers to a value determined by comparing two optimally aligned sequences over a comparison frame, where the polynucleotide or polynucleotide in the comparison frame. A portion of the peptide sequence may contain additions or deletions (ie gaps) when compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage determines the number of positions where nucleobases or amino acid residues identical in both sequences occur to obtain the number of matching positions, and divides the number of matching positions by the total number of positions in the comparison frame. Calculated by multiplying the result by 100 to obtain the percentage of sequence identity.

  Using the Basic Local Alignment Search Tool (BLAST) algorithm available online on the National Center for Biotechnology Information (NCBI) website, for example, two or more polynucleotide sequences disclosed herein (BLASTN algorithm) ) Or percent identity between polypeptide sequences (BLASTP algorithm). Alternatively, percent identity between sequences can be performed using a Clustal algorithm (eg, ClustalW or ClustalV). In the case of multiple alignment using the Clustal method of alignment, the default values may correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for calculating percent identity of protein sequences using pairwise alignment and Clustal method may be KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5 and DIAGONALS SAVED = 5. In the case of nucleic acids, these parameters may be KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4 and DIAGONALS SAVED = 4. Alternatively or additionally, percent identity between sequences can be determined using parameters such as GAP OPEN = 10, GAP EXTEND = 0.5, END GAP PENALTY = false, END GAP OPEN = 10, END GAP using the EMBOSS algorithm (eg, needle). This can be done using a BLOSUM matrix (eg BLOSUM62) with EXTEND = 0.5.

  Various polypeptide amino acid sequences are disclosed herein as a feature 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, the variant amino acid sequence has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% with the sequence disclosed herein. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98% or 99% identity. Variant amino acid sequences herein have the same function / activity of the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91 of the disclosed sequence. % / 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of function / activity.

  The term “isolated”, as used in certain embodiments, refers to any cellular component that is completely separated from its native origin (eg, an isolated polynucleotide or polypeptide molecule). In some examples, the isolated polynucleotide or polypeptide molecule is part of a larger composition, buffer system or reagent mixture. For example, an isolated polynucleotide or polypeptide molecule can be heterogeneously contained within a cell or organism. Another example is an isolated alpha-glucosidase (eg glucoamylase, transglucosidase) or glucosyltransferase enzyme. The enzymatic reactions disclosed herein (eg, alpha-glucosidase reaction, glucosyltransferase reaction) are synthetic, non-natural processes.

  Embodiments of the disclosed invention hydrolyze an alpha-1,3 or alpha-1,6 glucosyl-glucose bond in a sugar comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond. It relates to a method of disassembling. The sugar is a disaccharide or an oligosaccharide. This method involves contacting the sugar 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 bond of the sugar. Due to this hydrolysis, the amount of sugar is reduced compared to the amount of sugar present before the contacting step. Thus, this hydrolysis method may alternatively be referred to as a method of reducing the amount of sugar in the composition.

  Importantly, it is believed that it was not previously known that the alpha-glucosidase enzyme can hydrolyze alpha-1,3 and alpha-1,6 glucosyl-glucose bonds. Thus, the post-hydrolysis alpha-glucosidase reaction can be used to remove oligosaccharide by-products containing these glucose-glucose bonds from the glucan synthesis reaction and / or fractions obtained therefrom. Such removal represents an improvement over chemical processes of by-product removal, such as acid hydrolysis that can result in degradation of the glucan product. Finally, the glucan reaction fraction treated according to the above hydrolysis method is better suited for downstream applications such as fermentation, for example because the level of glucose monosaccharide is elevated in the fraction. Monosaccharides are generally easier to handle for downstream processes compared to oligosaccharide by-products.

  Alpha-glucosidase (EC 3.2.1.20), in embodiments herein, is alpha in a sugar comprising at least one of alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkages. Used to hydrolyze -1,3 and / or alpha-1,6 glucosyl-glucose bonds. It has been recognized that alpha-glucosidase enzymes so far catalyze the hydrolytic release of terminal non-reducing (1,4) -linked alpha-D-glucose residues from oligosaccharides (eg disaccharides) and polysaccharide substrates. It had been. It is also disclosed herein that these enzymes have hydrolytic activity on, for example, alpha-1,3 and alpha-1,6 glucosyl-glucose bonds.

  The alpha-glucosidase is from any source (eg plants, animals, microorganisms such as bacteria or fungi / yeast), such as from the sources disclosed below from which transglucosidase and / or glucoamylase can be derived. obtain. For example, the alpha-glucosidase can be a fungal alpha-glucosidase. Other examples of suitable alpha-glucosidases herein are described in US Pat. Nos. 6,355,467, 5,922,580, and 5,795,766, all of which are incorporated herein by reference. And those disclosed in US Pat. No. 5,763,252 and US Pat. No. 8,633,006.

  In certain embodiments herein, the alpha-glucosidase enzyme is SEQ ID NO: 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30. 32, 34, 36, 38, or DIAZYME RDF ULTRA (DuPont Industrial Biosciences). Alternatively, the alpha-glucosidase enzyme is SEQ ID NO: 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. Or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of DIAZYME RDF ULTRA; Has hydrolytic activity on alpha-1,3 and / or alpha-1,6 glucosyl-glucose bonds in sugars. Some of the aforementioned sequences are, for example, mature alpha-glucosidases that lack an N-terminal signal peptide. For such sequences, the N-terminal initiation methionine is typically used when expressing it without the use of a signal peptide (such as by an expression system where the enzyme is expressed intracellularly and obtained from cell lysates). Will be added (directly or via intervening heterologous amino acid sequences such as epitopes) (as appropriate).

  In certain embodiments herein, alpha-1,3 and / or alpha-1 in a sugar comprising at least one of alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkages. Transglucosidase (EC 2.4.1.24; 1,4-alpha-glucan 6-alpha-glucosyltransferase) can be used as an alpha-glucosidase to hydrolyze the 6-glucosyl-glucose bond. This class of enzymes has heretofore been recognized as a D-glucosyltransferase enzyme that catalyzes hydrolytic and transfer reactions upon incubation with certain alpha-D-gluco-oligosaccharides. Transglucosidases as disclosed herein also have hydrolytic activity for alpha-1,3 and alpha-1,6 glucosyl-glucose bonds.

  The transglucosidase enzyme herein may be from any microbial source, such as a bacterium or fungus. Examples of fungal transglucosidases include Trichoderma species (eg, T. reesei), Aspergillus species and Neosartoria species (eg, N. felis). )), But is not limited. Examples of Aspergillus species from which transglucosidase can be derived include: Niger, A. niger A. awamori, A. A. oryzae, A. oryzae A. terreus, A. A. clavatus, A. A. fumigatus and A. fumigatus Non-limiting examples include A. nidulans. Other examples of transglucosidase enzymes useful herein are all described by Barker et al., Which is incorporated herein by reference. (1953, J. Chem. Soc. 3588-3593); Pazur et al. (1986, Carbohydr. Res. 149: 137-147), Nakamura et al. (1997, J. Biotechnol. 53: 75-84) and US Patent Application Publication No. 2008/0229514. Still other examples of transglucosidase enzymes useful herein are those that are thermostable; US Pat. No. 4,689,296, incorporated herein by reference, is a thermostable transglucosidase. Disclose a process for generating. Still further examples of transglucosidase enzymes useful herein are any of those in the GENBANK database (NCBI), eg, accession number D45356 (GID: 2645159, A.I., which is incorporated herein by reference). Niger (A. niger), BAD06006.1 (GID: 4031328, A. awamori), BAA08125.1 (GID: 1045565, A. oryzae)), XP — 001210809.1 (GID: 115492363, A. terreus), XP_001216899.1 (GID: 11543524, A. terreus), XP_001271891.1 (GID: 121707620, A. terreus). A. clavatus), XP_751811.1 (GID: 70993928, A. fumigatus), XP_65921.1 (GID: 67523121, A. nidulans), XP_0012669999 (GID: 119500484, N. fischeri), XP_001258585.1 (GID: 119473371, N. fischeri), and the like. Alternatively, the transglucosidase herein may have an amino acid sequence that is at least 90% or 95% identical to any amino acid sequence of the previously disclosed transglucosidase sequence, and alpha-1, It may have hydrolytic activity on 3 and / or alpha-1,6 glucosyl-glucose bonds. All of the aforementioned transglucosidases, when used in the hydrolysis reactions herein, are preferably mature forms that lack an N-terminal signal peptide.

  In certain embodiments herein, the transglucosidase enzyme is A.I. It may include the amino acid sequence of SEQ ID NO: 1 (TG L-2000), which is an A. niger transglucosidase (US Patent Application Publication No. 2008/0229514). Alternatively, the 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; It may have hydrolytic activity on alpha-1,3 and / or alpha-1,6 glucosyl-glucose bonds in the sugar. For example, according to the disclosure of US Patent Application Publication No. 2008/0229514, which is incorporated herein by reference, either SEQ ID NO: 1 or a variant thereof can be generated. SEQ ID NO: 1 is a mature transglucosidase lacking an N-terminal signal peptide. Since SEQ ID NO: 1 does not start with a methionine residue, generally when expressing it without the use of a signal peptide (such as by an expression system in which the enzyme is expressed in a cell and obtained from a cell lysate) It is understood that an N-terminal initiation methionine is added to SEQ ID NO: 1 (directly or via an intervening heterologous amino acid sequence such as an epitope).

  Glucoamylase (EC 3.2.1.3; alpha-1,4-glucan glucohydrolase) can be used as an alpha-glucosidase in certain embodiments herein. For example, a glucoamylase can be included with a transglucosidase in each of the hydrolysis reaction settings / conditions disclosed herein. In this context, glucoamylases are (i) alpha-1,5 glucosyl-fructose linkages and / or (ii) alpha-1,3 and / or present in sugars containing any of these linkage types. Or it can be used to hydrolyze alpha-1,6 glucosyl-glucose bonds. This class of enzymes has heretofore been recognized as an exo-type enzyme that catalyzes the hydrolysis of both alpha-1,4 and alpha-1,6 glycosidic linkages from the non-reducing ends of glucose-containing disaccharides, oligosaccharides and polysaccharides. It has been. The glucoamylase disclosed herein also has hydrolytic activity for alpha-1,5 glucosyl-fructose linkages. In certain embodiments, the alpha-glucosidase is not a glucoamylase.

  The glucoamylase enzyme herein may be from any microbial source, such as bacteria or fungi. Examples of bacterial glucoamylases include Bacillus species (eg, B. alkalophilus, B. amyloliquefaciens, B. lentus, B. lentus, B. lentus) B. licheniformis, B. stearothermophilus, B. subtilis, B. thuringiensis and Streptomyces such as Streptomyces , S. lividans), but is not limited. Examples of fungal glucoamylases include Trichoderma species (eg, T. reesei, T. longibrachiatum, T. strictipilis, T. asperel). T. asperellum, T. konilangbra, T. hazianum), Aspergillus species (eg A. niger, A. oryzae, A. oryzae) A. terreus, A. clavatus, A. nidulans, A. kawachi, A A. awamori), Rhizopus species (eg, R. oryzae, R. niveus), Talaromyces species (eg, T. T. e. Emersonii), T. thermophilus, T. duponti), Mucor species, Hypocrea species (eg, H. gelatinosa, H. gelatosa, H. H. orientalis, H. vinosa, H. citrina), Fusarium species (E.g., F. oxysporum, F. roseum, F. venenatum), Neurospora species (e.g., N. crassa), Fumicola Genus (eg, H. grisea, H. insolens, H. lanuginose), Penicillium species (eg, P. notatum) .Notatum), P. chrysogenum) and Saccharomycopsis species (eg, S. cerevisiae). Examples include, but are not limited to, those of S. fibuligera. Examples of these bacterial and fungal glucoamylases for use herein are disclosed in US Patent Application Publication No. 2013/0102035, which is incorporated herein by reference. Other examples of glucoamylase enzymes useful herein are described by Svensson et al., All incorporated herein by reference. (1983, Carlsberg Res. Commun. 48: 529-544), Boel et al. (1984, EMBO J. 3: 1097-1102); Hayashida et al. (1989, Agric. Biol. Chem. 53: 923-929); U.S. Pat. No. 5,249,941, U.S. Pat. No. 4,794,175, U.S. Pat. No. 4,247,637, U.S. Pat. 6620924, Ashikari et al. (1986, Agric. Biol. Chem. 50: 957-964), Ashikari et al. (1989, Appl. Microbiol. Biotechnol. 32: 129-133), US Pat. No. 4,863,864; US Pat. No. 4,618,579, Houghton-Larsen et al. (2003, Appl. Microbiol. Biotechnol. 62: 210-217) and U.S. Pat. No. 7413887. Alternatively, the glucoamylase herein may have an amino acid sequence that is at least 90% or 95% identical to any amino acid sequence of any of the aforementioned disclosed glucoamylase sequences, and (i) alpha-1, It may have hydrolytic activity on 5 glucosyl-fructose linkages and / or (ii) alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkages. All of the aforementioned glucoamylases, when used in the hydrolysis reactions herein, are preferably mature forms lacking an N-terminal signal peptide. Commercially available glucoamylases useful herein include, for example, OPTIDEX L-400, GC147, GC321, GZYME G990 4X, OPTIMAX7525, DEXTROZYME, DISTLILLASE and GLUCZYME.

  In certain embodiments herein, the glucoamylase enzyme is It may comprise the amino acid sequence of SEQ ID NO: 2 (GC321), which is a T. reesei glucoamylase. Alternatively, the glucoamylase may comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: It may have hydrolytic activity on (i) alpha-1,5 glucosyl-fructose linkages and / or (ii) alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkages. For example, either SEQ ID NO: 2 or a variant thereof can be generated in accordance with the disclosure of US Pat. No. 7413887 or US Patent Application Publication No. 2013/0102035, which is incorporated herein by reference. SEQ ID NO: 2 is a mature glucoamylase lacking an N-terminal signal peptide. Since SEQ ID NO: 2 does not start with a methionine residue, generally when expressing it without the use of a signal peptide (such as by an expression system where the enzyme is expressed intracellularly and obtained from cell lysates), N It is understood that a terminal initiation methionine is added to SEQ ID NO: 2 (directly or via an intervening heterologous amino acid sequence such as an epitope).

  Alpha-glucosidase enzymes herein, such as transglucosidase or glucoamylase, can be commercially available (eg, DuPont Industrial Biosciences / Genencor, USA; Megazyme International, Ireland; Amano Enzyme Inc., Japan). Alternatively, such as those described in U.S. Patent Application Publication No. 2008/0229514, U.S. Pat. No. 7413887, or U.S. Patent Publication No. 2013/0102035, which are incorporated herein by reference. Such enzymes can be produced by any means known in the art. For example, alpha-glucosidase can be produced recombinantly in a heterologous expression system, such as a microbial or fungal heterologous expression system. Examples of heterologous expression systems include bacteria (eg, E. coli, Bacillus sp.) And eukaryotic systems. Eukaryotic systems include, for example, yeast (eg, Pichia sp., Saccharomyces sp.) Or fungi (eg, Trichoderma sp.), Such as T. reesei; An Aspergillus species, such as A. niger expression system may be used. For example, T.W. In a T. reesei host, the transglucosidase of SEQ ID NO: 1 and the glucoamylase of SEQ ID NO: 2 and variants thereof can be expressed.

  When used in the hydrolysis reactions herein, the alpha-glucosidase enzyme is preferably in the mature form lacking the N-terminal signal peptide. The expression system for producing the mature alpha-glucosidase enzyme herein may use a polynucleotide encoding the enzyme further comprising a sequence encoding an N-terminal signal peptide for directing extracellular secretion. The signal peptide in such embodiments is cleaved from the enzyme during the secretion process. The signal peptide can be either native or heterologous to transglucosidase or glucoamylase. Alternatively, the mature alpha-glucosidase enzyme can be provided by expressing the enzyme in a cell and expressing it without using a signal peptide, such as by an expression system obtained from cell lysates. In either scenario (secretion or intracellular expression), a heterologous amino acid sequence such as an epitope can optionally be included at the N-terminus of alpha-glucosidase.

  In certain embodiments, the alpha-glucosidase enzyme can be provided in the hydrolysis reactions herein by direct use of cells that express the enzyme. In other words, the alpha-glucosidase that is contacted with the sugar may be present by its expression from cells that have been subjected to conditions suitable for hydrolysis. Thus, such cells can be used instead of adding an isolated alpha-glucosidase preparation to the hydrolysis reaction. Cells for this purpose can be, for example, bacterial, yeast or fungal cells. Examples of yeast include the genus Saccharomyces (eg, S. cerevisiae), the genus Kluyveromyces, the genus Candida, the genus Pichia, the genus Schizosaccharomyces Those from the genus Hansenula, Kloeckera and Schwanniomyces. Other expression systems useful herein are disclosed in US Patent Application Publication No. 2013/0323822, which is incorporated herein by reference.

  Sugars herein include at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage. Thus, depending on the length of the sugar, it may contain, for example, 1, 2, 3, 4, 5, 6, 7 or 8 alpha-1,5 glucosyl-glucose bonds. The sugar preferably contains 1, 2 or 3 bonds of this type. The sugar in other preferred embodiments has only alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkages. In other embodiments, the sugar may have one or more alpha-1,5 glucosyl-fructose linkages.

  Since sugars herein contain at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage, the sugar contains at least two glucose units. In certain embodiments, the sugars herein include only glucose units or include both glucose and fructose units. Such compositions can characterize the disaccharide and oligosaccharide byproducts of the glucan synthesis reaction. Alternatively, the sugars herein may contain other monosaccharides such as galactose, ribose and xylose in addition to glucose and fructose.

  In certain embodiments of the disclosed invention, the hydrolyzed sugar can be an oligosaccharide. The oligosaccharides herein can have, for example, 2, 3, 4, 5, 6, 7, 8 or 9 monosaccharide units. As understood in the art, an oligosaccharide herein can be referred to in terms of its degree of polymerization (DP), which defines the number of monomer units in the oligosaccharide. The DP3 oligosaccharide has, for example, three monomer units. Thus, the oligosaccharide can be, for example, a DP3, DP4, DP5, DP6, DP7, DP8 or DP9 oligosaccharide. In certain embodiments, the sugar has a DP of 3-7 (ie, DP3-7).

  Oligosaccharides herein with more than 3 monosaccharide units may include other linkages in addition to, for example, at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage. For example, there may be one or more alpha-1,5 glucosyl-fructose linkages in an oligosaccharide that are also susceptible to hydrolysis by alpha-glucosidase as shown herein.

  In certain embodiments, the oligosaccharide comprises only glucose monomers that are linked by alpha-1,3 and / or alpha-1,6 glycosidic linkages. Accordingly, such oligosaccharides contain only alpha-1,3 glucosyl-glucose and / or alpha-1,6 glucosyl-glucose linkages. Examples of such oligosaccharides contain only alpha-1,3 linkages or alpha-1,6 linkages. In certain embodiments, the oligosaccharide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% or 99% glucosyl-glucose linkages. In other embodiments, there can be about 75-85% alpha-1,3 glucosyl-glucose linkage and about 15-25% alpha-1,6 glucosyl-glucose linkage in the oligosaccharides herein. . Alternatively, an oligosaccharide herein may be any percentage (any integer value between 1% and 99%) of alpha-1,3 glucosyl-so long as the sum of these percentages is 100% or less. It may include glucose binding and any percentage (any integer value from 1% to 99%) of alpha-1,6 glucosyl-glucose binding. Any of these oligosaccharides are in fractions from, for example, a glucan synthesis reaction that produces (i) insoluble alpha-glucan (eg, polyalpha-1,3-glucan) or (ii) a soluble alpha-glucan product. obtain. This binding content can characterize (i) each oligosaccharide individually or (ii) a group of oligosaccharides (ie average binding content). An oligosaccharide comprising only glucose monomers linked by alpha-1,3 and / or alpha-1,6 glycosidic bonds can be, for example, DP2-DP7 or DP3-DP7. It will be appreciated that the exact distribution of bonds in the oligosaccharide may vary depending on the conditions of the glucan synthesis reaction that produces the oligosaccharide byproduct (eg, the gtf enzyme). It will be further appreciated that the exact bond distribution is not critical to the method disclosed herein.

  The examples herein include (i) Leucrose and (ii) alpha-1,3 glucosyl-glucose where alpha-glucosidase (eg, transglucosidase and glucoamylase enzymes) comprises an alpha-1,5 glucosyl-fructose linkage. And / or reveal that both oligosaccharides containing only alpha-1,6 glucosyl-glucose linkages can be hydrolyzed. Thus, for example, using an alpha-glucosidase in a reaction to hydrolyze an alpha-1,5 glucosyl-fructose bond, an alpha-1,3 glucosyl-glucose bond and / or an alpha-1,6 glucosyl-glucose bond. Can do.

  At least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond in the sugars herein can be hydrolyzed by the alpha-glucosidase herein. Alternatively, it is believed that 2, 3, 4, 5 or more such linkages in the sugar can be hydrolyzed, for example by alpha-glucosidase. Hydrolysis of at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond can occur at the non-reducing end of the sugar in certain embodiments.

  Compared to the amount of sugar present prior to the contacting step, the amount of sugar is reduced in the disclosed hydrolysis method. This decrease is due to hydrolytic cleavage of at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond in the sugar. The amount of sugar (eg, concentration) after the contacting step in the hydrolysis method herein is the amount of sugar present before the contacting step (before contacting the saccharide with alpha-glucosidase under appropriate conditions). About 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or 1% -90% Less than some integer value).

  Compared to the amount of sugar present before the contacting step, the amount of sugar is reduced in the disclosed hydrolysis method. It will be appreciated that such a comparison can be made in any way. For example, the sugar concentration can be measured both before and after performing the hydrolysis method. Alternatively, a comparison can be made to a control reaction having the same conditions, except that no alpha-glucosidase as disclosed herein is added to the control reaction.

  In certain embodiments herein, the alpha-glucosidase can be immobilized. The enzyme may be obtained using any method and / or means known in the art, such as those disclosed in US Pat. Nos. 5,541,097 and 4,713,333, both of which are incorporated herein by reference. Can be immobilized. For example, contacting an enzyme with a solution of an amine-reactive substance (eg, glutaraldehyde) to form an adduct (eg, an enzyme-glutaraldehyde adduct), followed by a polyamine (eg, a polyethyleneimine such as EPOMIN P-1050). One or more enzymes can be immobilized by binding the adduct to the treated solid support.

  In certain embodiments, the solid support (solid support) on which the alpha-glucosidase enzyme can be immobilized can be an inorganic or organic material. Examples of such materials include gamma-alumina, titania, granular activated carbon, granular diatomaceous earth, glass beads, porous glass, pumice, silica gel, metal oxide, and aluminum oxide.

  To treat the solid support, a polyamine can be used such that subsequent exposure of the solid support to an adduct comprising an enzyme and an amine reactive material leads to the attachment of the enzyme to the solid support. Examples of polyamines useful herein include polyethylenediamine, polyethyleneimine (eg, polydiethylenetriamine, polytriethylenetetramine, polypentaethylenehexamine, polyhexamethylenediamine), polymethylenedicyclohexylamine, polymethylenediamine. Examples include aniline, polytetraethylenepentamine, polyphenylenediamine, and a mixture of two or more of these polyamine compounds. Preferred polyamines are water soluble and / or have a molecular weight of about 500 to 100,000 daltons. In certain embodiments, a polyethyleneimine such as EPOMIN P-1050 can be used.

  Amine-reactive materials useful for preparing adducts containing enzymes herein can be, for example, aldehydes, organic halides, anhydrides, azo compounds, isothiocyanates and / or isocyanates. Examples of these amine-reactive substances include glutaraldehyde, succindialdehyde, terephthalaldehyde, bis-diazobenzidine-2,2′-disulfonic acid, 4,4′-difluoro-3,3′-. Dinitrodiphenylsulfone, diphenyl-4,4′-dithiocyanate-2,2′-disulfonic acid, 3-methoxydiphenylmethane-4,4′-diisocyanate, toluene-2-isocyanate-4-isothiocyanate, toluene-2, − 4-diisothiocyanate, diazobenzidine, diazobenzidine-3,3′-dianisidine, N, N′-hexamethylenebisiodoacetamide, hexamethylene diisocyanate, cyanuric chloride and / or 1,5-difluoro-2,4-dinitrobe Zen and the like. Preferably, the amine reactive material is an aldehyde such as glutaraldehyde.

  The alpha-glucosidase enzyme to which the amine-reactive compound has been added can be contacted with the polyamine-treated solid support, thereby immobilizing the enzyme on the solid support. The immobilized enzyme herein may be used in a variety of reactor systems, such as in a column (eg packed column) or stirred tank reactor, to perform a hydrolysis reaction as disclosed herein. Can do.

  Appropriate conditions for contacting a sugar with an alpha-glucosidase (eg, transglucosidase) herein are one or more alpha-1,3 or alpha-1,6 glucosyl-in the sugar by the alpha-glucosidase. It is a condition that supports hydrolysis of glucose bonds. Examples of suitable conditions are disclosed in the examples below. Conditions for contacting alpha-glucosidase with the sugar substrate (eg, temperature, pH, time) are also described in US 2008/0229514, US 7413887 and US 2013/2013. No. 0102035, which are all incorporated herein by reference, may also be applicable to the disclosed hydrolysis method.

The disaccharides and oligosaccharides in the disclosed hydrolysis methods are generally soluble in water or aqueous solutions. Thus, contacting the sugar herein with alpha-glucosidase is preferably performed under suitable conditions that are aqueous, in which the sugar is dissolved. Aqueous conditions can characterize a solution or mixture comprising at least about 20 wt% water. Alternatively, the aqueous conditions herein are, for example, 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%). It is. Aqueous conditions may further include a buffer, such as an acidic, neutral or alkaline buffer, of appropriate concentration and selected based on the pH range provided by the buffer. Examples of buffers / buffering agents include citric acid, acetic acid (eg, sodium acetate), KH ₂ PO ₄ , MOPS, CHES, boric acid, sodium carbonate and sodium bicarbonate.

  The pH of the hydrolysis reaction herein can be, for example, about 3.0 to 9.0. The hydrolysis reaction pH is, for example, about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8 0.0, 8.5 or 9.0. Alternatively, the pH can be about 4-5. Techniques for setting the pH include, for example, the use of buffers, alkalis and / or acids and are well known in the art.

  The temperature of the hydrolysis reaction herein can be, for example, from about 20 ° C to about 80 ° C. The hydrolysis reaction temperature can be, for example, about 20, 30, 40, 50, 60, 70, or 80 ° C. (or any integer value between 20-80 ° C.). In certain embodiments, a hydrolysis temperature of about 60 ° C, 65 ° C or 60-65 ° C is preferred.

  The hydrolysis reaction herein can be conducted for at least, for example, about 10 minutes to about 90 hours. The time of the hydrolysis reaction is, 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, It can be 84 or 90 hours (or any integer value between 0.5 and 72 hours). In certain embodiments, the hydrolysis reaction can be performed, for example, for less than 4 hours (eg, 0.5-4 hours). The time required to achieve the desired level of hydrolysis will vary depending on the exact conditions used and will be understood by those skilled in the art. For example, increasing the amount of enzyme added to the reaction or immobilized on the solid support used in the reaction reduces the contact time.

In certain embodiments, one or more of the alpha-glucosidase enzymes herein may be used in the hydrolysis reaction. For example, both transglucosidase and glucoamylase can be used in the reaction. The amount of alpha-glucosidase in the hydrolysis reaction herein is, for example, ± 10% to 20% (or 5% to 10%) from any of the amounts used in the following examples (eg, Example 2). ). Alternatively, about 0.1-0.5 vol% or 0.1-1.0 vol% alpha-glucosidase can be used in the hydrolysis reaction. Alternatively, the alpha-glucosidase herein may be about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ppm in the hydrolysis reaction. Can be used. A transglucosidase unit (TGU) can be defined, for example, as the amount of transglucosidase enzyme that produces 1 micromole of panose per minute under the following assay conditions. Transglucosidase activity can be assayed, for example, as follows: transglucosidase in 100 mM sodium acetate buffer, pH 4.5, containing 4 mM para-nitrophenyl-alpha-glucoside and 1 mg / mL bovine serum albumin (BSA). Adjust. After incubating at 30 ° C. for 30 minutes, the reaction is stopped by the addition of an equal volume of 1M sodium carbonate and the OD ₄₀₅ is recorded. The glucoamylase unit (GAU) is, for example, as the amount of glucoamylase enzyme that produces 1 g of reducing sugar, calculated as glucose / hour from a soluble starch substrate (4% DS [degree of substitution]) at pH 4.2 and 60 ° C. Can be defined.

  In certain embodiments of the disclosed invention, the initial concentration of sugar during the hydrolysis reaction can be, for example, from about 1 wt% to 50 wt%. For example, the concentration of leucorose 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 (eg, DP2, DP3, DP4, DP2-DP7, DP3-DP7) during the hydrolysis reaction herein is about 1, 2, 3, It may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 wt%. One skilled in the art recognizes that the total concentration of sugars (including disaccharides and oligosaccharides) can affect the activity of the alpha-glucosidase enzyme; preferred sugars in the hydrolysis reaction to maximize enzyme activity The total concentration of can be less than 50 wt% dry solids (DS), and in some embodiments the most preferred concentration can be 20-35 wt% DS.

  Suitable conditions in certain embodiments for contacting a sugar with an alpha-glucosidase herein may include (i) a glucan synthesis reaction, or (ii) a fraction obtained from a glucan synthesis reaction, Here, sugar is a byproduct of glucan synthesis reaction. In other words, the hydrolysis reaction in the present specification can be performed in the situation of glucan synthesis reaction or fractionation of glucan synthesis reaction, but is generally performed in the latter. The glucan synthesis reaction herein can produce, for example, one or more insoluble and / or soluble alpha-glucan products. Thus, a glucan synthesis reaction may 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 may be present during the glucan synthesis reaction include fructose, glucose, leucus, soluble oligosaccharides (eg DP2-DP7) and soluble glucan products. One or more alpha-glucanohydrolase enzymes may also be included in the glucan synthesis reaction in some embodiments. It is understood that certain glucan products, such as polyalpha-1,3-glucan, with a DP of at least 8 or 9, can be insoluble in water and therefore do not dissolve in the glucan synthesis reaction, but rather can emerge from solution. Let's be done. Therefore, the glucan produced by the glucan synthesis reaction herein can be insoluble. The alpha-glucosidase enzyme herein may be added to the glucan synthesis reaction at any stage thereof, such as during initial preparation of the reaction or near the end of the reaction (eg, 80-90% completion) or at the end of the reaction, The latter two time points are preferred.

  The glucan synthesis reaction herein can generate byproducts such as leuco and / or soluble oligosaccharides in addition to generating a glucan product. The glucan is polyalpha-glucan in some embodiments. Thus, the glucan synthesis reaction herein, for example, to produce polyalpha-1,3-glucan or mutan that is generally co-produced with at least leucos and / or oligosaccharide by-products in the glucan synthesis reaction, for example. Can be.

  In certain embodiments, the glucan synthesis reaction includes a glucosyltransferase enzyme that produces a polyalpha-glucan, such as polyalpha-1,3-glucan. Examples of such glucosyltransferase enzymes useful herein include U.S. Patent No. 7000000 and U.S. Patent Application Publication Nos. 2013/0244288, 2013/0244287, and 2014/2014. No. 0087431, which is hereby incorporated by reference in its entirety.

  The glucosyltransferase enzyme herein may be derived from any microbial source, such as bacteria or fungi. Examples of bacterial glucosyltransferase enzymes are from the genus Streptococcus species, Leuconostoc species or Lactobacillus species. Examples of Streptococcus species include S. cerevisiae. S. salivarius, S. S. sobrinus, S. DENTIROUSSETTI, S. S.downei, S.D. S. mutans, S. mutans S. oralis, S. S. gallyticus and S. galoliticus. S. sanguinis is mentioned. Examples of Leuconostoc species include L. leuconostoc species. L. mesenteroides, L. L. amelibiosum, L. Argentinum, L. L. carnosum, L. carnosum. L. citreum, L. L. cremoris, L. L. dextranicum and L. dextranicum. Fructosum (L.fructosum) is mentioned. Examples of Lactobacillus species include L. lactobacillus species. L. acidophilus, L. L. delbrueckii, L. L. helveticus, L. L. salivarius, L. L. casei, L. L. curvatus, L. Plantarum, L. plantarum. L. sakei, L. Brevis (L. brevis), L. L. buchneri, L. Fermentum and L. fermentum. Reuteri (L. reuteri).

  The glucosyltransferase enzyme herein can be primer-independent or primer-dependent. Primer-independent glucosyltransferase enzymes do not require the presence of primers to perform glucan synthesis. Primer-dependent glucosyltransferase enzymes require 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 an initiator for a glucosyltransferase enzyme. Primers that can be used in certain embodiments include, for example, primers based on dextran and other carbohydrates, such as hydrolyzed glucans. US 2013/0244287, which is incorporated herein by reference, discloses the preparation of hydrolyzed glucan using polyalpha-1,3-glucan as a starting material. Dextran for use as a primer can be, for example, dextran T10 (ie, dextran having a molecular weight of 10 kD).

  The glucosyltransferase enzyme for the glucan synthesis reaction herein can be produced by any means known in the art. For example, the glucosyltransferase enzyme can be produced recombinantly in a heterologous expression system, such as a microbial heterologous expression system. Examples of heterologous expression systems include bacteria (eg, E. coli, such as TOP10 or MG1655; Bacillus sp.) And eukaryotes (eg, yeasts such as Pichia sp. And Saccharomyces). Expression systems (such as Saccharomyces sp.).

  The glucosyltransferase enzymes described herein can be used in any purification context (eg, pure or impure). For example, the glucosyltransferase enzyme can be purified and / or isolated prior to its use. Examples of impure glucosyltransferase enzymes include those in the form of cell lysates. Cell lysates or extracts can be prepared from bacteria (eg, E. coli) used to heterologously express the enzyme. For example, bacteria can be destroyed using a French press cell. In an alternative embodiment, the bacteria may be homogenized with a homogenizer (eg, APV, Rannie, Gaulin). Glucosyltransferase enzymes are generally soluble in these types of preparations. The bacterial cell lysate, extract or homogenate herein is used, for example, in a reaction solution to produce about 0.15 to about 0.1. Can be used at 3% (v / v).

  The temperature of the glucan synthesis reaction in the present specification can be adjusted as necessary. In certain embodiments, the temperature of the reaction is from about 5 ° C to about 50 ° C. In certain other embodiments, the temperature is from about 20 ° C to about 40 ° C.

  The initial concentration of sucrose in the glucan synthesis reaction herein can be, for example, from about 20 g / L to about 400 g / L. Alternatively, the initial concentration of sucrose can be about 75 g / L to about 175 g / L or about 50 g / L to about 150 g / L. Alternatively, the initial concentration of sucrose may be, for example, about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 g / L (or any of 40-160 g / L). Numerical value). “Initial concentration of sucrose” refers to the sucrose concentration in the gtf reaction solution immediately after all of the reaction solution components (at least water, sucrose, gtf enzyme) are added.

  The sucrose used in the glucan synthesis reactions herein can be very pure (≧ 99.5%) or any other purity or grade. For example, the purity of sucrose can be at least 99.0% or can be reagent grade sucrose. As another example, incompletely purified sucrose can be used. Incompletely purified sucrose herein refers to sucrose that has not been processed into white purified sucrose. Thus, incompletely purified sucrose can be completely unpurified or partially purified. Examples of unpurified sucrose are “crude sucrose” (“crude sugar”) and solutions thereof. Examples of partially purified sucrose have not gone through 1, 2, 3 or more crystallization steps. The ICUMSA (International Commission for Uniform Methods of Sugar Analysis) of incompletely purified sucrose herein may be, for example, greater than 150. The sucrose herein may be derived from any renewable sugar source, such as sugar cane, sugar beet, cassava, sorghum or corn. Suitable forms of sucrose useful herein are, for example, in crystalline or amorphous form (eg syrup, sugar cane juice, beet juice). Further suitable forms of incompletely purified sucrose are disclosed in US patent application Ser. No. 61 / 969,958.

  Methods for determining ICUMSA values for sucrose are well known in the art and are described, for example, by ICUMSA Methods of Sugar Analysis: Official and Tentative Methods in the United States of the United States, which are incorporated herein by reference. of Sugar Analysis (ICUMSA) (Ed. HC S. de Halleley, Elsevier Pub. Co., 1964), disclosed by International Commission for Uniform Methods of Sugar Analysis. ICUMSA is described, for example, in R.C., which is incorporated herein by reference. J. et al. McCowage, R.M. M.M. Urquart and M.M. L. Burger (Measured by the Deformation of the Solution Color of Raw Sugars, Brown Sugars and Colored Syrups at pH 7.0-Official, Verlag Dr Albert Barts, G it can.

  In certain embodiments, the pH of the glucan synthesis reaction can be 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 adjusted by the addition or incorporation of a suitable buffer including, but not limited to, phosphate, tris, citrate or combinations thereof. The buffer concentration in the glucan synthesis reaction can be, for example, 0 mM to about 100 mM or about 10, 20 or 50 mM.

  The polyalpha-1,3-glucan produced in the glucan synthesis reaction herein is at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% or 100% (or any integer value from 50% to 100%) of glycoside bonds that are alpha-1,3. Thus, in such embodiments, polyalpha-1,3-glucan is about 50%, 40%, 30%, 20%, 10%, 5%, 4% of glycoside linkages that are not alpha-1,3. 3%, 2%, 1% or 0% (or any integer value between 0% and 50%).

  The polyalpha-1,3-glucan in the present specification preferably has a skeleton that is linear / unbranched. In certain embodiments, the polyalpha-1,3-glucan is unbranched, or about 10%, 9%, 8%, 7%, 6%, as a percent of glycosidic bonds in the polymer, It has a branch point of less than 5%, 4%, 3%, 2% or 1%. Examples of branch points include alpha-1,6-branch points.

The molecular weight of the polyalpha-1,3-glucan produced in the glucan synthesis reaction herein can be measured as a number average molecular weight (M _n ) or a 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 poly-alpha-1,3-glucan polymer DP _w (weight average degree of polymerization) or DP _n (number average degree of polymerization).

The M _n or M _w of the polyalpha-1,3-glucan herein may be at least about 1000. Alternatively, M _n or M _w can be, for example, at least about 1000 to about 600000 (or any integer value from 1000 to 600000). Alternatively, the molecular weight of the poly-alpha-1,3-glucan can be at least about 100, or at least about 100 to 1000 (or any integer value of 100 to 1000) with DP _n or DP _w.

  The fraction of the glucan synthesis reaction can establish conditions suitable for contacting the sugar with an alpha-glucosidase as disclosed herein. The fraction can be part or all of the solution from the glucan synthesis reaction. In general, the fraction is separated from the soluble or insoluble glucan product synthesized in the reaction. For example, fractions falling from their in-situ solution can be separated from one or more glucan products that are insoluble in water (eg, polyalpha-1,3-glucan). The fraction in certain preferred embodiments of the present disclosure is derived from a polyalpha-1,3-glucan synthesis reaction.

  The volume of the fraction (optionally before diluting or concentrating the fraction, see below) is, in certain embodiments, at least about 10%, 20%, 30% of the volume of the glucan synthesis reaction from which it is obtained. , 40%, 50%, 60%, 70%, 80% or 90% (or any integer value between 10% and 90%). In general, in a glucan synthesis reaction that produces insoluble glucan (eg, polyalpha-1,3-glucan), the fraction will be part (but not all) of the solution components of the reaction. Fractions can be obtained at any stage of the glucan synthesis reaction, but are preferably obtained near completion of the reaction (eg, 80 or greater than 90% completion) or after completion of the reaction.

  Examples of fractions of glucan synthesis reactions include filtrate and supernatant in certain embodiments. Thus, in embodiments where an insoluble glucan product is synthesized, funnels, filters, (eg, press filters), centrifuges or any known in the art that allow some or all of the liquid to be removed from the solids. Other methods or instruments can be used to obtain (separate) the fractions herein from a glucan synthesis reaction. Filtration can be, for example, by gravity, vacuum or pressure filtration. Filtration preferably removes all or most of the insoluble glucan; using any filter material (eg filter paper) with an average pore size (eg about 40-50 microns) sufficient to remove solids from the liquid. it can. The fraction generally retains all or most of its dissolved components, such as byproducts of the glucan synthesis reaction.

  The fractions herein can be optionally diluted or concentrated as required. Concentration of the fraction can be performed using any other method or equipment known in the art suitable for concentrating the solution. For example, the fractions can be concentrated by evaporation, such as by using a rotary evaporator (eg, set to a temperature of about 40-50 ° C.). The fraction can be concentrated to a volume that in some embodiments herein is about 75%, 80%, 85%, 90% or 95% of the original fraction volume. A concentrated fraction (eg, concentrated filtrate) may optionally be referred to as a syrup.

  The fraction may in some embodiments comprise water in place of the water present in the composition from which the fraction was obtained. For example, sugar by-products from a glucan synthesis reaction can be separated in some chromatographic methods in which the original solvent is replaced with another solvent (eg, sugar by-products bound to the column [and thus removed from the original solvent). Can be eluted in a new solvent).

  The fraction may in some embodiments be treated to have any of the appropriate conditions disclosed above (eg, temperature, pH, and time) to contact the sugar with alpha-glucosidase. For example, the fraction can be modified to have a pH of about 4-5 prior to adding alpha-glucosidase to the fraction. As another example, the temperature of the hydrolysis reaction with the fraction can be about 55-65 ° C (eg, about 60 ° C). In another example, a fraction that has been concentrated to syrup in the hydrolysis reaction can be used.

  The fraction is, in certain preferred embodiments herein, derived from a polyalpha-1,3-glucan synthesis reaction; such a fraction is preferably a filtrate. The fraction of the polyalpha-1,3-glucan synthesis reaction herein includes at least water, fructose and one or more types of sugars (leucrose and / or oligosaccharides such as DP2-DP7). Other components that may be in this type of fraction include, for example, sucrose (ie, residual sucrose that was not consumed in the gtf reaction), one or more gtf enzymes, glucose, buffers, salts, FermaSure®. , Boric acid, sodium hydroxide, hydrochloric acid, cell lysate components, proteins and / or nucleic acids. At a minimum, the components of the fraction from the polyalpha-1,3-glucan synthesis reaction are, for example, water, fructose, glucose, one or more types of sugars (leucrose and / or oligosaccharides such as DP2-DP7) and Optionally includes sucrose. It is understood that the composition of the fraction depends in part on the conditions of the glucan synthesis reaction from which the fraction is obtained. In fractions containing one or more gtf enzymes, such one or more gtf enzymes may be inactivated (eg, heat inactivated) prior to use of the fraction in the hydrolysis reactions herein. Is preferred.

  It should be understood that the exact distribution of sugar by-products produced through the polymerization of sucrose in the glucan synthesis reaction can vary based on the reaction conditions used and the gtf enzyme, particularly temperature and sucrose concentration. It should also be understood that the exact composition of sugars in the fractions of the glucan synthesis reaction is not critical to the disclosed hydrolysis process. In general, increasing the amount of sucrose improves the selectivity of the reaction for both leucus and oligosaccharides. Conversely, when the temperature rises, the selectivity of the reaction against leucus tends to decrease, while the selectivity for oligosaccharides is not significantly affected. The ratio of sugar to water, ie wt% dry solid (DS) calculated by dividing the mass of sugar relative to the total solution weight, preferably evaporates the water at a temperature below 50 ° C. under vacuum. It should also be understood that either the addition of water or the addition of water can be adjusted without significantly affecting the relative distribution of sugars in the fractions of the glucan synthesis reaction. Stop the gtf reaction before full conversion (to glucan) is achieved, either by lowering the pH below the activity range for the gtf enzyme or by heat inactivation of the gtf enzyme. It is also possible to increase the percentage of sucrose in the fraction.

  In certain embodiments, the glucan synthesis reaction herein can produce one or more soluble alpha-glucan products. Soluble alpha-glucan product (or “soluble fiber”) is either (i) a direct product of glucosyltransferase, or (ii) one or more alpha-1,3-glycosidic linkages or one or more alpha-1, It can be the product of the concerted action of both glucosyltransferase and alpha-glucanohydrolase capable of hydrolyzing glucan polymers with 6-glycosidic linkages.

Soluble alpha-glucan herein may include, for example:
a) at least 75% alpha-1,3-glycosidic linkages;
b) Less than 25% alpha-1,6-glycosidic linkages;
c) Less than 10% alpha-1,3,6-glycosidic linkages;
d) M _w 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) dextrose equivalent (DE) in the range of 4-40;
g) Less than 10% digestibility as measured by Association of Analytical Communities (AOAC) method 2009.01;
h) Solubility of at least 20% (w / w) in water at pH 7 at 25 ° C .; and i) Polydispersity index (PDI) of less than 5.

  Such soluble alpha-glucans can be produced as disclosed in US Patent Application No. 62 / 004,290.

  By way of example, a soluble alpha-glucan fiber composition has at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95% alpha- (1,3 ) It may contain glycosidic bonds.

  As another example, in addition to the alpha- (1,3) glycosidic linkage embodiment described above, the soluble alpha-glucan fiber composition is less than 25%, preferably less than 10%, more preferably less than 5%, even more Preferably it may further comprise less than 1% alpha- (1,6) glycosidic linkage.

  As another example, in addition to the alpha- (1,3) and alpha- (1,6) glycoside bond content embodiments described above, the soluble alpha-glucan fiber composition is less than 10%, preferably less than 5%. Most preferably, it may further comprise less than 2.5% alpha- (1,3,6) glycosidic bonds.

  As another example, a soluble alpha-glucan fiber composition may comprise 93-97% alpha- (1,3) glycosidic linkages and less than 3% alpha- (1,6) glycosidic linkages, with 3-7 DP Having a weight average molecular weight corresponding to a mixture of In a further embodiment, the soluble alpha-glucan fiber composition may comprise about 95% alpha- (1,3) glycosidic linkage and about 1% alpha- (1,6) glycosidic linkage, wherein 3-7 DP Having a weight average molecular weight corresponding to a mixture of In a further aspect of the above embodiment, the soluble alpha-glucan fiber composition has 1 to 3% alpha- (1,3,6) linkage or preferably about 2% alpha- (1,3,6) linkage. Further may be included.

  As another example, in addition to the glycoside bond content embodiment described above, the soluble alpha-glucan fiber composition has less than 5%, preferably less than 1%, most preferably less than 0.5% alpha- (1, 4) It may further contain a glycosidic bond.

As another example, in addition to the glycoside bond content embodiments described above, the soluble alpha-glucan fiber composition is less than 5000 daltons, preferably less than 2500 daltons, more preferably 500-2500 daltons, most preferably about 500- A weight average molecular weight (M _w ) of about 2000 Daltons may be included.

  As another example, in addition to any of the above properties, the soluble alpha-glucan fiber composition is 12 wt% in water at 20 ° C., less than 250 centipoise (0.25 Pa · s), preferably 10 cP (0.01 Pa · s). 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), most preferably 3 cP (0.003 Pa · s). May contain a viscosity of less than s).

  The soluble alpha-glucan fiber composition has, in certain embodiments, less than 10% digestibility as measured by the Association of Analytical Communities (AOAC) method 2009.01, or preferably 9%, 8%, 7% , 6%, 5%, 4%, 3%, 2% or less than 1% digestibility. In another embodiment, the relative level of digestibility is alternatively determined using AOAC 20111.25 (Integrated Total Dietary Assay) (McCleary et al., 2012, J. AOAC Int., 95 (3), 824-844). Can be determined.

  In addition to any of the above embodiments, the soluble alpha-glucan fiber composition is at least 20% (w / w), preferably at least 30%, 40%, 50%, 60%, in water at pH 7 at 25 ° C. It can have a solubility of 70%.

  In one embodiment, the soluble alpha-glucan fiber composition may comprise a reducing sugar content of less than 10 wt%, preferably less than 5 wt%, most preferably 1 wt% or less.

  In one embodiment, the soluble alpha-glucan fiber composition may comprise a caloric content of less than 4 kcal / g, preferably less than 3 kcal / g, more preferably less than 2.5 kcal / g, most preferably about 2 kcal / g or less. .

As another example, the soluble alpha-glucan herein may include:
a) 10-30% alpha-1,3-glycoside 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 (M _w ) 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) dextrose equivalent (DE) in the range of 4-40, preferably 10-40;
g) Less than 10% digestibility as measured by Association of Analytical Communities (AOAC) method 2009.01;
h) Solubility of at least 20% (w / w) in water at pH 7 at 25 ° C .; and i) Polydispersity index (PDI) of less than 5.

  Such soluble alpha-glucans can be produced as disclosed in US Patent Application No. 62 / 004,308.

As another example, the soluble alpha-glucan herein may include:
a) an alpha-1,3-glycoside linkage of 25-35;
b) 55-75% alpha-1,6-glycosidic linkages;
c) 5-15% alpha-1,3,6-glycosidic linkage;
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) dextrose equivalent (DE) in the range of 4-40;
g) Less than 10% digestibility as measured by 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 polydispersity index of less than 5.

  Such soluble alpha-glucan can be produced as disclosed in US Patent Application No. 62 / 004,312.

As another example, the soluble alpha-glucan herein may include:
a) at least 95% alpha-1,6-glycosidic linkages;
b) 1% or less alpha-1,3-glycoside linkage;
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 20,000 daltons;
f) a viscosity of less than 0.25 Pascal second (Pa · s) at 12 wt% in water at 20 ° C .;
g) dextrose equivalent (DE) in the range of 1-30;
h) 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 water at pH 7 at 25 ° C .; and j) a polydispersity index of less than 5.

  Such soluble alpha-glucans can be produced as disclosed in US Patent Application No. 62 / 004,314.

As another example, the soluble alpha-glucan herein may include:
a) a series of
i) 1% to 50% alpha-1,3-glycoside linkage; or ii) greater than 10% but less than 40% alpha-1,4-glycoside linkage; or iii) any combination of i) and ii);
b) 1-50% alpha-1,2-glycosidic linkages;
c) 0-25% alpha-1,3,6-glycosidic linkage;
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) Less than 20% digestibility as measured by Association of Analytical Communities (AOAC) method 2009.01;
h) a solubility of at least 20% (w / w) in water at pH 7 at 25 ° C .; and i) a polydispersity index of less than 26, preferably less than 5.

  Such soluble alpha-glucans can be produced as disclosed in US Patent Application No. 62 / 004,305.

  In certain embodiments, the soluble alpha-glucan is a direct product of glucosyltransferase. Conditions for such glucosyltransferases and their use in suitable glucan synthesis reactions may be as disclosed herein or, for example, in US patent application 62 / 004,290, ibid. Disclosed in any of 62 / 004,308, 62 / 004,312, 62 / 004,314 and / or 62 / 004,305. It can be as follows.

  Soluble alpha-glucans are alternatively glucosyltransferases and alpha-glucano capable of hydrolyzing glucan polymers having, for example, one or more alpha-1,3-glycosidic linkages or one or more alpha-1,6-glycosidic linkages. It can be the product of both concerted actions of hydrolase. In some embodiments, the glucan synthesis reaction to produce a soluble alpha-glucan product can include both at least one glucosyltransferase and at least one alpha-glucanohydrolase. In other embodiments, the glucan synthesis reaction may initially include one or more glucosyltransferases as the only enzyme component. Such a reaction produces a first alpha-glucan product that has not yet been subjected to modification by alpha-glucanohydrolase. Then, at least one alpha-glucanohydrolase is added to the reaction for a time appropriate to allow modification of the first product to the soluble alpha-glucan product. Thus, there are various methods for synthesizing soluble alpha-glucan products through the concerted action of both glucosyltransferase and alpha-glucanohydrolase. Conditions for conducting a glucan synthesis reaction that includes one or more alpha-glucanohydrolase enzymes during and / or after the glucan synthesis reaction may be as disclosed herein or, for example, in the United States Patent applications 62 / 004,290, 62 / 004,308, 62 / 004,312, 62 / 004,314 and / or 62 / 004,305 may be as disclosed in any specification.

  As used herein, alpha-glucanohydrolase is, for example, dextranase (which can hydrolyze alpha-1,6-linked glycosidic bonds; EC 3.2.1.11), mutanase (alpha-1 , 3-linkable glycosidic bonds can be hydrolyzed; EC 3.2.2.1.59), mycodextranase (alpha containing both (1-3)-and (1-4) -linkages Endohydrolyzing (1-4) -alpha-D-glucoside bonds in D-glucan; EC 3.2.1.61), glucan 1,6-alpha-glucosidase (EC 3.2.1.70) And alternanase (alternan can be cleaved endo-hydrolyzable; EC 3.2.1.-; see US Pat. No. 5,786,196).

  A mutanase comprising SEQ ID NO: 47 can be used in certain embodiments. Alternatively, the mutanase may 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, eg, mutanase May have activity.

  The glucan synthesis reaction as disclosed herein to produce one or more soluble alpha-glucan products uses an alpha-glucosidase to hydrolyze alpha-1,5 glucosyl-fructose bonds. It can serve directly as a suitable condition for carrying out the hydrolysis reaction in the book. Such hydrolysis can be performed according to any of the conditions disclosed above for the hydrolysis treatment of a glucan synthesis reaction that produces, for example, polyalpha-1,3-glucan. Alternatively, fractions of the glucan synthesis reaction (eg, chromatographic fractionation) to produce one or more soluble alpha-glucan products can be obtained by alpha-glucosidase-mediated hydrolysis of alpha-1,5 glucosyl-fructose linkages. It can be used as an appropriate condition for performing.

  The fraction may be a chromatographic fraction of a glucan synthesis reaction in certain embodiments herein. 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 may optionally include one or more alpha-glucanohydrolases during and / or after completion of glucan synthesis. Fractionation in any of these types of embodiments generally results in all or most of the soluble alpha-glucan product from the reaction composition from which it was produced (eg, at least about 60%, 70%, 80% %, 90%, 95%). Once separated from all or most of the soluble alpha-glucan product, one or more alpha-glucanases are used to fractionate any of the alpha-1,5 glucosyl-fructose hydrolysis processes disclosed herein. Can be used.

  The chromatographic fractions herein can generally be obtained using any suitable type of liquid chromatography. Liquid chromatography can be performed using, for example, size exclusion chromatography (SEC), column chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, affinity chromatography, ultrafiltration, microfiltration or dialysis. .

  The disclosed invention also relates to a composition produced by contacting a sugar with an alpha-glucosidase (eg, transglucosidase), wherein (i) the sugar comprises at least one alpha-1,3 Or a disaccharide or oligosaccharide containing an alpha-1,6 glucosyl-glucose linkage, and (ii) the alpha-glucosidase contains at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the sugar. Hydrolyzes. The composition thus produced contains a reduced amount of sugar compared to the amount of sugar present prior to contact. Examples of compositions include any of those disclosed herein, such as a hydrolysis fraction from a glucan synthesis reaction or a hydrolysis fraction of a glucan synthesis reaction used to produce soluble alpha-glucan. Can be mentioned. Any of the properties and products disclosed above and in the examples regarding the hydrolysis method may characterize the composition. The following properties of the composition are examples.

  The alpha-glucosidase enzyme is, in certain embodiments of the composition, SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or DIAZYME RDF ULTRA (DuPont Industrial Biosciences) amino acid sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% It may contain amino acid sequences that are identical. The transglucosidase may comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 in certain embodiments of the composition. Alternatively, any of the alpha-glucosidases disclosed herein can be used to produce the disclosed compositions.

  The sugar in certain embodiments of the composition has a degree of polymerization of 3-7 prior to hydrolysis.

  The composition produced by the hydrolysis methods herein may have a sugar concentration of, for example, less than 50% of the sugar concentration present prior to contacting the sugar with alpha-glucosidase.

  The composition produced by the hydrolysis method in certain embodiments herein can be a glucan synthesis reaction or a fraction thereof, wherein the sugar byproduct of the glucan synthesis reaction is contacted with alpha-glucosidase. The fraction in this embodiment can be a glucan synthesis reaction filtrate or a fraction of a glucan synthesis reaction used to produce, for example, soluble alpha-glucan. The sugar in this embodiment can have a degree of polymerization of, for example, 3-7 before hydrolysis.

  It will be appreciated by those skilled in the art that the presently disclosed embodiments are useful, in part, for saccharifying disaccharides and oligosaccharides that may otherwise be difficult to degrade. This property can be exploited, for example, to perform (i) fructose enrichment and (ii) methods of promoting fermentation.

  In Example 6 below, chromatographic fructose concentration is enhanced when a glucan filtrate hydrolyzed with alpha-glucosidase (transglucosidase) is used compared to when a non-hydrolyzed filtrate is used. Make it clear.

  Accordingly, the disclosed invention further relates to a method for concentrating fructose present in a fraction of a glucan synthesis reaction. This method comprises (a) contacting a fraction obtained from a glucan synthesis reaction with alpha-glucosidase (eg, transglucosidase) under appropriate conditions, wherein the enzyme is a disaccharide or Hydrolyzing, contacting, at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond of the oligosaccharide; and (b) higher than the fructose concentration of the fraction of step (a). Separating fructose from the hydrolyzed fraction of step (a) to obtain a composition having a concentration of fructose.

  For example, characteristics of the disclosed fructose enrichment method for alpha-glucosidase (eg, transglucosidase) enzymes and fractionation of glucan synthesis reactions may follow any of the disclosures provided herein for each of these characteristics.

  The step (b) of separating fructose can be performed by any means known in the art. For example, chromatography can be used as disclosed in the Examples below, or by following the disclosure of European Patent Publication No. 2292803B1, which is incorporated herein by reference.

  Higher fructose compositions (eg, fructose solutions or fructose syrups) resulting from the disclosed concentration methods contain at least about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 wt% fructose. Can have.

  The fructose concentration method herein can be performed better than using a filtrate that has not been hydrolyzed with alpha-glucosidase as disclosed herein. Such performance improvement can be measured with respect to percent fructose recovery of at least 40%, 45% or 50%.

  The present disclosure includes (a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme (eg, transglucosidase or glucoamylase) under suitable conditions, wherein the alpha-glucosidase enzyme is fractionated. Hydrolyzing and contacting at least one alpha-1,5 glucosyl-fructose linkage of a disaccharide or oligosaccharide contained therein, and (b) using a microorganism to obtain a product of step (a) It further relates to a fermentation process comprising fermenting the fraction and (c) optionally isolating the product of (b). The fermentation process of (b) can be performed after process (a) or simultaneously with process (a). Importantly, this method can be used to produce ethanol, for example, by fermenting the hydrolysis filtrate of a glucan synthesis reaction. The ethanol yield from such a process is higher than the ethanol yield obtained when fermenting an unhydrolyzed glucan filtrate.

  Properties of the disclosed fermentation methods, disaccharides and oligosaccharides, glucan synthesis reactions and suitable contact conditions for alpha-glucosidase (eg, transglucosidase or glucoamylase) enzymes are described herein, for example, for each of these properties. Any of the disclosure provided in the document may be followed.

  Microorganisms for use in the fermentation methods herein can be, for example, bacteria, yeasts or fungi. Examples of bacteria useful herein include the genus Lactobacillus species, Streptococcus species, Bifidobacterium species, Leuconosestocia, Escherichia species (eg, E. coli) and Bacillus species. Examples of yeast useful herein include Saccharomyces species, such as S. cerevisiae. S. cerevisiae and S. cerevisiae Examples include S. bayanus.

  The fermentation methods herein can produce products such as ethanol or acids (eg lactic acid). However, it is believed that other products can be produced as needed. It will be appreciated by those skilled in the art that the production of certain products using fermentation methods as disclosed will depend on various conditions such as the microorganisms used in the fermentation. The conditions for fermentation herein are as disclosed in the examples below or, for example, El-Mansi et al., Both incorporated herein by reference. (2006, Fermentation Microbiology and Biotechnology, Second Edition, CRC Press) and Stanbury et al. (1999, Principles of Fermentation Technology, Second Edition, Butterworth-Heinemann).

  The yield of the product in certain embodiments of the fermentation process herein is the product yield obtained when fermenting the glucan filtrate not hydrolyzed with alpha-glucosidase herein. Higher than the rate. This comparison may be for a control fermentation using a non-hydrolyzed fraction of the glucan synthesis reaction, for example. Product yield of fermentation herein is improved, for example by at least about 10%, 20%, 40%, 60%, 80% or 100% (or any integer value between 10% and 100%) Can be made. Furthermore, the rate of product formation by fermentation in this specification can be improved.

  Example 7 below demonstrates that leucrose can be fermented to ethanol by yeast provided with a feed containing unhydrolyzed glucan filtrate. Accordingly, further disclosed herein is a method of using a microorganism to ferment leucus to produce a product (eg, ethanol). Such a method may comprise fermenting a (i) hydrolyzed or (ii) non-hydrolyzed glucan filtrate with an alpha-glucosidase as disclosed herein. Regardless of whether or not leucus is provided in the glucan filtrate or in another form (eg, semi-purified or concentrated form), the method for fermenting leuclos is a microorganism (eg, yeast, Acclimatizing S. cerevisiae, for example). Such adaptation can be achieved, for example, by allowing the microorganism to grow in the presence of leucos and optionally other sugars over at least 2 or 3 growth cycles, and then the microorganism to produce more leucos to ferment the product. Can be included. In certain embodiments, the microorganism is grown in a first feed comprising (i) leuclos (one cycle complete), (ii) recovered from the first feed, and (iii) a second comprising leuclos. Can be grown in feed (2 cycle completion), (iv) optionally removed from the second feed, and (v) optionally grown in the third feed (3 cycle completion) . Microorganisms adapted in this way may have improved leusucrose fermentability in certain embodiments.

  Example 9 below shows that when glucan filtrate is hydrolyzed with transglucosidase while simultaneously fermenting with yeast, almost all present in the glucan filtrate for fermentation by yeast (eg> 98% or> 99%). ) To make it possible to use Leucrose. Thus, facilitating the leuclos fermentation method herein may include simultaneously hydrolyzing leuclos with alpha-glucosidase (eg, transglucosidase or glucoamylase) while fermenting leuclos with a microorganism.

Non-limiting examples of compositions and methods disclosed herein include the following:
1. A method for hydrolyzing an alpha-1,3 or alpha-1,6 glucosyl-glucose bond in a sugar comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond, wherein the sugar is a disaccharide Or an oligosaccharide and the method comprises contacting the sugar with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme is at least one alpha-1,3 or alpha-1,6 glucosyl of the sugar. A method wherein the glucose bonds are hydrolyzed and the amount of sugar is reduced compared to the amount of sugar present before contact;
2. The method of embodiment 1, wherein the alpha-glucosidase enzyme is immobilized.
3. The method according to Embodiment 1 or 2, wherein the degree of polymerization of the sugar before hydrolysis is 3 to 7.
4). The method of embodiment 1, 2 or 3, wherein the concentration of sugar after the contacting step is less than 50% of the concentration of sugar present before contacting.
5. Appropriate conditions are
The method according to Embodiment 1, 2, 3 or 4, comprising (i) a glucan synthesis reaction, or (ii) a fraction obtained from a glucan synthesis reaction, wherein the sugar is a byproduct of the glucan synthesis reaction.
6). Embodiment 6. The method of embodiment 5, wherein the glucan synthesis reaction produces at least one insoluble alpha-glucan product.
7). The method according to embodiment 6, wherein the fraction is a filtrate of a glucan synthesis reaction.
8). Glucan synthesis reaction
It is possible to hydrolyze a product of (i) a glucosyltransferase, or (ii) one or more alpha-1,3-glycosidic bonds or one or more alpha-1,6-glycosidic bonds 6. The method of embodiment 5, wherein at least one soluble alpha-glucan product is produced that is the product of the concerted action of both glucosyltransferase and alpha-glucanohydrolase.
9. The method according to embodiment 8, wherein the fraction is a chromatographic fraction of a glucan synthesis reaction.
10. Embodiment 10. The method of any one of embodiments 1-9, wherein the alpha-glucosidase enzyme is a transglucosidase.
11. A composition produced by contacting a sugar with an alpha-glucosidase enzyme, wherein the sugar is a disaccharide or an oligosaccharide and has at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage. The enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage of the sugar, and the composition has a reduced amount compared to the amount of sugar present prior to contact. A composition comprising sugar.
12 The composition according to embodiment 11, wherein the degree of polymerization of the sugar before hydrolysis is 3-7.
13. The composition according to embodiment 11 or 12, wherein the sugar is in (i) a glucan synthesis reaction, or (ii) a fraction obtained from the glucan synthesis reaction, and the sugar is a byproduct of the glucan synthesis reaction.
14 A method for concentrating fructose present in a fraction of a glucan synthesis reaction,
(A) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme is at least one of a disaccharide or an oligosaccharide contained in the fraction; Hydrolyzing, contacting, alpha-1,3 or alpha-1,6 glucosyl-glucose bonds;
(B) separating the fructose from the hydrolyzed fraction of step (a) to obtain a composition having a higher fructose concentration compared to the fructose concentration of the fraction of step (a). .
15. (A) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme is at least one of a disaccharide or an oligosaccharide contained in the fraction; Hydrolyzing, contacting, alpha-1,3 or alpha-1,6 glucosyl-glucose bonds;
(B) fermenting the fraction of step (a) with microorganisms to obtain a product, wherein the fermentation is performed after step (a) or simultaneously with step (a) When,
(C) optionally isolating the product of (b), wherein the yield of the product of (b) is fermenting a fraction of the glucan synthesis reaction that is not in contact with the alpha-glucosidase enzyme. Fermentation process that is increased compared to product yield.

  The disclosed invention is further defined in the following examples. It should be understood that these examples are given by way of illustration only, while showing certain preferred embodiments of the present invention. From the above discussion and these examples, one of ordinary skill in the art can ascertain the basic features of the present invention and to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Various changes and modifications of the present invention can be made.

Abbreviations As used herein, some abbreviations have the following meanings: “g” means grams, “h” means hours, “mL” means milliliters, “ “psi” means pounds per square inch, “wt%” means weight percent, “μm” means micrometer, “%” means percent, “° C.” means Celsius temperature. , “Mg” means milligrams, “mm” means millimeters, “mL / min” means milliliters / minute, “m” means meters, and “μL” means microliters. , “Mmol” means millimolar, “min” means minutes, “mol%” means mole percent, “M” means molar concentration, “mg / g” means milligram / gram. Meaning "rpm" is the number of revolutions per minute Taste, and "MPa" means mega pascal.

General Methods All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Sucrose was obtained from VWR (Radnor, PA).

Preparation of crude extract of glucosyltransferase (gtf) enzyme Streptococcus salivarius in E. coli strain DH10B using an isopropyl beta-D-1-thiogalactopyranoside (IPTG) inducible expression system The gtfJ enzyme (SEQ ID NO: 3) was expressed. SEQ ID NO: 3 is the S. GENBANK recognition number 47527. There is a 42 residue deletion at the N-terminus when compared to the S. salivarius gtfJ amino acid sequence, but it contains the starting methionine. Briefly, E. coli DH10B cells were transformed to express SEQ ID NO: 3 from a codon-optimized DNA sequence for expressing the gtfJ enzyme in E. coli. This DNA sequence was contained in the expression vector, pJexpress404® (DNA2.0, Menlo Park CA). Transformed cells are inoculated in LB medium (10 g / L tryptone; 5 g / L yeast extract, 10 g / L NaCl) to an initial optical density of 0.025 (OD at 600 _nm ) and shaken at 250 rpm And grown at 37 ° C. in an incubator. When the OD _{600 of the} culture reached 0.8-1.0, induction was performed on the culture by adding 1 mM IPTG. The induced culture was placed on a shaker and collected 3 hours after induction.

  The GtfJ enzyme (SEQ ID NO: 3) is recovered by centrifuging the cultured cells (25 ° C., 16000 rpm) in an Eppendorf® centrifuge, and the cells are resuspended in 5.0 mM phosphate buffer (pH 7.0). Suspended and cooled to 4 ° C. on ice. Cells were disrupted using a bead beater with 0.1 mm silica beads and then centrifuged at 16000 rpm at 4 ° C. to pellet unbroken cells and cell debris. The crude extract (soluble GtfJ enzyme, containing SEQ ID NO: 3) was separated from the pellet and analyzed by Bradford protein assay to determine protein concentration (mg / mL).

  A Streptococcus sp. C150gtf-S enzyme (SEQ ID NO: 40) was prepared as follows. SG1184 is a Bacillus subtilis expression strain that expresses a truncated form of glycosyltransferase Gtf-S ("GTF0459") from Streptococcus sp. C150 (GENBANK (R) GI: 32127321). . A gene encoding the N-terminal truncated protein GTF0459 (SEQ ID NO: 42) from the E. coli expression plasmid pMP79 is inserted into a Bacillus subtilis integrated expression plasmid p4JH under NheI and HindIII under the aprE promoter. Cloned into the site and B. Fused with B. subtilis AprE signal peptide. The construct was first transformed into E. coli DH10B and selected on LB plates with ampicillin (100 g / mL). Subsequently, nine protease deletions (containing amyE :: xylRPxylAcomK-ermC, degUHy32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ, Δmpr-ybfJ, Δmpr-ybfJ, B. subtilis BG6006 was transformed with the confirmed construct pDCQ984 expressing GTF0459 and selected on LB plates with chloramphenicol (5 μg / mL). Colonies grown on LB plates with 5 μg / mL chloramphenicol were streaked several times on LB plates with 25 μg / mL chloramphenicol. B. obtained. A B. subtilis expression strain, SG1184, was first grown in LB medium containing 25 μg / mL chloramphenicol and then subcultured in Grants II medium with 25 μg / mL chloramphenicol at 30 ° C. Grown for 2-3 days. The culture was spun at 15,000 g for 30 minutes at 4 ° C. and the supernatant was filtered through a 0.22 μm filter. The filtered supernatant was dispensed and frozen at -80 ° C.

  B. expressing GTF0459 (SEQ ID NO: 42) by conventional fed-batch fermentation. B. subtilis SG1184 strain was grown under aerobic submerged conditions. 0-0.25% corn steep solid (Roquette), 5-25 g / L sodium and potassium phosphate, 0.3-0.6 M ferrous sulfate solution, manganese chloride and calcium chloride; A nutrient medium containing a solution of 0.5-4 g / L magnesium sulfate and 0.01-3.7 g / L zinc sulfate, copper (I) sulfate, boric acid and citric acid was used. An antifoam, FOAMBLAST 882, was added at 2-4 mL / L to control foam formation. If the initial glucose in the batch was undetectable, a 10% fermentation was given a 50% (w / w) glucose feed. The glucose feed rate was gradually changed over several hours. The fermentation was controlled at 30 ° C. and 20% DO and with an initial agitation of 750 rpm. The pH was adjusted to 7.2 using 50% (v / v) ammonium hydroxide. Fermentation parameters such as pH, temperature, airflow and DO% were monitored throughout the two day fermentation run. At the end of the fermentation, the culture broth was collected and centrifuged to obtain a supernatant. The supernatant containing GTF0459 (SEQ ID NO: 42) was then stored frozen at -80 ° C.

  S. A S. mutans MT-4239gtf-C enzyme (SEQ ID NO: 43) was prepared as follows. From GENBANK® GI: 3130088 (SEQ ID NO: 43; S. mutans MT-4239, using codons optimized for expression in Bacillus subtilis A gene encoding a truncated form of the glucosyltransferase (gtf) enzyme identified as gtf-C) was synthesized by GenScript. A gene (SEQ ID NO: 44) encoding GTF0088BsT1 (SEQ ID NO: 45) with N-terminal shortening and C-terminal T1 shortening was amplified from the GENSCRIPT plasmid and transformed into the Bacillus subtilis integrated expression plasmid p4JH under the aprE promoter. Cloned into NheI and HindIII sites and B. Fused with B. subtilis AprE signal peptide. The construct was first transformed into E. coli DH10B and selected on LB with ampicillin (100 μg / mL). Subsequently, nine protease deletions (containing amyE :: xylRPxylAcomK-ermC, degUHy32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ, Δmpr-ybfJ, Δmpr-ybfJ, B. subtilis BG6006 was transformed with the identified construct pDCQ1021, expressing GTF0088BsT1, and selected on LB plates with chloramphenicol (5 μg / mL). Colonies grown on LB plates with 5 μg / mL chloramphenicol were streaked several times on LB plates with 25 μg / mL chloramphenicol. B. obtained. B. subtilis expression strain SG1221 is first grown in LB medium with 25 μg / mL chloramphenicol, then subcultured to Grants II medium containing 25 μg / mL chloramphenicol, Grow for ~ 3 days. The culture was spun at 15,000 g for 30 minutes at 4 ° C. and the supernatant was filtered through a 0.22 μm filter. The filtered supernatant was dispensed and frozen at -80 ° C.

  B. expressing GTF0088BsT1 (SEQ ID NO: 45) by conventional fed-batch fermentation. B. subtilis SG1221 strain was grown under aerobic submerged conditions. 0-0.25% corn steep solids (Roquette), 5-25 g / L sodium and potassium phosphate, 0.3-0.6 M ferrous sulfate solution, manganese chloride and calcium chloride, 0.5-4 g A nutrient medium containing a solution of / L magnesium sulfate and 0.01 to 3.7 g / L zinc sulfate, copper (I) sulfate, boric acid and citric acid was used. An antifoam, FOAMBLAST 882, was added at 2-4 mL / L to control foam formation. If the initial glucose in the batch was undetectable, the 2L fermentation was given a 50% (w / w) glucose feed. The glucose feed rate was gradually changed over several hours. Fermentation was controlled at 30 ° C. and 20% DO and with initial stirring at 400 rpm. The pH was adjusted to 7.2 using 50% (v / v) ammonium hydroxide. Fermentation parameters such as pH, temperature, airflow and DO% were monitored throughout the two day fermentation run. At the end of the fermentation, the culture broth was collected and centrifuged to obtain a supernatant. The supernatant containing GTF088BsT1 (SEQ ID NO: 45) was then stored frozen at -80 ° C.

Determination of glucosyltransferases GTF0459 and GTF0088BsT1 activity 25 mM or 50 mM at pH 5.5 at 37 ° C. and 125 rpm orbital shaking in the presence or absence of 25 g / L dextran (MW ca 1500, Sigma-Aldrich, Cat. # 31394) The glucosyltransferase activity assay was performed by incubating 1-10% (v / v) crude protein extract containing GTF enzyme with 200 g / L sucrose in sodium acetate buffer. One aliquot of the reaction mixture was taken at 1 hour, 2 hours and 3 hours and heated at 90 ° C. for 5 minutes to inactivate GTF. Insoluble material was removed by centrifugation at 13,000 × g for 5 minutes, followed by filtration through a 0.2 μm RC (regenerated cellulose) membrane. The resulting filtrate was analyzed by HPLC to quantify the sucrose concentration using two AMINEX HPX-87C column series (Bio-Rad, Hercules, CA) at 85 ° C. 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 required to consume 1 micromole of sucrose per minute under assay conditions.

Preparation of a crude extract of alpha- (1,3) -glucanohydrolase (mutanase) The gene coding for was synthesized by GenScript (Piscataway, NJ). A vector designed to express the gene of interest in Trichoderma reesei under the control of the CBHI promoter and terminator using Aspergillus niger acetamidase for selection The nucleotide sequence (SEQ ID NO: 46) encoding the protein sequence (MUT3325; SEQ ID NO: 47) was subcloned into the plasmid pTrex3 at the SacII and AscI restriction sites. The resulting plasmid was transformed into T. pylori. T. reesei was transformed by gene gun injection. Detailed methods of gene gun transformation are described in PCT Patent Application Publication No. WO 2009/126773 A1, which is incorporated herein by reference. A production medium (below) was inoculated using 1 cm ² agar plugs with spores from stable clone TRM05-3. Cultures were grown in shake flasks at 28 ° C. and 220 rpm for 4-5 days. To recover the secreted protein, cell clumps were first removed by centrifugation at 4000 g for 10 minutes and the supernatant was filtered through a 0.2 μm sterile filter. Expression of mutanase MUT3325 (SEQ ID NO: 47) was confirmed by SDS-PAGE.

  The components of the production medium are listed below.

  A fermented seed culture was prepared by inoculating a 0.5 L minimal medium in a 2 L baffled flask with a 1.0 mL frozen spore suspension of MUT3325 expression strain TRM05-3 (minimum medium was 5 g / L). Ammonium sulfate, 4.5 g / L monobasic potassium phosphate, 1.0 g / L magnesium sulfate heptahydrate, 14.4 g / L citric anhydride, 1 g / L calcium chloride dihydrate, 0.4375 g / 25 g / L glucose and trace elements containing L citric acid, 0.5 g / L ferrous sulfate heptahydrate, 0.04 g / L zinc sulfate heptahydrate, 0.008 g / L copper sulfate pentahydrate , 0.0035 g / L manganese sulfate monohydrate and 0.002 g / L boric acid, pH was 5.5). The culture was grown for 48 hours at 32 ° C. and 170 rpm and then transferred to 8 L production medium in a 14 L fermentor. Production medium is 75 g / L glucose, 4.5 g / L monobasic potassium phosphate, 0.6 g / L calcium chloride anhydrous, 1.0 g / L magnesium sulfate heptahydrate, 7.0 g / L ammonium sulfate, 0.5 g / L citric anhydride, 0.5 g / L ferrous sulfate heptahydrate, 0.04 g / L zinc sulfate heptahydrate, 0.00175 g / L copper sulfate pentahydrate, Consists of 0035 g / L manganese sulfate monohydrate, 0.002 g / L boric acid and 0.3 mL / L FOAMBLAST 882.

  Fermentation was performed initially on batch growth on glucose for 24 hours at 34 ° C. and 500 rpm. At the end of 24 hours, the temperature was reduced to 28 ° C. and the stirring speed was increased to 1000 rpm. The fermenter was then charged with a mixture of glucose and sophorose (62% w / w) at a specific feed rate of 0.030 g glucose-sophorose solids / g biomass / hr. At the end of the fermentation run, the biomass was removed by centrifugation and the MUT3325 mutanase (ultrafiltration using a 10 kD molecular weight cut-off ultrafiltration cartridge (UFP-10-E-35; GE Healthcare, Little Chaflon, Buckinghamshire, UK) ( The supernatant containing SEQ ID NO: 47) was concentrated about 10 times. Concentrated protein was stored at −80 ° C.

Determination of alpha-glucanohydrolase (mutanase) activity Insoluble mutane required to determine mutanase activity using the secreted enzyme produced by Streptococcus sobrinus ATCC® 33478 ™ A polymer was prepared. Specifically, S.M. One loop of a glycerol stock of S. sobrinus ATCC® 33478 ™ was streaked onto a BHI agar plate (Brain Heart Infusion Agar, Teknova, Hollister, Calif.) For 2 days at 37 ° C. Plates were incubated. Several colonies were picked using a loop and inoculated into 2 × 100 mL BHI liquid medium in an original medium bottle from Teknova, and the culture was incubated at 37 ° C. and allowed to stand for 24 hours. The resulting cells were removed by centrifugation and the resulting supernatant was filtered through a 0.2 μm sterilized filter; 2 × 101 mL filtrate was collected. 2 × 11.2 mL of 200 g / L sucrose was added to the filtrate (final sucrose 20 g / L). The reaction was incubated at 37 ° C. without stirring for 67 hours. The resulting polysaccharide polymer was recovered by centrifugation at 5000 × g for 10 minutes. The supernatant was carefully decanted. The insoluble polymer was washed 4 times with 40 mL sterile water. The resulting mutan polymer was lyophilized for 48 hours. Mutane polymer (390 mg) was suspended in 39 mL of sterile water to give a 10 mg / mL suspension. Mutane suspension was homogenized by sonication (40% amplitude until large lumps disappeared, about 10 minutes total). The homogenized suspension was dispensed and stored at 4 ° C.

  The mutanase assay was initiated by incubating the appropriate amount of enzyme with 0.5 mg / mL mutan polymer (prepared as described above) at 37 ° C. in 25 mM KOAc buffer at pH 5.5. Aliquots of the reaction mixture were taken at various time points and inactivated with an equal volume of 100 mM glycine buffer (pH 10). Insoluble material in each inactivated sample was removed by centrifugation at 14,000 × g for 5 minutes. The p-hydroxybenzoic acid hydrazide solution (PAHBAH) assay (Lever M., Anal. Biochem., (1972) 47: 273-279) quantifies the reducing end of the oligosaccharide and polysaccharide polymer produced at each time point, The initial velocity was determined from the slope of the linear plot of the first 3 or 4 time points of the time course. PAHBAH assay was performed by adding 10 μL of reaction sample supernatant to 100 μL of PAHBAH diluted standard solution and heating at 95 ° C. for 5 minutes. Mix 1 part Reagent A (0.05 g / mL p-hydroxybenzoic acid hydrazide and 5% by volume concentrated hydrochloric acid) and 4 parts Reagent B (0.05 g / mL NaOH, 0.2 g / mL potassium sodium tartrate) A diluted standard solution was prepared. The concentration at the reducing end was calculated by recording the absorbance at 410 nm, subtracting the appropriate background absorbance and using a standard curve generated with various concentrations of glucose as a standard.

Analysis of reaction profile by HPLC Samples were periodically taken from the reaction and analyzed using an Agilent® 1260 HPLC equipped with a refractive index detector. Aminex® HP-87C column (BioRad, Hercules, Calif.) With a flow rate of 0.6 mL / min and deionized water at 85 ° C. is used to quantify sucrose, glucose, leucrose and fructose levels in the gtf reaction did. Soluble oligosaccharide byproducts (DP2-DP7) in the gtf reaction were quantified using an Aminex® HP-42A column (BioRad) with a flow rate of 0.6 mL / min and deionized water at 85 ° C.

  A Dionex® UltimateMate ™ 3000 HPLC (Thermo Scientific) equipped with a refractive index detector was used for the sample containing the immobilized enzyme (Example 4). Sugar was analyzed using a Phenomenex® Rezex ™ calcium monosaccharide column with a flow rate of 0.3 mL / min and deionized water at 85 ° C.

Analysis of oligosaccharide bonds by NMR NMR data were obtained on an Agilent DD2 spectrometer operating at 500 MHz for ¹ H using a 5 mm low temperature triple resonance pulsed field gradient (PFG) probe. Carefully place the observer transmitter frequency on resonance to the residual water signal in the “presat” experiment, then use the first slice of the NOESY experiment with a full phase cycle (multiple of 32) and a mixing time of 10 ms. By doing so, water suppression was obtained. One-dimensional ¹ H spectra were obtained with a spectral width of 6410 Hz, a capture time of 5.1 s, 65536 data points, 4 s presaturation and a 90 ° pulse of 5.85 μs. The sample temperature was maintained at 25 ° C. 5 mm NMR with 60 μL D ₂ O containing 450 μL D ₂ O and 12.4 mM DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt) internal reference with methyl resonance set to 0 ppm Samples were prepared by adding 50 μL to the tube. Chemical shift assignments for the different anomeric bonds were obtained from Goffin et al. (2009, Bull Korean Chem. Soc. 30: 2535-2541. Peak assignments were 5.35 ppm for alpha (1,3) bonds and for Leucrose. 5.1 ppm, and 4.95 for alpha (1,6) linkage, reducing end (RE) assigned 5.2 for alpha RE and 4.65 for beta RE It was.

Example 1
Generation of Sugar Syrup by Sucrose Polymerization This example discloses a general method in which a mixture of soluble sugars was generated by polymerization of sucrose using the gtf enzyme in a glucan synthesis reaction. Specifically, a glucan synthesis reaction filtrate was prepared and then concentrated to a syrup.

Sucrose (3000 g) was added to a clean 5 gallon polyethylene bucket. To this bucket was added water (18.1 L) and Fermasure ™ (10 mL) and the pH was adjusted to 7.0 by adding ₅ vol% NaOH and 5 vol% H ₂ SO ₄ . The final volume was about 20 L and the initial sucrose concentration as measured by HPLC was 152.5 g / L. The glucan polymerization reaction was initiated by the addition of 0.3 vol% crude gtf enzyme (SEQ ID NO: 3) extract prepared as described in the General Methods section. This extract contained approximately 2.9 mg / mL protein. An overhead mechanical motor equipped with a glass shaft and PTFE blade was used to stir the reaction solution.

  After 48 hours, HPLC analysis revealed that 96% of the sucrose had been consumed and the reaction was considered complete. The reaction insoluble poly-alpha-1,3-glucan product was removed by filtration through a Buchner filter funnel using a 325 mesh steel screen and 40 micron filter paper. The mother liquor (filtrate) was then concentrated using a rotary evaporator (bath temperature 40-50 ° C.) to give a total sugar concentration of about 320 g / L of sugar. The composition of the concentrated filtrate is shown in Table 2.

  Table 2 shows that the concentrated filtrate of the glucan synthesis reaction contains sucrose, fructose, glucose, leucorose and DP2-DP7 oligosaccharides.

Example 2
Effect of Enzymes on Sugar Hydrolysis in the Glucan Synthesis Reaction Filtrate This example illustrates the use of various glucoamylases to reduce the concentration of leucine and / or oligosaccharide by-products in the concentrated filtrate of the glucan synthesis reaction. (EC 3.2.1.3), transglucosidase (EC 2.4.1.24), beta-glucosidase (EC 3.2.1.21), alpha-amylase (EC 3.2.1.1) and glucosidase ( EC 3.2.1) Measure enzyme activity. Certain enzymes, such as DIAZYME RDF ULTRA, transglucosidase (EC 2.4.1.24) and glucoamylase (EC 3.2.1.3), all alpha-glucosidases, reduce the amount of these by-products. It was found to be particularly effective in, as a result of which a corresponding increase in monosaccharides (glucose and fructose) occurs in the treated filtrate.

  The filtrate for the glucan synthesis reaction was first prepared and concentrated to a syrup according to the procedure outlined in Example 1. The composition of this concentrated filtrate is shown in Table 3. NMR analysis revealed that the ratio of alpha (1,3) to alpha (1,6) linkage present in the syrup was 78:22.

  The syrups in Table 3 were used to test the hydrolytic activity of various enzymes on leucine and oligosaccharide byproducts of the glucan synthesis reaction. Because Leucrose contains unusual linkages [alpha (1,5) -glucosyl fructose] and oligosaccharides mainly contain alpha (1,3) and alpha (1,6) glucosyl-glucose linkages. In view of which enzymes could be used to hydrolyze both of these byproducts, it was not clear at the beginning of these experiments. Enzymes with various activities were selected for this analysis (Table 4).

  The conditions for treating the syrup of Table 3 with each of the above enzymes are shown in Table 5 (enzyme load, time, temperature, pH, sugar concentration). The syrup was diluted with water to the sugar concentration used in each hydrolysis reaction. Table 5 further provides the percent hydrolysis of Leucrose and DP3 + (at least DP3-DP7) oligosaccharides by each enzyme. The percent DP3 + hydrolysis was calculated as (1- (wt% DP3 + oligosaccharide in the final syrup) / (wt% DP3 + oligosaccharide in the first syrup)). Similarly, the percent leuco hydrolysis was calculated as (1- (wt% leuco in the final syrup) / (wt% leuco in the initial syrup)).

  Table 5 shows that 1,4-alpha-glucosidase and 1,6-alpha-glucosidase have some (least 2.1) or little (least 2.2) hydrolysis of leucrose (example 2.2). It shows that some glucose was released from the oligosaccharide. The use of alpha-amylase (Example 2.3 and Example 2.4) showed little activity against the compound of interest. Similarly, the use of pullulanase (Example 2.5) showed little activity.

  Cellulases (Examples 2.14 and 2.15) were very poor in hydrolyzing leucus but some hydrolyzed oligosaccharides.

  Despite the oligosaccharides containing no beta linkages, surprisingly, the beta-glucosidase enzyme was also changed from very low levels (Accelerase BG, Example 2.9) to very high levels (NOVO188, run Hydrolytic transformations in the range of Examples 2.10 and 2.11) were also shown. The relative potency of these enzymes varied very dramatically. In some cases, the amount of hydrolyzed oligosaccharides was much higher (Example 2.11) or close to (% Example 2) than the percentage of hydrolyzed leucus. In other cases, leucus was well hydrolyzed by beta-glucosidase, while oligosaccharide hydrolysis was moderate (Example 2.13). Due to the large differences between the results observed with beta-glucosidase, the presence of other enzymes in the tested beta-glucosidase formulations, such as glucoamylase or another type of alpha-glucosidase, was observed. It is suggested that it may be a cause of active activity.

  Conversely, the results in Table 5 indicate that transglucosidase (TG L-2000, Example 2.6) showed very high activity in terms of hydrolyzing both oligosaccharides and leucus. Leucrose hydrolysis by transglucosidase appeared to be quantitative under certain circumstances, with over 95% of DP3 + material hydrolyzed to glucose and DP2 at high enzyme loading (Example 2.7). The use of purified type transglucosidase reveals a similar activity (Example 2.8) indicating that the observed activity is due to the transglucosidase enzyme and not the background activity.

  Glucoamylase (Examples 2.16-2.18) showed a range of activities against leucorose and oligosaccharides. Only one test glucoamylase (Example 2.18) had less than 30% hydrolysis of both Leucrose and oligosaccharides.

  The results in Table 5 indicate that alpha-glucosidases such as DIAZYME RDF ULTRA, glucoamylase and transglucosidase can hydrolyze leuco by-products present in the glucan reaction filtrate. The leucine hydrolytic ability of alpha-glucosidase indicates that these enzymes can hydrolyze alpha-1,5 glucosyl-fructose linkages. While this activity has been shown above using leucine as a substrate, it is believed that this activity can be extended to oligosaccharides containing alpha-1,5 glucosyl-fructose linkages.

  The results in Table 5 further show that alpha-glucosidases such as glucoamylase and transglucosidase can hydrolyze oligosaccharide by-products present in the glucan reaction filtrate. Since these oligosaccharides are composed mostly of glucose monomer units linked by alpha-1,3 and / or alpha-1,6 linkages (Example 3), the data in Table 5 shows that the alpha-glucosidase enzyme It shows that alpha-1,3 glucosyl-glucose and / or alpha-1,6 glucosyl-glucose bonds can be hydrolyzed.

  Since alpha-glucosidase enzymes were generally effective in hydrolyzing leuco and / or oligosaccharide by-products of glucan synthesis reactions, these enzymes have increased glucan reactions with increased amounts of monosaccharides and decreased amounts of sugar by-products. Can be used alone or in combination to reduce the processing time required to produce high purity syrup from the filtrate. An example of an effective enzyme combination may be a transglucosidase, such as TG L-2000, and a glucoamylase (eg, GC321) enzyme that efficiently hydrolyzes oligosaccharide by-products for leucohydrolysis.

  Thus, the alpha-glucosidase enzyme hydrolyzes (i) alpha-1,5 glucosyl-fructose bonds and (ii) alpha-1,3 and alpha-1,6 glucosyl-glucose bonds individually in certain sugars. can do.

Example 3
Comparison of binding distribution of glucan reaction filtrate components before and after enzymatic hydrolysis This example shows transglucosidase (EC 2.4.1.24) and beta for leucos and oligosaccharide by-products present in the concentrated filtrate of glucan synthesis reaction. -Determine the hydrolytic activity of the glucosidase (EC 3.2.1.21) enzyme. Transglucosidase has been found to reduce the amount of these by-products, resulting in a corresponding increase in monosaccharides (glucose and fructose) in the treated filtrate.

  The oligosaccharide by-product present in the filtrate of the glucan synthesis reaction contains> 90% glucose-glucose bond as measured by NMR (general method). Of the glucose-glucose bonds, about 78% corresponds to alpha-1,3 bonds and about 22% corresponds to alpha-1,6 bonds.

  NMR was used to determine the binding profile of the material produced in Example 2.11. Above after hydrolysis. As shown in FIG. 1, the peak corresponding to the alpha-1,3 bond is reduced by 86%, the drop of the peak corresponding to the alpha-1,6 bond is only 2.3%, corresponding to the leucus peak. The peak to be reduced was 21%. While sucrose was hydrolyzed almost quantitatively by this enzyme, Novo 188 appears to be unable to hydrolyze alpha-1,6 linkages.

NMR was similarly used to determine the binding profile of the material produced with TG L-2000 (SEQ ID NO: 1) transglucosidase (Figure 2). 210 μL of concentrated filtrate from the materials in Table 3, 300 μL of D ₂ O and 90 μL of D ₂ O containing 12.4 mM DSS as internal reference are mixed in an NMR tube to a total sugar concentration of 300 g / L. And heated to 60 ° C. A time zero spectrum (starting material in FIG. 2) was obtained after thermal equilibration at 60 ° C., then 0.5 vol% enzyme was added. Samples were re-equilibrated in the probe at 60 ° C., calibrated, and measurements were taken within minutes of analysis. After 10 hours of treatment with TG L-2000 enzyme (treated material in FIG. 2), the peak corresponding to alpha-1,3 binding is reduced by 41% and the peak corresponding to alpha-1,6 binding is 36%. % And the peak corresponding to Leucrose was reduced by> 95% (FIG. 2). An increase in both the alpha-reducing end and the beta-reducing end peak was observed, corresponding to increases in fructose and glucose (FIG. 2).

  These results reveal that transglucosidase can convert oligosaccharides containing alpha-1,3 and alpha-1,6 linkages to glucose, and can convert leucine to fructose and glucose. Thus, transglucosidase can hydrolyze (i) alpha-1,5 glucosyl-fructose linkages and (ii) alpha-1,3 and alpha-1,6 glucosyl-glucose linkages in certain sugars. .

Example 4
Hydrolysis of Leucrose and Oligosaccharides in Glucan Reaction Filtrate Using Immobilized Enzyme This example shows immobilization to hydrolyze leuclos and other oligosaccharides present in the filtrate obtained from glucan synthesis reaction The use of activated glucoamylase (EC 3.2.1.3) and transglucosidase (EC 2.4.1.24) is described. Specifically, immobilized transglucosidase TG L-2000 (SEQ ID NO: 1, Genencor / DuPont Industrial) in hydrolysis of leucos and oligosaccharides DP2, DP3 and HS (higher saccharide, DP4 +) in the filtrate of glucan synthesis reaction Bioscenses) and immobilized glucoamylase GC-147 (obtained from Genencor / DuPont Industrial Biosciences) were examined.

  Immobilization of glucoamylase and transglucosidase enzymes was performed according to the method described in US Pat. No. 5,541,097, which is incorporated herein by reference.

  In a typical process for immobilizing glucoamylase or transglucosidase, two batches of porous granular diatomaceous earth (EP Minerals, Reno, NV) at about 8.0 g / batch are hydrated with distilled water and then with a diameter of 1 Transferred to a glass column reactor of 5 cm and 30 cm height. Water was pumped upstream at approximately 6-7 mL / min to remove particulates from all three columns. In general, the water effluent was free of particulates within 1 hour. Water was drained from the column to the top of the granular diatomaceous earth bed and replaced with a 0.1% w / v aqueous polyethyleneimine solution (PEI, EPOMIN P-1050). 3500 mL of PEI solution was then pumped upstream and the effluent was recycled through the bed for 2 hours. The granular diatomaceous earth bed was then washed upward with distilled water for 2 hours to remove free PEI at room temperature. In this way, a granular diatomaceous earth-PEI carrier was obtained.

  Meanwhile, 3.5 mL glucoamylase GC-147 having the activity defined in Table 4 was added to 315 mL 0.02 M acetate buffer (pH 4.5). Then 1.575 g of 50% w / w glutaraldehyde (Protectol® GA-50) is slowly added to the aqueous glucoamylase solution with gentle mixing, and the glutaraldehyde is added to the aqueous glucoamylase solution at 20-25 ° C. At 4 ° C. for 4 hours with gentle agitation resulting in the formation of a treated enzyme-glutaraldehyde adduct containing the treated glucoamylase. Independently, these steps were repeated using transglucosidase TG L-2000 having the activity defined in Table 4 instead of glucoamylase, thereby treating the treated enzyme-glutaraldehyde containing the treated transglucosidase An adduct was formed.

  Each of the treated enzyme-glutaraldehyde adducts was then circulated for 4 hours (20-25 ° C.) in its own column prepared as above containing granular diatomaceous earth-PEI support. The excess treated adduct was then washed from the carrier with water. In this way, a column containing immobilized glucoamylase or transglucosidase was prepared.

  The 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 immobilized enzyme. The column temperature was adjusted to 60 ° C. Samples were taken periodically at different flow rates after 16 hours of column equilibration. The sugar composition of the hydrolysis reaction product was determined by HPLC (Table 6). Each time, the flow rate setting was changed and the column was re-equilibrated over at least 1-2 bed volumes before sampling. Using the method described in Example 2, the degree of hydrolysis of leucus and oligosaccharides was calculated. Three column arrangements were tested: 1) immobilized glucoamylase, 2) immobilized transglucosidase and 3) immobilized glucoamylase, followed by immobilized transglucosidase.

  Table 6 shows that the degree of hydrolysis of leucos and oligosaccharides generally improved due to the extended average contact time (defined as the nominal column volume divided by the average flow rate). The use of immobilized transglucosidase to hydrolyze leucos was particularly effective as no significant difference was observed when using the maximum flow rates tested. Each column individually showed reasonable conversion, while the combination of glucoamylase and transglucosidase maximized oligosaccharide hydrolysis.

  Thus, the use of immobilized glucoamylase or transglucosidase or both types of immobilized enzyme is effective for hydrolyzing oligosaccharides containing alpha-1,3 and alpha-1,6 glucosyl-glucose linkages as well as leucrose. Corresponds to technology. These results are consistent with the results of Example 2. Immobilization of other alpha-glucosidase enzymes should give similar results.

Example 5
Concentration of fructose from glucan reaction filtrate by chromatography This example discloses how fructose in glucan reaction filtrate can be further concentrated through chromatography.

  In general, when separating sugar molecules by chromatography, the components elute inversely with the molecular size, so the largest molecule elutes first. Therefore, with respect to the filtrate of glucan synthesis reaction, oligosaccharide is eluted first, followed by disaccharide and then monosaccharide. In the separation using a sodium cation resin, fructose and glucose are not separated well, and leucus, sucrose and DP2 are all eluted simultaneously. The use of an ion exchange resin whose cation is calcium is preferred for separating glucose and fructose.

  The filtrate for the glucan synthesis reaction was first prepared and concentrated to a syrup according to the procedure outlined in Example 1. The composition of this concentrated filtrate is shown in Table 7.

  The syrup of Table 7 was filtered, diluted with ion exchange water to a 25 g dry solid / 100 g solution and added to a column containing a calcium-type cross-linked strongly acidic ion exchange resin. The physical parameters of this column are shown in Table 8. Diluted syrup (15.8 L) was added to the column, which was maintained at 65 ° C., after which the column was eluted with water at a flow rate of 30 L / hr.

  In this separation, Leucrose remained on the column longer than sucrose, probably because it was complexed with calcium cations and actually eluted with glucose. Two fractions containing fructose were isolated. Fraction 5.1 eluted at 47-120 minutes and fraction 5.2 eluted at 120-172 minutes. Of the fructose added to the chromatographic separation, 95.7% fructose was isolated with> 90% purity. Table 9 shows the product distribution in each fraction (5.1 and 5.2) as measured by HPLC.

  Since the feed composition for this separation contained 36.0% fructose, a total of 34.5% total stream was recovered as fructose syrup with> 90 wt% DS fructose. Because sucrose in the feed was ignored, 40.7% of the sugar was recovered as fructose syrup with> 90 wt% DS fructose.

  Therefore, fructose in the glucan reaction filtrate can be further concentrated through chromatography. Example 6 below demonstrates that glucan filtrate hydrolyzed with transglucosidase can be used to facilitate this process.

Example 6
Concentration of fructose from hydrolyzed glucan reaction filtrate by chromatography This example shows the isolation of fructose from non-hydrolyzed glucan filtrate as a result of fructose isolation from glucan filtrate hydrolyzed with oligosaccharides and leucus We show that the yield of high-purity fructose syrup increases compared with

  A syrup was prepared by concentrating (vacuum at 50 ° C.) the glucan filtrate treated with 1 vol% transglucosidase TG L-2000 (SEQ ID NO: 1) at 60 ° C. and pH 4.5 for 24 hours. Some oligosaccharide formation was observed during the concentration process, which was expected because the transglucosidase enzyme is known to produce oligosaccharides at high concentrations of monosaccharides. The syrup resulted in the final product distribution listed in Table A.

  The syrup described in Table A was filtered, diluted with ion-exchanged water to a 25.4 g DS / 100 g solution, and a column containing a calcium-type crosslinked strong acidic cation exchange resin was added. The physical parameters of this column are shown in Table B. Diluted syrup (169 g) was then added to the column and maintained at 65 ° C., after which the column was eluted with water at a flow rate of 50 mL / min.

  Two fractions containing fructose were isolated. Fraction 6.1 was eluted from 73 minutes to 103 minutes and Fraction 6.2 was eluted from 103 minutes to 120 minutes. Of the fructose added to the chromatographic separation, 93.0% of the fructose added to the column was isolated in fraction 6.2 with> 90% purity. The product distribution in each fraction (6.1 and 6.2) as measured by HPLC is shown in Table C.

  The reduced separation efficiency in this example compared to Example 5 can be attributed to column scale differences and higher glucose fractions of the sample. Even so, as a result of chromatographic purification of this material, the yield was higher compared to the yield achieved in Example 5 where syrup was chromatographically prepared from glucan filtrate not hydrolyzed by transglucosidase. The yield of pure fructose syrup was improved. Since the feed composition for this separation contained 47% fructose (Table A), 43.7% of the total stream was recovered as fructose syrup with> 90 wt% DS fructose. This 43.7% recovery is significantly better than the 34.5% recovery in Example 5.

  Therefore, as a result of fructose isolation from the glucan filtrate hydrolyzed with transglucosidase, the yield of fructose is improved as compared with the case where fructose is isolated from the non-hydrolyzed glucan filtrate.

Example 7
Production of Ethanol by Fermenting the Glucan Synthesis Reaction Filtrate This example discloses yeast fermentation of glucan filtrate to ethanol.

  Suspend yeast (S. cerevisiae) cream (Tonon mill, Brazil) in tap water (2.4 L, optical density of 65 at 600 nm), then use a LEGEND XTR centrifuge (Thermo Scientific) to 4500 g. The yeast cream was washed by centrifuging the yeast cream for 5 minutes. After decanting the supernatant, the yeast cells were resuspended and concentrated by further centrifugation. After the third washing, the pH was adjusted to 2 by adding 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 the addition of tap water. The adjusted yeast cream (1.5 L) was added to a 7.5 L BIOFLO 310 fermenter vessel (New Brunswick). The fermentor was set to maintain stirring at a temperature of 30 ° C. and 100 rpm. Although pH was measured during fermentation, no adjustment was made by addition of acid or base solution.

  A feed solution containing 200 g / L sugar from yeast extract (10 g / L), peptone (20 g / L) and glucan filtrate is prepared and sterilized using a PHOENIX AV-250 PLUS autoclave at 121 ° C. for 15 minutes. did. After the feed solution was cooled to 25 ° C. (room temperature), fermentation started. Sterile feed solution (3.5 L) was added to the fermentor at a rate of 684 mL / hr over approximately 5 hours and the fermentation was allowed to proceed for 22 hours.

  Samples were taken periodically during fermentation and analyzed for optical density using Brix using a GENESYS 20 4001 spectrophotometer, PAL-3 refractometer (Atago), and sugar and ethanol concentrations by HPLC (general method) Was analyzed. These results are summarized in Table 10.

  When fermentation was complete, yeast cells were separated by centrifugation using a LEGEND XTR centrifuge at 4500 g for 5 minutes. After decanting the supernatant, the yeast was resuspended and concentrated twice more by centrifugation. After the third washing, the pH was adjusted to 2 by adding 5 wt% sulfuric acid. The optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 at 600 nm by the addition of tap water. According to the same conditions as above, two additional fermentation cycles, each with a fresh feed, were performed using recycled yeast cells from the previous fermentation. The fermentation results obtained using the first and second reused yeast are shown in Tables 11 and 12, respectively.

  Although only a small amount of leucus was consumed in the first fermentation, the yeast cells adapted to the second reuse and began to consume leucine. After three fermentation cycles with recycled yeast, the ethanol fermentation titer increased from 33 g / L (Table 10, 22 hours) to 54 g / L (Table 12, 21 hours), but even after the last cycle There was also a significant amount of leucus in the medium.

  Thus, the glucan filtrate can be used in a fermentation process to produce ethanol.

Example 8
Production of ethanol by fermenting hydrolyzed glucan filtrate This example demonstrates that fermenting a glucan filtrate pre-saccharified with leuco- and oligosaccharide by-product components results in an increase in ethanol yield To do.

  Fermentation was performed following the procedure outlined in Example 7, but using a glucan filtrate previously treated with transglucosidase (TG L-2000, SEQ ID NO: 1). Hydrolyzed glucan filtrate was prepared as follows. The glucan filtrate was adjusted to 300 g sugar / 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 6.75L. 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 (135 mL) as described in Table 4 was mixed with the sterile filtrate and the solution was incubated in an incubator shaker (IKA KS4000) at 60 ° C. and 100 rpm for 72 hours. Thus, a hydrolyzed glucan filtrate was prepared.

  Suspend yeast (S. cerevisiae) cream (Bom Retiro mill, Brazil) in tap water (2.4 L, 65 nm optical density at 600 nm), then at 4500 g using a LEGEND XTR centrifuge. The yeast cream was washed by centrifuging for 5 minutes. After decanting the supernatant, the yeast was resuspended and concentrated twice more by centrifugation. After the third wash, the pH was adjusted to 4.5 by adding 5 wt% sulfuric acid, the optical density was measured using a GENESYS 20 4001 spectrophotometer, and adjusted to 100 at 600 nm by adding tap water. The adjusted yeast cream (1.5 L) was added to a 7.5 L BIOFLO 310 fermentor vessel. The fermenter was set to maintain stirring at a temperature of 30 ° C. and 100 rpm, and maintain the pH at 4.5 using a 4M ammonium hydroxide aqueous solution or a 5 wt% sulfuric acid aqueous solution.

  A feed solution containing yeast extract (10 g / L), peptone (20 g / L) and 200 g / L sugar from the hydrolysis filtrate is prepared and used at 121 ° C. for 15 minutes using a PHOENIX AV-250 Plus autoclave. Sterilized. After the feed solution was cooled to 25 ° C. (room temperature), fermentation started. Sterilized feed solution (3.5 L) was added to the fermentor at a rate of 684 mL / hr over approximately 5 hours and the fermentation was allowed to proceed for 22 hours.

  Samples were periodically taken during fermentation and analyzed for optical density using Brix with a GENESYS 20 4001 spectrophotometer, PAL-3 refractometer, and analyzed for sugar and ethanol concentrations by HPLC (general method) . These results are summarized in Table 13.

  When fermentation was complete, 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 twice more by centrifugation. After the third washing, the pH was adjusted to 2 by adding 5 wt% sulfuric acid. The optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 at 600 nm by the addition of tap water. According to the same conditions as above, two additional fermentation cycles, each with a fresh feed, were performed using recycled yeast cells from the previous fermentation. The fermentation results obtained using the first and second reused yeast are shown in Tables 14 and 15, respectively.

  All fermentations were basically completed within about 6 hours from the start of the fermentation, resulting in an ethanol titer of 57-60.0 g / L. Comparing these fermentations with the fermentations of Example 7, by hydrolyzing the glucan filtrate before subjecting it to fermentation, ethanol production is more than that obtained from fermentation using non-hydrolyzed glucan filtrate. It becomes clear that it gets faster and more.

  Thus, fermenting glucan filtrates with saccharified leuco- and oligosaccharide by-product components increased ethanol production at a faster rate. This saccharification can be performed using, for example, transglucosidase.

Example 9
Simultaneous Saccharification and Fermentation of Glucan Filtrate Solution This example discloses that simultaneous saccharification and fermentation of a feed containing glucan filtrate results in improved fermentation characteristics.

  Suspend yeast (S. cerevisiae) cream (Bom Retiro mill, Brazil) in tap water (2.4 L, 65 nm optical density at 600 nm), then at 4500 g using a LEGEND XTR centrifuge. The yeast cream was washed by centrifuging for 5 minutes. After decanting the supernatant, the yeast was resuspended and concentrated twice more by centrifugation. After the third wash, the pH was adjusted to 4.5 by adding 5 wt% sulfuric acid, the optical density was measured using a GENESYS 20 4001 spectrophotometer, and adjusted to 100 at 600 nm by adding tap water. The adjusted yeast cream (1.5 L) was added to a 7.5 L BIOFLO 310 fermentor vessel. The fermenter was set to maintain stirring at a temperature of 30 ° C. and 100 rpm, and maintain the pH at 4.5 using a 4M ammonium hydroxide aqueous solution or a 5 wt% sulfuric acid aqueous solution.

  A feed solution containing 200 g / L sugar from yeast extract (10 g / L), peptone (20 g / L) and glucan filtrate is prepared and sterilized at 121 ° C. for 15 minutes using a PHOENIX AV-250 PLUS autoclave. did. After the feed solution was cooled to 25 ° C. (room temperature), fermentation started. TG L-2000 transglucosidase enzyme extract as described in Table 4 (1% v / v) was added to the sterilized feed solution, and then the solution was immediately added to the fermentor. A feed solution (3.5 L) containing TG L-2000 enzyme at 684 mL / hr over approximately 5 hours was added to the fermentor and the fermentation was allowed to proceed for 48 hours.

  Samples were taken periodically during fermentation and analyzed for optical density using Brix using a GENESYS 20 4001 spectrophotometer, PAL-3 refractometer (Atago), and sugar and ethanol concentrations by HPLC (general method) Was analyzed. These results are summarized in Table 16.

  This fermentation is nominally completed in 6 hours, similar to the fermentation (Example 8) where the filtrate was hydrolyzed before the fermentation process, compared to the case where a non-hydrolyzed filtrate (Example 7) was used. The ethanol titer was slightly superior (62 g / L). Furthermore, almost all leucorose was consumed by 6 hours (compare Table 16 with Tables 13-15). Similar results should be obtained when the saccharifying enzyme is added directly to the fermentation in addition to the addition of saccharifying enzyme such as TG L-2000 to the feed containing the glucan filtrate just prior to fermentation.

  Thus, as a result of simultaneous saccharification and fermentation of feeds containing glucan filtrate, fermentation characteristics such as (i) increased consumption of glucan filtrate components (eg, leuclos) and (ii) improved ethanol yield and production rate It can improve.

Example 10
Preparation of various alpha-glucosidases This example prepares various alpha-glucosidases in addition to those alpha-glucosidases (transglucosidase, glucoamylase, DIAZYME RDF ULTRA) used in some of the previous examples. To disclose. In Examples 11, 12, 15 and 16 provided below, hydrolysis to oligosaccharides comprising alpha-1,5 glucosyl-fructose linkages or alpha-1,3 and / or alpha-1,6 glucosyl-glucose linkages These additional alpha-glucosidases were tested for activity.

Discovery of Aspergillus clavatus alpha-glucosidase (Aclglu1) A strain of Aspergillus clavatus was selected as a potential source of other enzymes that could be useful in various industrial applications. One of the genes identified in Aspergillus clavatus encodes alpha-glucosidase and the sequence of this gene called “Aclglu1” is provided in SEQ ID NO: 4. The corresponding protein encoded by SEQ ID NO: 4 is provided in SEQ ID NO: 5. Acgllu1 belongs to glycosyl hydrolase family 31 based on PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO: 5) has a 19 amino acid long signal peptide as predicted by SignalP version 4.0 (Nordahhl Petersen et al., 2011, Nature Methods, 8: 785-786). The presence of the signal sequence suggests that Acgllu1 is a secreted enzyme. The predicted mature amino acid sequence of Aclglu1 is shown in SEQ ID NO: 6.

Expression of Aspergillus clavatus alpha-glucosidase Aclglu1 The synthetic Aclglu1 gene was cloned into a pTrex3gM expression vector (described in US Patent Application Publication No. 2011/0136197, incorporated herein by reference). The plasmid was named pJG294. The sequence of the Acgllu1 gene was confirmed by DNA sequencing.

  Using the gene gun method (Te'o VS et al., J Microbiol Methods, 51: 393-9, 2002), the plasmid pJG294 was transformed into a four-deletion Trichoderma reesei strain (WO 05/001036). No. pamphlet). A protein predicted to contain SEQ ID NO: 6 was secreted into the extracellular medium and filtered cultures were used to perform SDS-PAGE and alpha-glucosidase activity assays to confirm enzyme expression.

Discovery of Neosartoria fischeri alpha-glucosidase Nfigllu As a potential source of other enzymes that may be useful in a variety of industrial applications, a strain of Neosartoria fischeri was selected. One of the genes identified in Neosartoria fischeri encodes alpha-glucosidase and the sequence of this gene called “Nfigllu1” is provided in SEQ ID NO: 7. The corresponding protein encoded by SEQ ID NO: 7 is provided in SEQ ID NO: 8. Nfigllu belongs to the glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO: 8) has a 19 amino acid long signal peptide as predicted by SignalP version 4.0 (Nordahhl Petersen et al., 2011, Nature Methods, 8: 785-786). The presence of the signal sequence suggests that Nfigl1 is a secreted enzyme. The predicted amino acid sequence of the mature form of Nfigl1 is shown in SEQ ID NO: 9.

Expression of Neosartoria fischeri alpha-glucosidase Nfigllu The synthetic Nfigllu1 gene was cloned into a pTrex3gM expression vector (described in US Patent Application Publication No. 2011/0136197) and the resulting plasmid was named pJG295. The sequence of the Nfiglu1 gene was confirmed by DNA sequencing.

  Using the gene gun method (Te'o VS et al., J Microbiol Methods, 51: 393-9, 2002), the plasmid pJG295 was transformed into a quadruple deletion Trichoderma reesei strain (WO 05/001036). No. pamphlet). A protein predicted to contain SEQ ID NO: 9 was secreted into the extracellular medium and filtered cultures were used to perform SDS-PAGE and alpha-glucosidase activity assays to confirm enzyme expression.

Discovery of Neurospora crassa alpha-glucosidase Ncrgllu A strain of Neurospora crassa was selected as a potential source of other enzymes that could be useful in various industrial applications. One of the genes identified in Neurospora crassa encodes alpha-glucosidase and the sequence of this gene called “Ncrglu1” is provided in SEQ ID NO: 10. The corresponding protein encoded by SEQ ID NO: 10 is provided in SEQ ID NO: 11. Ncrglu1 belongs to the 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 22 amino acid long signal peptide as predicted by SignalP version 4.0 (Nordahl Petersen et al., 2011, Nature Methods, 8: 785-786). The presence of the signal sequence suggests that Ncrglu1 is a secreted enzyme. The predicted mature amino acid sequence of Ncrglu1 is shown in SEQ ID NO: 12.

Expression of Neurospora crassa alpha-glucosidase Ncrglu1 The synthetic Ncrglu1 gene was cloned into a pTrex3gM expression vector (described in US Patent Application Publication No. 2011/0136197) and the resulting plasmid was named pJG296. The sequence of the Ncrgllu1 gene was confirmed by DNA sequencing.

  Using the gene gun method (Te'o VS et al., J Microbiol Methods, 51: 393-399, 2002), the plasmid pJG296 was transformed into a four-deficient Trichoderma reesei strain (WO 05/001036). No. pamphlet). The protein predicted to contain SEQ ID NO: 12 was secreted into the extracellular medium, and filtered cultures were used to perform SDS-PAGE and alpha-glucosidase activity assays to confirm enzyme expression.

Discovery of Rasamonia composticola alpha-glucosidase TauSec098 As a potential source of other enzymes that could be useful in a variety of industrial applications, a strain of Rasamonia composticola was selected. One of the genes identified in Rasamonia composticola encodes alpha-glucosidase and the sequence of this gene called “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 is based on the PFAM search (pfam.sanger.ac.uk web link) and belongs to the glycosyl hydrolase family 31 and contains an N-terminal CBM20 domain. At the N-terminus, the protein (SEQ ID NO: 14) has a 22 amino acid long signal peptide as expected in SignalP version 4.0 (Nordahhl Petersen et al., 2011, Nature Methods, 8: 785-786). The presence of the signal sequence suggests that TauSec098 is a secreted enzyme. The predicted mature amino acid sequence of TauSec098 is shown in SEQ ID NO: 15.

Expression of Rasamonia composticola alpha-glucosidase TauSec098 Generay Biotech Co. (Shanghai, China) cloned the synthetic TauSec098 gene into the Trichoderma reesei expression vector pGXT (pTTT-derived plasmid) and named the resulting plasmid as pGX256-TauSec098. The sequence of the TauSec098 gene was confirmed by DNA sequencing.

Using protoplast transformation (Te'o et al., Et al., J. Microbiol. Methods 51: 393-399, 2002), the plasmid pGX256-TauSec098 was transformed into the quadruple deletion Trichoderma reesei strain (International Publication No. 1). 05/001036 pamphlet). Medium containing acetamide as the only nitrogen source (acetamide 0.6 g / L; cesium chloride 1.68 g / L; glucose 20 g / L; potassium dihydrogen phosphate 15 g / L; magnesium sulfate heptahydrate 0.6 g / L; calcium chloride dihydrate 0.6 g / L; iron (II) sulfate 5 mg / L; zinc sulfate 1.4 mg / L; cobalt (II) chloride 1 mg / L; manganese (II) sulfate 1.6 mg / L Transformants were selected on agar 20 g / L; pH 4.25). Transformed colonies (about 50-100) appeared in about one week. After growth on acetamide plates, transformant spores were collected and transferred onto fresh acetamide agar plates. After 5 days of growth on acetamide plates, 1 × 10 ⁸ spores were inoculated into 30 mL glucose / sophorose limited medium in a 250 mL shake flask. The shake flask was shaken 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 Rasamonia composticola Alpha-glucosidase TauSec099 A strain of Rasamonia composticola was selected as a potential source of other enzymes that may be useful in a variety of industrial applications. One of the genes identified in Rasamonia composticola encodes 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 the glycosyl hydrolase family 31 based on the PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, this protein (SEQ ID NO: 17) has a 17 amino acid long signal peptide as predicted by SignalP version 4.0 (Nordahhl Petersen et al., 2011, Nature Methods, 8: 785-786). The presence of the signal sequence suggests that TauSec099 is a secreted enzyme. The predicted mature amino acid sequence of TauSec099 is shown in SEQ ID NO: 18.

Expression of Rasamonia composticola alpha-glucosidase TauSec099 Generay Biotech Co. The synthetic TauSec099 gene was cloned into the Trichoderma reesei expression vector pGXT (pTTT-derived plasmid) by (Shanghai, China), and the resulting plasmid was named pGX256-TauSec099. The sequence of the TauSec0998 gene was confirmed by DNA sequencing.

Using protoplast transformation (Te'o et al., J. Microbiol. Methods 51: 393-399, 2002), the plasmid pGX256-TauSec099 was transformed into the quadruple deletion Trichoderma reesei strain (International Publication No. 05). / 001036 pamphlet). Medium containing acetamide as the only nitrogen source (acetamide 0.6 g / L; cesium chloride 1.68 g / L; glucose 20 g / L; potassium dihydrogen phosphate 15 g / L; magnesium sulfate heptahydrate 0.6 g / L; calcium chloride dihydrate 0.6 g / L; iron (II) sulfate 5 mg / L; zinc sulfate 1.4 mg / L; cobalt (II) chloride 1 mg / L; manganese (II) sulfate 1.6 mg / L Transformants were selected on agar 20 g / L; pH 4.25). Transformed colonies (about 50-100) appeared in about one week. After growth on acetamide plates, transformant spores were collected and transferred onto fresh acetamide agar plates. After 5 days of growth on acetamide plates, 1 × 10 ⁸ spores were inoculated into 30 mL glucose / sophorose limited medium in a 250 mL shake flask. The shake flask was shaken 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).

Sequence of Bifidobacterium longum alpha-glucosidase BloGlu1 An alpha-glucosidase gene, “BloGlu1”, was identified from Bifidobacterium longum subsp. Longum JDM301. The nucleic acid sequence for the BloGlu1 gene (SEQ ID NO: 19, GENBANK, accession number NC014169.1, complementary sequence at positions 140600 to 142414) and the amino acid sequence of the hypothetical protein encoded by SEQ ID NO: 19 (SEQ ID NO: 20) is the GENBANK accession number Found in YP_0036604432.1.

Expression of Bifidobacterium longum alpha-glucosidase BloGlu1 The DNA sequence encoding the entire BloGlu1 protein (SEQ ID NO: 20) Optimized for expression in B. subtilis and then synthesized (resulting in SEQ ID NO: 21), Generay Biotech Co. Was inserted into the p3JM plasmid by (Shanghai, China) to obtain p3JM-BloGlu1. The p3JM-BloGlu1 plasmid contains the aprE promoter to drive expression of the optimized BloGlu1 sequence (SEQ ID NO: 21).

B. To transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), plasmid p3JM-BloGlu1 was used and supplemented with 5 ppm chloramphenicol. Transformed cells were seeded on agar plates. In order to select colonies with the correct insertion as confirmed by PCR and sequencing and to express the BloGlu1 protein (SEQ ID NO: 20), MBD medium (limited medium based on MOPS supplemented with additional 5 mM CaCl ₂ ) was included. Subjected to fermentation in a 250 mL shake flask.

Sequence of Bifidobacterium longum alpha-glucosidase BloGlu2 An alpha-glucosidase, BloGlu2, was identified from Bifidobacterium longum. The amino acid sequence of BloGlu2 (SEQ ID NO: 22) was found in the NCBI database (GENBANK accession number WP_0070546665.1).

Expression of Bifidobacterium longum alpha-glucosidase BloGlu2 The DNA sequence encoding the BloGlu2 protein is represented by Optimized for expression in B. subtilis and then synthesized (resulting in SEQ ID NO: 23), Generay Biotech Co. Was inserted into the p3JM plasmid to obtain p3JM-BloGlu2. SEQ ID NO: 23 encodes the amino acid sequence of SEQ ID NO: 24. The p3JM-BloGlu2 plasmid contains the aprE promoter to drive expression of the optimized BloGlu2 sequence (SEQ ID NO: 23).

B. To transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), plasmid p3JM-BloGlu2 was used and supplemented with 5 ppm chloramphenicol. Transformed cells were seeded on agar plates. In order to select colonies with the correct insertion as confirmed by PCR and sequencing and to express the BloGlu2 protein (SEQ ID NO: 24), MBD medium (a limited medium based on MOPS supplemented with additional 5 mM CaCl ₂ ) was included. Subjected to fermentation in a 250 mL shake flask.

Sequence of Bifidobacterium longum alpha-glucosidase BloGlu3 An alpha-glucosidase gene, "BloGlu3" was identified from Bifidobacterium longum subspecies longum F8. The nucleic acid sequence for the BloGlu3 gene (SEQ ID NO: 25, GENBANK accession number NC — 021008.1, positions 2130627-2132441) and the amino acid sequence of the hypothetical protein encoded by SEQ ID NO: 25 (SEQ ID NO: 26) is GENBANK accession number YP — 007768249.1. It was found.

Expression of Bifidobacterium longum alpha-glucosidase BloGlu3 The DNA sequence encoding the entire BloGlu3 protein (SEQ ID NO: 26) is expressed in B. cerevisiae. Optimized for expression in B. subtilis, then synthesized (resulting in SEQ ID NO: 27) and generated by Generay Biotech Co. Was inserted into the p3JM plasmid to obtain p3JM-BloGlu3. The p3JM-BloGlu3 plasmid contains the aprE promoter to drive expression of the optimized BloGlu3 sequence (SEQ ID NO: 27).

B. To transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), plasmid p3JM-BloGlu3 was used and supplemented with 5 ppm chloramphenicol. Transformed cells were seeded on agar plates. In order to select colonies with the correct insertion when confirmed by PCR and sequencing and to express the BloGlu3 protein (SEQ ID NO: 26), MBD medium (limited medium based on MOPS supplemented with additional 5 mM CaCl ₂ ) was included. Subjected to fermentation in a 250 mL shake flask.

Sequence of Bifidobacterium pseudolongum alpha-glucosidase BpsGlu1 An alpha-glucosidase, BpsGlu1, was identified from Bifidobacterium pseudolongum. The amino acid sequence of BpsGlu1 (SEQ ID NO: 28) was found in the NCBI database (GENBANK accession number WP_022858408.1).

Expression of Bifidobacterium pseudolongum alpha-glucosidase BpsGlu1 The DNA sequence encoding the BpsGlu1 protein Optimized for expression in B. subtilis and then synthesized (resulting in SEQ ID NO: 29), Generay Biotech Co. Was inserted into the p3JM plasmid to obtain p3JM-BpsGlu1. SEQ ID NO: 29 encodes the amino acid sequence of SEQ ID NO: 30. The p3JM-BpsGlu1 plasmid contains the aprE promoter to drive expression of the optimized BpsGlu1 sequence (SEQ ID NO: 29).

B. To transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), plasmid p3JM-BpsGlu1 was used and supplemented with 5 ppm chloramphenicol. Transformed cells were seeded on agar plates. In order to select colonies with correct insertions as confirmed by PCR and sequencing, and to express the BpsGlu1 protein (SEQ ID NO: 30), MBD medium (limited medium based on MOPS supplemented with additional 5 mM CaCl ₂ ) was included. Subjected to fermentation in a 250 mL shake flask.

Sequence of Bifidobacterium thermofilm alpha-glucosidase BthGlu1 An alpha-glucosidase gene, “BthGlu1” was identified from Bifidobacterium thermophilum RBL67. The nucleic acid sequence of the BthGlu1 gene (SEQ ID NO: 31, GENBANK accession number NC — 0205466.1, positions 150690 to 152495) and the hypothetical protein amino acid sequence (SEQ ID NO: 32) encoded by SEQ ID NO: 31 are represented by the GENBANK accession number YP — 007592840.1 Found in

Expression of Bifidobacterium thermophilum alpha-glucosidase BthGlu1 Optimized for expression in B. subtilis and then synthesized (resulting in SEQ ID NO: 33), Generay Biotech Co. Was inserted into the p3JM plasmid to obtain p3JM-BthGlu1. The p3JM-BthGlu1 plasmid contains the aprE promoter to drive expression of the plasmid with the optimized BthGlu1 sequence (SEQ ID NO: 33).

B. To transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), plasmid p3JM-BthGlu1 was used and supplemented with 5 ppm chloramphenicol. Transformed cells were seeded on agar plates. MBD medium (limited medium based on MOPS supplemented with additional 5 mM CaCl ₂ ) was included to select colonies with the correct insertion as confirmed by PCR and sequencing and to express the BthGlu1 protein (SEQ ID NO: 32) Subjected to fermentation in a 250 mL shake flask.

Sequence of Bifidobacterium breve alpha-glucosidase BbrGlu2 An alpha-glucosidase, BbrGlu2, was identified from Bifidobacterium breve. The amino acid sequence of BbrGlu2 (SEQ ID NO: 34) was found in the NCBI database (GENBANK accession number WP_003827971.1).

Expression of Bifidobacterium breve alpha-glucosidase BbrGlu2 The DNA sequence encoding the BbrGlu2 protein is expressed in B. cerevisiae. Optimized for expression in B. subtilis and then synthesized (resulting in SEQ ID NO: 35), Generay Biotech Co. Was inserted into the p3JM plasmid to obtain p3JM-BbrGlu2. SEQ ID NO: 35 encodes the amino acid sequence of SEQ ID NO: 36. The p3JM-BbrGlu2 plasmid contains the aprE promoter to drive expression of the optimized BbrGlu2 sequence (SEQ ID NO: 35).

B. To transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), plasmid p3JM-BbrGlu2 was used and supplemented with 5 ppm chloramphenicol. Transformed cells were seeded on agar plates. In a 250 mL shake flask containing MBD medium (a limited medium based on MOPS supplemented with additional 5 mM CaCl ₂ ) to select colonies with the correct insertion as confirmed by PCR and sequencing and to express SEQ ID NO: 36 It was subjected to fermentation.

Sequence of Bifidobacterium breve alpha-glucosidase BbrGlu5 An alpha-glucosidase gene “BbrGlu5” was identified from Bifidobacterium breve ACS-071-V-Sch8b. The nucleic acid sequence of the BbrGlu5 gene (SEQ ID NO: 37, GENBANK accession number NC — 07218.11, the complement of the sequence at positions 2241705-2224895) and the amino acid sequence of the hypothetical protein encoded by SEQ ID NO: 37 (SEQ ID NO: 38) Found with the number YP_005583701.1.

Expression of Bifidobacterium breve alpha-glucosidase BbrGlu5 Optimized for expression in B. subtilis and then synthesized (resulting in SEQ ID NO: 39), Generay Biotech Co. Was inserted into the p3JM plasmid to obtain p3JM-BbrGlu5. The p3JM-BbrGlu5 plasmid contains the aprE promoter to drive expression of the optimized BbrGlu5 sequence (SEQ ID NO: 39).

B. To transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), plasmid p3JM-BbrGlu5 was used and supplemented with 5 ppm chloramphenicol. Transformed cells were seeded on agar plates. In order to select colonies with the correct insertion as confirmed by PCR and sequencing and to express the BbrGlu5 protein (SEQ ID NO: 38), MBD medium (limited medium based on MOPS supplemented with additional 5 mM CaCl ₂ ) was included. Subjected to fermentation in a 250 mL shake flask.

Purification of alpha-glucosidases AclGlu1 and NcrGlu1 from expression cultures Two chromatographic steps were used to purify both AclGlu1 (SEQ ID NO: 6) and NcrGlu1 (SEQ ID NO: 12) alpha-glucosidases. For each purification, the crude broth from the shake flask was concentrated, followed by the addition of ammonium sulfate to a final concentration of 2M. The solution was loaded onto a 50 mL phenyl HP column pre-equilibrated with 20 mM Tris pH 8.0, 2M ammonium sulfate. The target protein (SEQ ID NO: 6 or SEQ ID NO: 12) was eluted from the column with 1M ammonium sulfate, 20 mM Tris pH 8.0. Individual fractions were pooled, concentrated and the buffer was changed to 20 mM Tris pH 8.0 (Buffer A) using a VIVAFLOW 200 ultrafilter (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 NaCl in buffer A. Fractions containing the target protein were then pooled, concentrated using a 10K AMICON ULTRA-15 instrument, and stored in 40% glycerol at −20 ° C. until use.

NfiGlu1
NfiGlu1 alpha-glucosidase (SEQ ID NO: 9) was purified using two hydrophobic interaction chromatography steps. The crude broth from the shake flask was concentrated and then ammonium sulfate was added to a final concentration of 1M. The solution was loaded onto a 50 mL phenyl HP column pre-equilibrated with 20 mM Tris pH 8.0, 1M ammonium sulfate. The target protein (SEQ ID NO: 9) passed through the column. The flow-through fractions were pooled and then ammonium sulfate was added to a final concentration of 2M. The solution was loaded onto the same phenyl HP column pre-equilibrated with 20 mM Tris pH 8.0, 2M ammonium sulfate. The target protein was eluted from the column with 1M ammonium sulfate, 20 mM Tris pH 8.0. Fractions containing the target protein were then pooled, concentrated using a 10K AMICON ULTRA-15 instrument, and stored in 40% glycerol at −20 ° C. until use.

TauSec098 and TauSec099
Both TauSec098 (SEQ ID NO: 15) and TauSec099 (SEQ ID NO: 18) alpha-glucosidase were purified via hydrophobic interaction chromatography. For each purification, ammonium sulfate was added to about 180 mL concentrated crude broth from a 7 L fermentor to a final concentration of 1M. This solution was then loaded onto a 50 mL HIPREP phenyl-FF Sepharose column (GE Healthcare) pre-equilibrated with 20 mM sodium acetate pH 5.0, 1 M ammonium sulfate (Buffer A). After washing with 3 column volumes (CV) with the same buffer, the column was eluted stepwise with 75%, 50% and 0% buffer A, followed by ₂ CV MILLIQ H ₂ O with each 3 CV. All fractions were analyzed by SDS-PAGE. The target protein (SEQ ID NO: 15 or SEQ ID NO: 18) is predominantly present in the flow-through fraction and is concentrated and the buffer exchanged to remove excess ammonium sulfate using a 10 KDa AMICON ULTRA-15 instrument. . The final product exceeding 90% purity was stored in -40 ° C. in 40% glycerol until use.

BloGlu1, BloGlu2 and BloGlu3
BloGlu1 (SEQ ID NO: 20), BloGlu2 (SEQ ID NO: 24) and BloGlu3 (SEQ ID NO: 26) alpha-glucosidase were all purified in three steps. For each purification, the crude broth from the 1 L DASGIP fermentor was concentrated and then ammonium sulfate was added to 60% saturation. The solution was stirred at 4 ° C. for 1 hour and then centrifuged at 8000 × g for 30 minutes. The resulting pellet was resuspended in 20 mM Tris pH 8.0 (Buffer A). Ammonium sulfate is added to the resulting solution to a final concentration of 1M; the preparation is then loaded onto a 40 mL HiPrep ™ Phenyl FF column pre-equilibrated with 20 mM Tris pH 8.0, 1M ammonium sulfate (Buffer B). I put it. After washing, the column was eluted stepwise with 75%, 50% and 0% buffer B and H ₂ O in 3 column volumes each. All fractions were analyzed using SDS-PAGE, activity assay. Fractions containing the target protein (SEQ ID NO: 20, SEQ ID NO: 24 or SEQ ID NO: 26) are pooled, concentrated and subsequently pre-equilibrated with 20 mM sodium phosphate pH 7.0, 0.15 M NaCl. It was loaded onto a 26/60 Superdex ™ 75 column. The flow-through fractions containing the target protein were then pooled, concentrated using a 10K AMICON ULTRA-15 instrument, and stored in 40% glycerol at -20 ° C until use.

BpsGlu1 and BthGlu1
Both BpsGlu1 (SEQ ID NO: 30) and BthGlu1 (SEQ ID NO: 32) were purified in two steps. For each purification, the crude broth from the 1 L DASGIP fermentor was concentrated and then ammonium sulfate was added to 60% saturation. The solution was stirred at 4 ° C. for 1 hour and then centrifuged at 8000 × g for 30 minutes. The resulting pellet was resuspended in 20 mM Tris pH 8.0 (Buffer A). Ammonium sulfate is added to the resulting solution to a final concentration of 1M; the preparation is then loaded onto a 40 mL HiPrep ™ Phenyl FF column pre-equilibrated with 20 mM Tris pH 8.0, 1M ammonium sulfate (Buffer B). I put it. After washing, the column was eluted stepwise with 75%, 50% and 0% buffer B and H ₂ O, each at 3 column volumes. 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; the eluate was pooled and concentrated using a 10K AMICON ULTRA-15 instrument. The final product, which was higher than 95% purity, was stored in 40% glycerol at −20 ° C. until use.

BbrGlu2 and BbrGlu5
Both BbrGlu2 (SEQ ID NO: 36) and BbrGlu5 (SEQ ID NO: 38) alpha-glucosidases were purified in four steps. For each purification, the crude broth from the 1 L DASGIP fermentor was concentrated and then ammonium sulfate was added to 60% saturation. The solution was stirred at 4 ° C. for 1 hour and then centrifuged at 8000 × g for 30 minutes. The resulting pellet was resuspended in 20 mM HEPES pH 7.0 (Buffer A). Ammonium sulfate was added to the resulting solution to a final concentration of 1M; the preparation was then loaded onto a HiPrep ™ phenyl FF column pre-equilibrated with 20 mM HEPES pH 7.0, 1M ammonium sulfate. The target protein (SEQ ID NO: 36 or SEQ ID NO: 38) was eluted from the column with 0.5M ammonium sulfate. Individual fractions were pooled, concentrated, and the buffer was changed to buffer A using a VIVAFLOW 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to a HiPrep ™ Q FF 16/10 column pre-equilibrated with buffer A. The target protein was eluted from the column with a linear gradient of 0-0.5 M NaCl in buffer A. Fractions containing the 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 NaCl. Fractions containing the target protein were then pooled, concentrated using a 10K AMICON ULTRA-15 instrument, and stored in 40% glycerol at −20 ° C. until use.

  In this way, various additional alpha-glucosidases were expressed and purified. In Examples 11, 12, 15 and 16 provided below, these hydrolytic activities for alpha-1,5 glucosyl-fructose and alpha-1,3 and / or alpha-1,6 glucosyl-glucose bonds Alpha-glucosidase was tested.

Example 11
Testing of Alpha-Glucosidase for Hydrolytic Activity on Various Glycoside Bonds This example demonstrates that hydrolytic activity of alpha-glucosidase exceeds that previously associated with this class of enzymes (EC 3.2.1.20). It is disclosed to test whether it can have. The alpha-glucosidase from Example 10 was shown to have hydrolytic activity on alpha-1,5 glucosyl-fructose linkages and alpha-1,3 and alpha-1,6 glucosyl-glucose linkages.

Alpha-Glucosidase Substrate Specificity Each of the disclosed in Example 10 based on the release of glucose from a specific substrate (Isomaltose, Maltose, Panose, Leucrose or Nigerose) when the substrate is incubated with alpha-glucosidase The substrate specificity of alpha-glucosidase was assayed. Glucose release rate was measured using a conjugated 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-ethylbenzothiazoline-6-sulfonic acid (ABTS) by peroxide generated from conjugated GOX / HRP enzyme reacted with glucose.

Prepare individual substrate solutions by mixing 9 mL substrate solution (1% in water, w / v) with 1 mL 0.5 M pH 5.0 sodium acetate buffer and 40 μL 0.5 M calcium chloride in a 15 mL conical tube. did. A conjugate enzyme (GOX / HRP) solution with ABTS was prepared in 50 mM sodium acetate buffer (pH 5.0) at a final concentration 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 standards were prepared in MILLIQ water. In the case of nigerose, alpha-glucosidase samples were tested at a dose of only 10 ppm due to the limited storage of substrate solutions. Each alpha-glucosidase sample (10 μL) was transferred to a new microtiter plate (Corning 3641) containing 90 μL substrate solution pre-incubated at 600 rpm for 5 minutes at 50 ° C. The reaction was performed at 50 ° C. for 10 minutes (for isomaltose, maltose, panose and nigerose substrates) or 60 minutes (for leuclose substrates) with shaking (600 rpm) in THERMOMIXER (Eppendorf). Then 10 μL of each reaction mixture as well as 10 μL serial dilutions of glucose standards were quickly transferred to each new microtiter plate (Corning 3641), and then 90 μL ABTS / GOX / HRP solution was added appropriately thereto. The microtiter plate containing the reaction mixture was immediately measured using a SOFMAX PRO plate reader (Molecular Devices) for 5 minutes at 11 second intervals at 405 nm. Output is the reaction rate for each enzyme concentration was V _o. Using a linear regression to determine the slope of the plot V _o versus enzyme dosages. Formula 1:
Specific activity (unit / mg) = slope (enzyme) / slope (std) × 1000 (1)
(Wherein 1 unit = 1 μmol glucose / min)
Was used to calculate the specific activity of each alpha-glucosidase based on the glucose standard curve. In the case of nigerose, reaction rate values at 10 ppm enzyme dose were used directly to show enzyme activity.

  Using the previous method, the specificity of each alpha-glucosidase was determined for each substrate. The activities of oligo-1,6-glucosidase (purchased from Megazyme, see Table 4) and transglucosidase (TG L-2000, see Table 4) for each substrate were also measured. The results of this analysis are shown in Table 17.

  Interestingly, alpha-glucosidase, besides exhibiting hydrolytic activity on alpha-1,4 glucosyl-glucose bond (maltose), alpha-1,6 glucosyl-glucose bond (isomaltose), alpha-1,3 It was also shown to exhibit hydrolytic activity on glucosyl-glucose bonds (nigerose) and alpha-1,5 glucosyl-fructose bonds (leucus) (Table 17).

  Thus, alpha-glucosidase has a hydrolytic activity that exceeds that previously associated with the EC 3.2.1.20 enzyme. Specifically, alpha-glucosidase has hydrolytic activity on alpha-1,5 glucosyl-fructose bonds and alpha-1,3 and alpha-1,6 glucosyl-glucose bonds.

Example 12
Hydrolysis of Leucrose and Oligosaccharides in Glucan Reaction Filtrate Using Alpha-Glucosidase This example illustrates the use of alpha-to hydrolyze leucus and other oligosaccharides present in the filtrate obtained from glucan synthesis reaction The use of glucosidase is described. Specifically, alpha disclosed in Example 10 in the hydrolysis of leucos and oligosaccharides DP2, DP3 and HS (higher saccharide, DP4 +) in the filtrate of insoluble glucan (polyalpha-1,3-glucan) synthesis reaction -The effect of glucosidase was tested.

Isolation and analysis of oligosaccharides to test for alpha-glucosidase activity First, a concentrated filtrate of glucan synthesis reaction was prepared as in Example 1.

Briefly, oligosaccharides were isolated from the concentrated filtrate by chromatographic separation and analyzed for glycoside binding profiles. The oligosaccharide fraction of the concentrated filtrate was isolated using chromatographic separation using a strong acid cation exchange resin. The physical parameters of the column used for this separation were as follows: FINEX CS11GC, # 227 resin; Na ⁺ ion type; 5% divinylbenzene (crosslinked); 0.34 mm particle size; 1.64 m bed length 0.093 m column diameter.

  More specifically, the concentrated sugar solution (that is, the concentrated filtrate) described in Table 3 was filtered and diluted with tap water to a 25 g dry solid / 100 g solution. Before this sugar solution is added to the column resin, the resin is washed with 6 bed volumes (BV) of sodium chloride solution (3 BV with 10 wt% sodium chloride followed by 3 BV with 5 wt% sodium chloride) to remove the resin from the sodium form. Converted to. The sugar solution (0.6 L) was then added to the column and then the column was eluted with water at a flow rate of 50 mL / min. The operating conditions for chromatographic separation are summarized as follows: 0.6 L feed size, 25 g dry solid / 100 g solution, 65 ° C. column temperature, 50 mL / min flow rate. The oligosaccharide solution was eluted in 11-21 minutes. A small amount of salt was eluted at the same time as indicated by the increase in conductivity. The oligosaccharide fraction thus prepared was analyzed by HPLC to determine its product distribution. Overall, the fractions contained> 89% oligosaccharides containing 3 or more hexose units and less than 1.5% distinguishable mono and disaccharides. Using a thin film evaporator (LCI Corporation, Charlotte, NC) followed by a rotary evaporator with ROTAVAPOR (R-151; Buchi, New Castle, DE), this fraction was brought to a total dry weight of 317 g / L. Concentrated. Table 18 shows the product distribution of the concentrated fraction as measured by HPLC.

Primary screening of alpha-glucosidase in glucan oligomer hydrolysis Activity of 11 different alpha-glucosidases (Example 10) and two benchmark enzymes, oligo-1,6-glucosidase (purchased from Megazyme) and transglucosidase (TG L -2000) activity was individually assessed against the purified oligosaccharide fractions prepared above (Table 18). Each alpha-glucosidase (prepared at 1 mg / mL) was incubated in a solution containing oligosaccharide substrate (2.9% dry solids) at pH 5.0 and 2 mM calcium chloride at 60 ° C. Each reaction was quenched after 24 hours of incubation by adding 50 μL of 0.5 M NaOH.

The oligosaccharide / monosaccharide content of the stopped reaction was determined as follows. Samples for each reaction were diluted 5-fold in water for HPLC analysis. HPLC separation was performed using an Agilent 1200 series HPLC system on an AMINEX HPX-42A column (300 mm x 7.8 mm) at 85 ° C. Sample (10 μL) was applied to the HPLC column and separated with a homogeneous gradient of MILLI-Q water as the mobile phase at a flow rate of 0.6 mL / min. The oligosaccharide product was detected using a refractive index detector. Numbers shown in Table 19 reflects the average (two sets of each sample) of the peak area percentage of each DP _n as the sum of the fractions of DP1～DP7.

  As shown by the shaded in Table 19, the oligosaccharide content of the reaction is generally shifted towards a smaller size sugar compared to a control reaction without enzyme (“blank”). did. These results indicate that alpha-glucosidase can be used to hydrolyze the oligosaccharides contained within the glucan synthesis reaction and its fractions. Also, considering the oligosaccharide binding profile (Examples 3 and 4) and the activity of alpha-glucosidase on various glycosidic bonds (Example 11) in addition to alpha-1,4 linkages, alpha-1,5 glucosyl- It would be possible to use alpha-glucosidase to degrade oligosaccharides with fructose linkages and / or alpha-1,3 and alpha-1,6 glucosyl-glucose linkages. The results shown in Table 19 also suggest that fungal alpha-glucosidase has better hydrolytic activity on soluble oligosaccharides compared to bacterial alpha-glucosidase.

Confirmation of Alpha-Glucosidase Hydrolysis Activity for Oligosaccharide Product of Glucan Synthesis Reaction A reaction product containing concentrated filtrate obtained from one or two types of alpha-glucosidase and polyalpha-1,3-glucan synthesis reaction was prepared ( Table 3). The alpha-glucosidase reaction was performed with 4 ppm of enzyme or, in the case of a mixture, each enzyme was used at a final ratio of 4 ppm in a 1: 1 ratio. In each reaction, the concentrated filtrate was loaded with 10% dry solids. Each reaction further contained 2 mM calcium chloride at pH 5.0 and was performed at 60 ° C. or 65 ° C. After incubation for 23 hours, the reaction was stopped by adding 50 μL of 0.5 M NaOH.

The oligosaccharide / monosaccharide content of the stopped reaction was determined as follows. Samples for each reaction were diluted 25-fold in water for HPLC analysis. HPLC separation was performed using an Agilent 1200 series HPLC system on an AMINEX HPX-42A column (300 mm x 7.8 mm) at 85 ° C. Sample (10 μL) was applied to the HPLC column and separated with a homogeneous gradient of MILLI-Q water as the mobile phase at a flow rate of 0.6 mL / min. The oligosaccharide product was detected using a refractive index detector. The numbers shown in Table 20 below reflect the average (from duplicates for each sample) peak area percentage for each DP _n as the overall fraction. The results shown in Table 20 generally confirm the activity of certain alpha-glucosidases, as discussed above with respect to the results shown in Table 19.

Thus, alpha-glucosidase is used to hydrolyze leucrose and other oligosaccharides present in fractions (eg, filtrate) obtained from glucan synthesis reactions such as polyalpha-1,3-glucan synthesis reactions can do.

Example 13
Isolation of the oligomer / leucus fraction from the gtf-S / MUT3325 reaction Dissolve sucrose (4.50 kg) in distilled deionized water in a final total volume of 9.5 L and stir at 80 ° C. for 5 minutes. The resulting solution was heated and then cooled to 47 ° C. While stirring, 500 grams of 15.0 mL crude extract containing 0.6 g / L gtf-S enzyme (GTF0459, SEQ ID NO: 42) and 10 g / L mutanase (MUT3325, SEQ ID NO: 47) The crude extract was added with stirring (see general method for enzyme preparation). The pH of the resulting mixture was immediately adjusted to pH 5.5-pH 6.0 by slowly adding a 1:10 (v / v) dilution of 37 wt% HCl with stirring. The reaction temperature and pH are maintained at 47 ° C. and pH 5.5-6.0, respectively, until sucrose conversion is> 95% by HPLC analysis, after which the reaction mixture is immediately adjusted to pH 7.0-7.5, Heated to 90 ° C. for 20 minutes, then cooled to 25 ° C. for immediate filtration to remove particles and precipitate. The following resins and conditions: FIN ²⁺ CS 11 GC SAC of the Ca ²⁺ type, 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. The resulting filtrate was held at 5 ° C. prior to IEX / SEC column chromatography using 1 g / 100 g, sample interval = 3 minutes. Column fractions collected at 30-67 minutes were combined, concentrated by evaporation to 66% dissolved solids and analyzed by HPLC as described in General Methods. Table 21 shows the oligosaccharide and monosaccharide components of the isolated fraction thus prepared.

  In this example, a glucan synthesis reaction was used to produce at least one soluble alpha-glucan product. This soluble product was obtained from the concerted action of both glucosyltransferase (GTF0459, SEQ ID NO: 42) and alpha-glucanohydrolase (MUT3325, SEQ ID NO: 47), both present during the glucosyltransferase reaction. This example also revealed the preparation of a chromatographic fraction from the glucan synthesis reaction. This fraction was used in Examples 15 and 16 below to test the activity of alpha-glucosidase against it.

Example 14
Isolation of Oligomer / Leucrose Fraction from Gtf-C Reaction Dissolve sucrose (4.50 kg) in distilled deionized water to a final total volume of 9.5 L and stir the resulting solution at 80 ° C. for 5 minutes And then cooled to 47 ° C. While stirring, 500 grams of crude extract containing 0.41 g / L 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 pH 5.5-pH 6.0 by slowly adding a 1:10 (v / v) dilution of 37 wt% HCl with stirring. The reaction temperature and pH are maintained at 47 ° C. and pH 5.5-6.0, respectively, until sucrose conversion is> 95% by HPLC analysis, after which the reaction mixture is immediately adjusted to pH 7.0-7.5, Heated to 90 ° C. for 20 minutes, then cooled to 25 ° C. for immediate filtration to remove particles and precipitate. The following resins and conditions: FIN ²⁺ CS 11 GC SAC of the Ca ²⁺ type, 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. The resulting filtrate was kept at 5 ° C. before IEX / SEC column chromatography using 8 g / 100 g, sample interval = 3 minutes. Column fractions collected at 34-72 minutes were combined, concentrated by evaporation to 67% dissolved solids and analyzed by HPLC as described in General Methods. Table 22 shows the oligosaccharide and monosaccharide components of the isolated fraction thus prepared.

  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-glucosidase against it.

Example 15
Primary Screening for Alpha-Glucosidase Using Oligomeric / Leucrose Fractions from Gtf-S / MUT3325 and Gtf-C Reactions Describes the use of alpha-glucosidase to hydrolyze leucrose and other oligosaccharides present in the fraction. Specifically, the effect of the alpha-glucosidase disclosed in Example 10 on the hydrolysis of leucus and oligosaccharides in the fractions prepared in Examples 13 and 14 was tested.

  A total of 12 alpha-glucosidases and 2 benchmark enzymes (oligos) using the oligomer / leucrose fraction from the gtf-S / MUT3325 (Example 13) and gtf-C (Example 14) reactions as substrate materials -1,6-glucosidase and TG L-2000 transglucosidase). All enzymes (alpha-glucosidase and benchmark enzyme) were divided at equal protein concentrations. Each alpha-glucosidase (divided at 100 ppm) was incubated in a solution containing an oligomer / leucus substrate (10% dry solids) at pH 5.5 and 10 mM dry solids and 2 mM calcium chloride. Each reaction was stopped after 21 hours of incubation by adding 50 μL of 0.5 M NaOH.

The oligosaccharide / monosaccharide content of the stopped reaction was determined as follows. Samples from each reaction were centrifuged and the supernatant from them was diluted 25-fold in water for HPLC analysis (general method). The percentages reported in Table 23 reflect the average (from duplicate of each sample) of the peak area percentage for each DP _n as the overall fraction. The results show that fungal alpha-glucosidase had better hydrolytic activity on glucan oligomers when compared to bacterial alpha-glucosidase.

  As shown by the shaded in Table 23, the oligosaccharide content of the reaction is generally shifted towards smaller size sugars compared to the control reaction without enzyme (“blank”). did. From these results, alpha-glucosidase was used to hydrolyze the oligosaccharides contained within the chromatographic fraction of the glucan synthesis reaction and its fractions, especially the glucan synthesis reaction that produced the soluble alpha-glucan product. It can be shown that Also, considering the oligosaccharide binding profile (Examples 13 and 14) and the activity of alpha-glucosidase on various glucoside linkages (Example 11) in addition to alpha-1,4 linkages, alpha-1,5 glucosyl- It appears that alpha-glucosidase can be used to degrade oligosaccharides that also have fructose linkages and also possibly alpha-1,3 and alpha-1,6 glucosyl-glucose linkages. The results shown in Table 23 also suggest that fungal alpha-glucosidase has better hydrolytic activity on soluble oligosaccharides compared to bacterial alpha-glucosidase.

  Thus, alpha-glucosidase hydrolyzes leucus and other oligosaccharides present in fractions obtained from glucan synthesis reactions, such as those that synthesize soluble alpha-glucan products (eg, chromatographic fractions). Can be used for.

Example 16
Selective Screening for Alpha-Glucosidase Using Oligomer / Leucrose Fractions from Gtf-S / MUT3325 and Gtf-C Reactions Describes the use of alpha-glucosidase to hydrolyze leucus and other oligosaccharides present in the chromatographic fractions obtained from the reaction.

  By analyzing the sugar composition that causes a reaction containing an enzyme separated at equal protein concentrations, against hydrolysis of the oligomer / leucus fraction from the gtf-S / MUT3325 and gtf-C reactions (Example 15) The alpha-glucosidase with the highest activity was evaluated. In the presence of 2 mM calcium chloride at pH 5.5, alpha-glucosidase (separated by 4 ppm at 60 ° C. and 65 ° C. respectively; in the case of the mixture, the ratio of the two enzymes was 1: 1 and the total dose was 4 ppm. ) And oligomer / leucus substrate (10% ds). The reaction was stopped after 23 hours of incubation by adding 50 μL of 0.5 M NaOH.

The oligosaccharide / monosaccharide content of the stopped reaction was determined as follows. Samples from each reaction were centrifuged and the supernatant from it was diluted 25-fold in water for HPLC analysis (general method). The percentages reported in Table 24 (below) reflect the average (from duplicates for each sample) of the peak area percentage for each DP _n as the overall fraction. The results show that TauSec098 is effective for hydrolysis of DP2-DP7 oligomers and that TauSec099 was superior to TG L-2000 for leuco hydrolysis when incubated at 65 ° C. Indicated. A mixture of TauSec098 and TauSec099 (or TG L-2000) was effective against the hydrolysis of oligomers and leucus for DP1 production.

  Thus, alpha-glucosidase hydrolyzes leucus and other oligosaccharides present in fractions obtained from glucan synthesis reactions, such as those that synthesize soluble alpha-glucan products (eg, chromatographic fractions). Can be used for

Claims (15)

  A method of hydrolyzing an alpha-1,3 or alpha-1,6 glucosyl-glucose bond in a sugar comprising at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond, said sugar being A sugar or an oligosaccharide, wherein the method comprises contacting the sugar with an alpha-glucosidase enzyme under suitable conditions, wherein the alpha-glucosidase enzyme is at least one alpha-1,3 or alpha of the sugar. A method wherein the 1,6-glucosyl-glucose bond is hydrolyzed and the amount of the sugar is reduced compared to the amount of the sugar present before the contact.   The method of claim 1, wherein the alpha-glucosidase enzyme is immobilized.   The method according to claim 1, wherein the degree of polymerization of the sugar before hydrolysis is 3-7.   The method of claim 1, wherein the sugar concentration after the contacting step is less than 50% of the sugar concentration present before the contacting. The appropriate condition is
The method according to claim 1, comprising (i) a glucan synthesis reaction, or (ii) a fraction obtained from the glucan synthesis reaction, wherein the sugar is a byproduct of the glucan synthesis reaction.   6. The method of claim 5, wherein the glucan synthesis reaction produces at least one insoluble alpha-glucan product.   The method according to claim 6, wherein the fraction is a filtrate of the glucan synthesis reaction. The glucan synthesis reaction is
It is possible to hydrolyze a product of (i) a glucosyltransferase, or (ii) one or more alpha-1,3-glycosidic bonds or one or more alpha-1,6-glycosidic bonds 6. The method of claim 5, wherein at least one soluble alpha-glucan product is produced that is the product of the concerted action of both glucosyltransferase and alpha-glucanohydrolase.   The method according to claim 8, wherein the fraction is a chromatographic fraction of the glucan synthesis reaction.   The method of claim 1, wherein the alpha-glucosidase enzyme is a transglucosidase.   A composition produced by contacting a sugar with an alpha-glucosidase enzyme, wherein the sugar is a disaccharide or oligosaccharide and at least one alpha-1,3 or alpha-1,6 glucosyl-glucose linkage Wherein the enzyme hydrolyzes at least one alpha-1,3 or alpha-1,6 glucosyl-glucose bond of the sugar and the composition is compared to the amount of sugar present prior to the contact. And a reduced amount of said sugar.   The composition according to claim 11, wherein the degree of polymerization of the sugar before hydrolysis is 3-7.   The composition according to claim 11, wherein the sugar is (i) a glucan synthesis reaction, or (ii) a fraction obtained from the glucan synthesis reaction, and the sugar is a byproduct of the glucan synthesis reaction. A method for enriching fructose present in a fraction of a glucan synthesis reaction,
(A) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme is at least a disaccharide or an oligosaccharide contained in the fraction. Hydrolyzing one alpha-1,3 or alpha-1,6 glucosyl-glucose bond, contacting;
(B) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition having a higher fructose concentration compared to the fructose concentration of the fraction of step (a). ,Method. (A) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme is at least a disaccharide or an oligosaccharide contained in the fraction. Hydrolyzing one alpha-1,3 or alpha-1,6 glucosyl-glucose bond, contacting;
(B) Fermenting the fraction of step (a) with a microorganism to obtain a product, wherein the fermentation is performed after step (a) or simultaneously with step (a) And letting
(C) optionally isolating the product of (b), wherein the yield of the product of (b) is a fraction of the glucan synthesis reaction not contacted with the alpha-glucosidase enzyme. The fermentation method, which is increased compared to the yield of the product that ferments the painting.