BR112016019820B1 - GLUCANE SYNTHESIS METHOD AND HYDROLYSIS OF LEUCROSE BY-PRODUCT - Google Patents

Abstract

HYDROLYSIS METHOD, COMPOSITION, FRUCTOSE ENRICHMENT METHOD AND FERMENTATION METHOD A method of hydrolyzing an alpha-1,5 glycosylfructose linkage in a saccharide (disaccharide or oligosaccharide) such as leucrose is described. This method comprises contacting the saccharide with an alpha-glucosidase enzyme such as transglucosidase or glucoamylase under appropriate conditions and, during this contacting step, the enzyme hydrolyses at least one alpha-1,5-glycosylfructose linkage of the saccharide. This method is useful, for example, to reduce the amount of leucrose in a filtrate isolated from a glucan synthesis reaction.

Description

[001] This application claims the benefit of US Provisional Orders No. 61/945,233 (filed on February 27, 2014), 61/945,241 (filed on February 27, 2014), 62/004,290 (filed on 29 May 2014), 62/004308 (filed May 29, 2014), 62/004312 (filed May 29, 2014), 62/004300 (filed May 29, 2014), 62/004314 (filed on May 29, 2014) and 62/004305 (filed on May 29, 2014), all of which are incorporated in full herein by reference.

FIELD OF THE INVENTION

[002] The present invention is in the field of enzymatic hydrolysis of small sugar polymers. Specifically, the present invention relates to hydrolyzing oligosaccharides and disaccharides comprising one or more alpha-1,5 glycosylfructose linkages with an alpha-glucosidase enzyme.

REFERENCE TO THE ELECTRONICLY DEPOSITED SEQUENCE LISTING

[003] The official copy of the sequence listing is presented electronically through EFS-Web in the form of a sequence listing in ASCII format with a file called CL6115USNP_SequenceListing_ST25.txt created on February 10, 2015, which has a size of 266 kilobytes and deposited simultaneously with this descriptive report. The listing of sequences contained in this document in ASCII format is part of this descriptive report and is fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

[004] Glucoamylases (EC 3.2.1.3, alpha-1,4-glucan glucohydrolase) are enzymes with exo action that catalyze the hydrolysis of alpha-1,4 and alpha-1,6 glycosidic bonds of non-reducing ends of di , oligo- and polysaccharides that contain glucose, releasing glucose units one at a time (1960, Pazur and Ando, J. Biol. Chem. 235: 297-302). The split occurs at the glycosidic bond connecting the anomeric carbon with oxygen (1962, Fleetwood and Weigel, Nature 196: 984). Alpha-1,4, alpha-1,6 and alpha-1,3 bonds are the only bonds hydrolyzed at significant rates by glucoamylase (1957, Barker et al, J. Chem. Soc. 4865-4871).

[005] Glucoamylase is also capable of hydrolyzing the glycosidic bond between the two glycosyl units linked by alpha-1,2 (eg, Kojibiose) or alpha-1,1 (eg, trehalose). This enzymatic activity occurs, however, at a much slower rate and in more dilute substrate concentrations compared to glucoamylase activity for disaccharides with alpha-1,4 (maltose) or alpha-1,6 (isomaltose) linkages.

[006] Glucoamylase has been widely used in the production of starch syrup with high glucose content. High glucose syrup is useful as a raw material for the production of several value-added compounds such as fuel alcohol, high fructose corn syrup, organic acids, amino acids and vitamins. Glucoamylase has been isolated from various microorganisms, animals and plants and, among microorganisms, many fungi are good sources of this enzyme. Glucoamylase produced in fungal organisms such as Aspergillus niger is commonly used for commercial applications such as the production of high glucose syrup.

[007] Transglucosidases (EC.2.4.1.24, 1,4-alphaglucan 6-alphaglucosyltransferase) are D-glycosyltransferase enzymes that catalyze hydrolytic and transfer reactions upon incubation with alpha-D-glyco-oligosaccharides (1951, Pazur and French, J. Amer. Chem. Soc. 73: 3536). Maltose is the most preferred substrate for transglycosylation reactions with this enzyme. Transfer most frequently occurs to HO-6, producing isomaltose from D-glucose, or panose (6-O-alphaglycosyl maltose) from maltose. Transglucosidase can also transfer a glycosyl residue to HO-2 or HO-3 from another D-glycosyl moiety to form Kojibiose or Nigerosis. This enzyme can additionally transfer a D-glycosyl unit back to HO-4 to reform maltose.

[008] As a result of transglycosylation reactions with transglycosidase, maltooligosaccharide residues are converted to isomaltooligosaccharides (IMO) that contain a higher proportion of glycosyl residues linked by alpha-D-1,6 glycosidic bonds from the non-reducing end . IMO sugars are used in many food and beverage formulations in Asia. Brier et al (US Patent Application Published No. 2003/0167929) described the use of transglycosidase to produce IMO from barley wort.

[009] Poulose et al (US Patent Application Published No. 2008/0229514) described the use of transglycosidase to degrade polysaccharides such as xanthan and guar gums. Xanthan gum comprises a cellulosic backbone in which alternating glucoses are 1,3-linked to branches containing mannose and glucuronic acid. The backbone of guar gum comprises mannose residues linked by beta-1,4 to which galactose residues are linked by alpha-1,6 in alternating mannoses.

[0010] Lantero et al (US Patent No. 5,770,437) described the use of transglycosidase to degrade sucrose, melezitose and trehalulose. These sugars comprise glucose linked to fructose via 1,2-(sucrose), 1,3-(melezitose) or 1,1-(trehalulose) linkages.

[0011] Although several hydrolytic activities of glucoamylase and transglycosidase have been described, these enzymes are generally considered alpha-glucosidases, considering their ability to hydrolyze alpha bonds between two glycosyl residues. Glucoamylase and transglucosidase are associated, for example, with maltase activity (hydrolysis of the alpha-1,4 glycosidic bond between the two glycosyl residues of maltose), which is a type of alpha-glucosidase activity.

[0012] Notwithstanding the above descriptions, it has surprisingly been found that alpha-glucosidases such as transglucosidase (EC 2.4.1.24), glucoamylase (EC 3.2.1.3) and other alpha-glucosidases can hydrolyze alpha-1,5 glycosidic bond of glycosyl fructose. Alpha-glucosidases are described herein as being useful for degrading disaccharides and oligosaccharides which contain glycosyl alpha-1,5-fructose.

SHORT DESCRIPTION OF THE INVENTION

[0013] In one embodiment, the present invention relates to a method of hydrolyzing an alpha-1,5 glycosylfructose bond in a saccharide comprising at least one alpha-1,5 glycosylfructose bond, wherein the saccharide is a disaccharide or oligosaccharide and the method comprises: contacting the saccharide with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose linkage of the saccharide and the amount of the saccharide is reduced compared to the amount of the saccharide that was present before the contact step.

[0014] In another embodiment, the alpha-glucosidase enzyme of the hydrolyzation method is immobilized.

[0015] In another embodiment, the saccharide of the hydrolyzation method is leucrose. In another embodiment, the leucrose concentration after the contacting step is less than 50% of the leucrose concentration that was present before the contacting step.

[0016] In another embodiment, the appropriate conditions of the hydrolyzation method comprise (i) glucan synthesis reaction; or (ii) a fraction obtained from the glucan synthesis reaction; wherein the saccharide is a by-product of the glucan synthesis reaction. The glucan synthesis reaction generates at least one insoluble alpha-glucan product in another embodiment. The fraction is a filtrate from the glucan synthesis reaction in another embodiment. In another embodiment, the glucan synthesis reaction generates at least one soluble alpha-glucan product that is (i) a product of glycosyltransferase or (ii) a product of the concerted action of glycosyltransferase and alpha-glucan hydrolase capable of hydrolyzing glucan polymers that contain a or more alpha-1,3-glycosidic linkages or one or more alpha-1,6-glycosidic linkages. The fraction is a chromatographic fraction of the glucan synthesis reaction in another embodiment in which the glucan synthesis reaction generates at least one soluble alpha-glucan product.

[0017] In another embodiment, the alpha-glucosidase enzyme is a transglycosidase or glucoamylase. In another embodiment, (i) the transglycosidase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1; or (ii) the glucoamylase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2.

[0018] In another embodiment, the present invention relates to a composition produced by contacting a saccharide with an alpha-glucosidase enzyme, wherein the saccharide is a disaccharide or oligosaccharide and comprises at least one alpha-1,5 glycosylfructose bond , the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosyl fructose linkage of the saccharide and the composition comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to the contacting step.

[0019] In another embodiment, the saccharide of the composition is leucrose. The leucrose concentration in the composition is, for example, less than 50% of the leucrose concentration that was present prior to the contacting step.

[0020] In another embodiment, the saccharide of the composition is in (i) a glucan synthesis reaction; or (ii) a fraction obtained from the glucan synthesis reaction; wherein the saccharide is a by-product of the glucan synthesis reaction. In another embodiment, the fraction is a glucan synthesis reaction filtrate or a glucan synthesis reaction chromatographic fraction.

[0021] In another embodiment, the present invention relates to a method of enriching fructose present in a fraction of a glucan synthesis reaction, comprising: (a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose linkage of a disaccharide or oligosaccharide comprised in the fraction; and (b) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition that contains a higher concentration of fructose compared to the fructose concentration of the fraction of step (a).

[0022] In another thirteenth embodiment, the present invention relates to a fermentation method comprising: (a) contacting a fraction obtained from a glucan synthesis reaction with an alphaglucosidase enzyme under appropriate conditions, in that the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose bond of a disaccharide or oligosaccharide comprised in the fraction; (b) fermenting the fraction from step (a) with a microbe to generate a product, wherein the fermentation can be carried out after step (a) or simultaneously with step (a); and (c) optionally isolating the product of (b); wherein the yield of product from (b) increases compared to the yield of product from fermentation of a fraction from the glucan synthesis reaction which has not been contacted with the alpha-glucosidase enzyme.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCES

[0023] Figure 1: 1H NMR spectrum of glucan reaction filtered material before (starting material) and after (treated material) hydrolysis treatment with NOVO 188 enzyme (see Examples 2-3).

[0024] Figure 2: 1H NMR spectrum of glucan reaction filtered material before (starting material) and after (treated material) hydrolysis treatment with TG L-2000 transglycosidase (see Examples 23).

TABLE 1

[0025] Summary of protein and nucleic acid sequence identification numbers:

DETAILED DESCRIPTION OF THE INVENTION

[0026] The descriptions of all patent and non-patent literature mentioned are incorporated in full herein by reference.

[0027] As used herein, the expression "invention" or "present invention" is not intended to be limiting, but applies generally to any of the inventions defined in the claims or described herein. interchangeable at present.

[0028] The terms "saccharide", "saccharide molecule" and "carbohydrate" are used interchangeably herein and mean disaccharide or oligosaccharide, unless otherwise indicated. "Disaccharide" herein means a carbohydrate containing two monosaccharides linked by a glycosidic linkage. "Oligosaccharide" herein designates a carbohydrate consisting of two to nine monosaccharides, for example, linked by glycosidic linkages. Oligosaccharide may also be referred to herein as an "oligomer." Monosaccharides that are comprised of a disaccharide or oligosaccharide they may be called "monosaccharide units" or "monomeric units", for example.Preferred monosaccharides herein are fructose and glucose.

[0029] The terms "glycosidic bond" and "glycosidic bond" are used interchangeably herein and designate the type of covalent bond that links a carbohydrate molecule to another carbohydrate molecule.

[0030] The expressions “alpha-1,3 glycosylglucose bond”, “alpha-1,3 glucose-glucose bond” and “glucose-alpha 1,3-glucose” mean alpha-1,3-glycosidic bond between two alpha-D-glucose molecules. The expressions "alpha-1,6 glycosylglucose bond", "alpha-1,6 glucose-glucose bond" and "glucose-alpha 1,6-glucose" designate alpha-1,6-glycosidic bond between two alpha molecules -D-glucose. Alpha-1,3 glycosylglucose linkage(s) and/or alpha-1,6 glycosylglucose linkage(s) herein is(are) comprised in a disaccharide or oligosaccharide in certain embodiments.

[0031] The terms “alpha-1,5 glycosylfructose bond”, “alpha-1,5 glucose-fructose bond” and “glucose-alpha-1,5-fructose” herein mean alpha-1,5-glycosidic bond between one alpha-D-glucose molecule and one fructose molecule. Alpha-1,5 glycosylfructose linkage is herein comprised in a disaccharide or oligosaccharide in certain embodiments.

[0032] “Alpha-D-glucose” in the present can also be called “glucose”.

[0033] Disaccharide containing an alpha-1,5 linkage glycosylfructose is termed leucrose in this. The terms "leucrose" and "D-glucopyranosyl alpha(1-5)-D-fructopyranose" are used interchangeably herein. Leucrose has the following structure:

[0034] The terms "alphaglucosidase", "alpha-1,4-glucosidase" and "alpha-D-glucoside glucohydrolase" are used interchangeably herein. Alpha-glucosidases (EC 3.2.1.20) (“EC” designates the Enzyme Commission number) were previously recognized as enzymes that catalyze the hydrolytic release of terminal non-reducing alpha-D-glucose (1,4)-linked residues from substrates of polysaccharides and oligosaccharides (eg disaccharides). Alpha-glucosidases are now described as also exhibiting hydrolytic activity for alpha-1,5 glycosylfructose linkages and hydrolytic activity for alpha-1,3 and alpha-1,6 glycosylglucose linkages. Transglucosidase and glucoamylase enzymes are examples of alpha-glucosidases with this activity.

[0035] The terms "transglycosidase" (TG), "transglycosidase enzyme" and "1,4-alphaglucan 6-alphaglycosyltransferase" are used interchangeably herein. Transglycosidases (EC.2.4.1.24) were previously recognized as D-glycosyltransferase enzymes that catalyze hydrolytic and transfer reactions upon incubation with certain alpha-D-glyco-oligosaccharides. Transglycosidases are now described at present as also exhibiting hydrolytic activity for alpha-1,5 glycosylfructose linkages and hydrolytic activity for alpha-1,3 and alpha-1,6 glycosylglucose linkages.

[0036] The terms “glucoamylase” (GA), “glucoamylase enzyme” and “alpha-1,4-glucan glucohydrolase” are used interchangeably. Glycoamylases (EC 3.2.1.3) have been recognized as exo-acting enzymes that catalyze hydrolysis of alpha-1,4 and alpha-1,6 glycosidic bonds from non-reducing ends of di, oligo and polysaccharides containing glucose. Glucoamylases are now described at present to also exhibit hydrolytic activity for alpha-1,5 glycosylfructose linkages.

[0037] Enzymatic hydrolysis is a process in which an enzyme enables the cleavage of bonds in molecules with the addition of water elements. "Hydrolysis", "hydrolysis of" or "hydrolytic activity for" an alpha-1,5 glycosylfructose linkage herein means enzymatic hydrolysis of the alpha-1,5 glycosidic linkage between glucose and fructose by an alpha-glucosidase such as glucoamylase or transglucosidase. This hydrolysis occurs during contact of a disaccharide or oligosaccharide containing an alpha-1,5 glycosylfructose linkage with an alpha-glucosidase in the present under appropriate conditions. "Hydrolysis reaction" herein therefore comprises at least (i) a disaccharide or oligosaccharide containing an alpha-1,5-glycosylfructose linkage and (ii) alpha-glucosidase.

[0038] The term "saccharification" in the present designates a process of breaking down a saccharide (disaccharide or oligosaccharide) into its monosaccharide components. Saccharides can be saccharified in a hydrolysis reaction at present.

[0039] "Appropriate conditions" for contacting saccharides (disaccharides or oligosaccharides) comprising at least one alpha-1,5 glycosylfructose bond with alpha-glucosidase herein designate conditions (such as temperature, pH, time) that support the hydrolysis of a or more alpha-1,5-glycosylfructose linkages on the saccharide by alpha-glucosidase. Appropriate conditions may comprise "aqueous conditions", for example, comprising at least 20% by weight of water. Aqueous conditions can characterize a solution or mixture. The solution or mixture in which a saccharide comprising at least one alpha-1,5-glycosylfructose bond is brought into contact with an alpha-glucosidase, for example, may be termed an alpha-glucosidase reaction (e.g., transglucosidase or glucoamylase reaction).

[0040] Enzyme "mobilized" herein means enzyme that is bound to an inert insoluble material. Methods of preparing immobilized enzymes are described, for example, in US Patent No. 5,541,097, which is incorporated herein by reference.

[0041] The terms "glucan" and "glucan polymer" are used interchangeably herein and designate a polysaccharide of glucose monomers linked by glycosidic bonds. "Alphaglucan" herein means a polymer of glucan comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% alpha-glycosidic bonds.

[0042] "Insoluble glucan" herein means a glucan polymer that is not soluble under aqueous conditions. An example of insoluble glucan herein is poly alpha-1,3-glucan with DP of at least 8 or 9. Glycosyltransferase reaction in certain embodiments described herein generates at least one insoluble glucan product.

[0043] The terms "soluble glucan", "soluble alpha-glucan", "soluble fiber", "soluble glucan fiber", "soluble dietary fiber" and the like are used interchangeably herein to mean a glucan polymer that is soluble in aqueous conditions. Examples of soluble glucan herein are certain oligosaccharides, such as polyalpha-1,3-glucan with DP less than 8 and certain oligosaccharides described in the Examples provided below. Glycosyltransferase reaction in certain embodiments described herein generates at least one soluble glucan product. Another set of characteristics that characterize soluble alpha-glucan compounds in certain embodiments of the present invention is that they are (i) water-soluble glucose oligomers having a degree of polymerization of 3 or more; (ii) resistant to digestion (ie, exhibit very slow or no digestion capacity) with little or no absorption in the human small intestine and (iii) at least partially fermentable in the lower gastrointestinal tract. The digestibility of a soluble glucan fiber composition can be measured using, for example, the AOAC 2009.01 method.

[0044] The terms "alpha-1,3-glucan poly" and "alpha-1,3-glucan polymer" are used interchangeably herein. Poly alpha-1,3-glucan is a polymer comprising monomeric units of glucose linked together by glycosidic linkages, with at least about 50% of the glycosidic linkages being alpha-1,3-glycosidic linkages. The expression "alpha-1,3-glycosidic bond", as used herein, designates the type of covalent bond that links alpha-D-glucose molecules to each other through carbons 1 and 3 on alpha-D-glucose rings adjacent.

[0045] The "molecular weight" of glucan in the present can be represented as number average molecular weight (Mn) or as weight average molecular weight (Mw). Alternatively, molecular weight can be represented in Daltons, grams/mol, DPw (weight average degree of polymerization) or DPn (number average degree of polymerization). Various means are known in the art for calculating such molecular weight measurements such as high pressure liquid chromatography (HPLC), size exclusion chromatography (SEC) or gel permeation chromatography (GPC).

[0046] The terms "glycosyltransferase enzyme", "gtf enzyme", "gtf enzyme catalyst", "gtf", "glucan sucrase" and the like are used interchangeably herein. The gtf enzyme activity in this catalyzes the sucrose substrate reaction to elaborate the products glucan and fructose. Other products (by-products) of a gtf reaction can include glucose (results from hydrolysis of glucose from the glycosyl-gtf enzyme intermediate complex), various soluble oligosaccharides (e.g., DP2-DP7), and leucrose (results from binding of glucose from glycosyl-gtf enzyme intermediate complex to fructose). Wild-type forms of glycosyltransferase enzymes usually contain (in the N-terminal to C-terminal direction) a signal peptide, variable domain, catalytic domain, and glucan-binding domain. Glycosyltransferase at present is classified under the glycoside hydrolase 70 (GH70) family according to the CAZy (Active Carbohydrate Enzymes) database ( Cantarel et al, Nucleic Acids Res. 37: D233238, 2009 ).

[0047] The term "sucrose" herein designates a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,2-glycosidic bond. Sucrose is commonly known as table sugar.

[0048] The expressions "glucan synthesis reaction", "glucan reaction", "gtf reaction" and the like are used interchangeably herein and designate a reaction that is carried out by a glycosyltransferase enzyme. Glucan synthesis reaction used herein generally designates a solution comprising at least one active glycosyltransferase enzyme in a solution comprising sucrose and water and, optionally, other components. Other components that may be present in a glucan synthesis reaction in the present include fructose, glucose, leucrose, soluble oligosaccharides (eg DP2-DP7) and soluble glucan product(s), for example. Furthermore, one or more alpha-glucan hydrolase enzymes may be comprised in a glucan synthesis reaction in some aspects. It would be understood that certain glucan products, such as poly alpha-1,3-glucan with a degree of polymerization (DP) of at least 8 or 9, are insoluble in water and therefore are not dissolved in a reaction of glucan. glucan synthesis, but they can be present out of solution.

[0049] The terms "alphaglucanhydrolase" and "glucanhydrolase" are used interchangeably herein and designate an enzyme capable of hydrolyzing alphaglucan oligomer. Alphaglucanhydrolase can be defined by its endohydrolysis activity for certain alpha-D-glycosidic bonds. Examples of alpha-glucanhydrolase enzymes in the present include dextransases (EC 3.2.1.11; capable of endo-hydrolyzing alpha-1,6-linked glycosidic bonds), mutanases (EC 3.2.1.59; capable of endo-hydrolyzing alpha-1-glycosidic bonds, 3-linked) and alternanases (EC 3.2.1, able to split endohydrolytically alternan). Several factors, including but not limited to level of branching, type of branching, and the relative branch length in certain alpha-glucans, can impair the ability of alpha-glucanhydrolase to endohydrolyze some glycosidic bonds.

[0050] The glucan synthesis reaction “percent dry solids” designates the weight percent of all sugars in a glucan synthesis reaction. The percent dry solids of a gtf reaction can be calculated, for example, based on the amount of sucrose used to prepare the reaction.

[0051] “Fraction” of a glucan synthesis reaction herein means a part of liquid solution of a glucan synthesis reaction. Fraction may be a part or all of the liquid solution from a glucan synthesis reaction and has been separated from a soluble or insoluble glucan product synthesized in the reaction. Fraction may optionally be referred to as "original liquid" in embodiments in which the product is an insoluble (solid) glucan product. An example of a fraction is a filtrate from a glucan synthesis reaction. As a fraction may contain dissolved sugars such as sucrose, fructose, glucose, leucrose and soluble oligosaccharides (eg DP2-DP7), a fraction may also be termed a "mixed sugar solution" derived from a glucan synthesis reaction. "Hydrolyzed fraction" herein means a fraction which has been treated with an alpha-glucosidase herein to hydrolyze leucrose and/or oligosaccharides present in the fraction.

[0052] The expressions “filtrate”, “glucan reaction filtrate”, “glucan filtrate” and the like are used interchangeably herein and designate a fraction that was removed by means of filtration from a solid glucan product synthesized in a glucan synthesis reaction. "Hydrolyzed filtrate" herein means a filtrate which has been treated with an alpha-glucosidase herein to hydrolyze leucrose and/or oligosaccharides present in the filtrate.

[0053] The expressions “percentage by volume”, “percentage volume”, “% vol”, “% v/v” and the like are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute)/(volume of solution)] x 100%.

[0054] The expressions “percent by weight”, “percent by weight (% weight)”, “percent weight to weight (% w/w)” and the like are used interchangeably herein. Percent by weight designates the percentage of a material based on mass as comprised in a composition, mixture or solution. All percentages herein are weight percentages unless otherwise noted.

[0055] As used herein, “polydispersion index”, “PDI”, “heterogeneity index”, “dispersion” and the like designate a measure of molecular weight distribution in a given polymer sample (for example, oligomer of glucose such as soluble alpha-glucan) and can be calculated by dividing the weight average molecular weight by the number average molecular weight (PDI = Mw/Mn).

[0056] The terms “increase”, “enlargement” and “enhancement” are used interchangeably herein. These terms designate a greater amount or activity, such as an amount or activity slightly greater than the original amount or activity, or an amount or activity in great excess compared to the original amount or activity, including all amounts or activities in between. Alternatively, these terms may designate, for example, quantity or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% greater than the quantity or activity to which the greater quantity or activity is being compared.

[0057] The expressions "sequence identity" or "identity", as used herein with respect to polypeptide or polynucleotide sequences, designate the amino acid residues or nucleic acid bases in two sequences that are identical when aligned for maximum correspondence over a specified comparison window. Thus, "percent sequence identity" or "percent identity" indicates the value determined by comparing two ideally aligned sequences along a comparison window, where the portion of the polynucleotide or polypeptide sequence in the sequence window The comparison may comprise additions or deletions (i.e. gaps) compared to the reference sequence (which comprises no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the identical amino acid residue or nucleic acid base occurs in the two sequences to generate the number of matching positions, dividing the number of matching positions by the total number of positions in the matching window. comparison and multiplying the results by one hundred to generate the percent sequence identity.

[0058] The Basic Local Alignment Search Tool (BLAST) algorithm, which is available online on the website of the National Center for Biotechnology Information (NCBI), can be used, for example, to measure the percentage of identity between two or more of the polynucleotide sequences (BLASTN algorithm) or polypeptide sequences (BLASTP algorithm) described herein. Alternatively, percent identity between sequences can be performed using a Clustal algorithm (eg, ClustalW or ClustalV). For multiple alignments using the Clustal alignment method, the default values may correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. Default parameters for pairwise alignments and calculation of percent protein sequence identity using the Clustal method can be WORD LENGTH = 1, RANGE PENALTY = 3, BEST RESULT DISPLAY = 5, and DIAGONAL SPACING = 5. For nucleic acids, these parameters can be WORD LENGTH = 5, BEST RESULT DISPLAY = 4, and SPACING AT DIAGONALS = 4. Still alternatively, percent identity between sequences can be performed using an EMBOSS algorithm (eg needle), with parameters such as GAP OPENING = 10, GAP EXTENSION = 0.5, END OF BATCH PENALTY = false, RANGE END OPENING = 10, RANGE END EXTENSION = 0.5 using a BLOSUM array (for example, BLOSUM62).

[0059] Various amino acid sequences of polypeptides are described herein as characteristic of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90-95% identical to the sequences described herein may be used. Alternatively, variant amino acid sequences can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of identity with a sequence described herein. A variant amino acid sequence of the present has the same function/activity as a described sequence or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% of the function/activity of a described sequence.

[0060] The term "isolated", as used in certain embodiments, means any cellular component that is completely separated from its native source (eg, isolated polypeptide or polynucleotide molecule). In some cases, an isolated polypeptide or polynucleotide molecule is part of a larger composition, buffer system, or reagent mixture. The isolated polypeptide or polynucleotide molecule can be comprised, for example, in a cell or organism in a heterologous form. Another example is an isolated alpha-glucosidase enzyme (eg, glucoamylase, transglycosidase) or glycosyltransferase. Enzyme reactions (eg, alpha-glucosidase reaction, glycosyltransferase reaction) described herein are synthetic and not naturally occurring processes.

[0061] Embodiments of the present invention relate to a method of hydrolyzing an alpha-1,5 glycosylfructose linkage in a saccharide comprising at least one alpha-1,5 glycosylfructose linkage. The saccharide is disaccharide or oligosaccharide. This method comprises contacting the saccharide with an alpha-glucosidase enzyme under appropriate conditions. In the contact step, the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose linkage of the saccharide. Due to this hydrolysis, the amount of saccharide is reduced compared to the amount of saccharide that was present before the contacting step. This hydrolyzation method can therefore alternatively be called the method of reducing the amount of saccharides in compositions.

[0062] Significantly, it is believed to be previously unknown that alpha-glucosidase enzymes can hydrolyze alpha-1,5 glycosylfructose linkages. Alpha-glucosidase reactions following this hydrolysis method can therefore be used to remove leucrose and other oligosaccharide by-products that contain alpha-1,5-glycosylfructose linkages from a glucan synthesis reaction and/or a fraction obtained therefrom. This removal represents an improvement over chemical by-product removal processes, such as acid hydrolysis, which can result in product glucan degradation. Finally, a glucan reaction fraction that is treated according to the above hydrolyzation method is more suitable for downstream applications such as fermentation, for example, as the level of fructose and glucose monosaccharides increases in the fraction. Monosaccharides are generally more amenable to downstream processes compared to leucrose by-products and oligosaccharides.

[0063] Alpha-glucosidase (EC 3.2.1.20) is used in embodiments of the present to hydrolyze an alpha-1,5 glycosylfructose linkage in a saccharide comprising at least one alpha-1,5 glycosylfructose linkage. It was previously recognized that alpha-glucosidase enzymes catalyze the hydrolytic release of terminal non-reducing alpha-D-glucose (1,4)-linked residues from oligosaccharide (eg, disaccharide) and polysaccharide substrates. These enzymes are now described, for example, as also exhibiting hydrolytic activity for alpha-1,5 glycosylfructose linkages.

[0064] Alpha-glucosidase may be from any source (e.g., plant, animal, microbe such as bacteria or fungus/yeast), for example, such as the sources described below, from which a transglycosidase and/or glucoamylase may be derived. Alpha-glucosidase, for example, can be fungal alpha-glucosidase. Other examples of suitable alpha-glucosidases herein include those described in US Patent Nos. 6,355,467, 5,922,580, 5,795,766, 5,763,252 and 8,633,006, all of which are incorporated herein by reference.

[0065] Alpha-glucosidase enzyme, in certain embodiments of the present, may comprise the amino acid sequence of 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, an alpha-glucosidase enzyme can comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or the amino acid sequence of DIAZYME RDF ULTRA and has hydrolytic activity for alpha-1,5-glycosylfructose linkages in saccharides. Several of the above sequences, for example, are mature alpha-glucosidases that do not contain an N-terminal signal peptide. For such sequences, it would be understood that an initial N-terminal methionine would typically be added (if needed) (either directly or via an intervening heterologous amino acid sequence such as an epitope) if expressing it without using a signal peptide ( such as with an expression system in which the enzyme is expressed intracellularly and obtained from a cell lysate).

[0066] Transglycosidase (EC 2.4.1.24; 1,4-alphaglucan 6-alphaglucosyltransferase) can be used in certain embodiments of the present as alpha-glucosidase to hydrolyze an alpha-1,5-glycosylfructose linkage in a saccharide comprising at least one alpha-linkage 1,5 glycosylfructose. This class of enzymes was previously recognized as D-glycosyltransferase enzymes that catalyze hydrolytic and transfer reactions upon incubation with certain alpha-D-glyco-oligosaccharides. Transglycosidases are now described at present as also exhibiting hydrolytic activity for alpha-1,5 glycosylfructose linkages.

[0067] Transglycosidase enzyme in the present can be derived from any microbial source such as bacteria or fungus. Examples of fungal transglycosidases include, but are not limited to, those from Trichoderma species (eg, T. reesei), Aspergillus species, and Neosartorya species (eg, N. fischeri). Examples of Aspergillus species from which a transglycosidase can be derived include, but are not limited to, A. niger, A. awamori, A. oryzae, A. terreus, A. clavatus, A. fumigatus, and A. nidulans. Other examples of transglycosidase enzymes useful in the present are described in Barker et al (1953, J. Chem. Soc. 3588-3593); Pazur et al (1986, Carbohydr. Res. 149: 137-147), Nakamura et al (1997, J. Biotechnol. 53: 75-84) and U.S. Patent Application Published No. 2008/0229514, all of which are incorporated here as a reference. Yet other examples of transglycosidase enzymes useful herein are the thermostable; US Patent No. 4,689,296, which is incorporated herein by reference, describes a process for producing thermostable transglycosidase. Still other examples of transglycosidase enzymes useful in the present can be any from the GENBANK database (NCBI), such as accession numbers: D45356 (GID: 2645159, A. niger), BAD06006.1 (GID: 4031328, A. awamori ), BAA08125.1 (GID: 1054565, A. oryzae), XP_001210809.1 (GID: 115492363, A. terreus), XP_001216899.1 (GID: 115433524, A. terreus), XP_001271891.1 (GID: 12170 7620, A . clavatus), XP_751811.1 (GID: 70993928, A. fumigatus), XP_659621.1 (GID: 67523121, A. nidulans), XP_001266999.1 (GID: 119500484, N. fischeri) and XP_001258585.1 (GID: 1) 19473371 , N. fischeri), all of which are incorporated herein by reference. Alternatively, transglycosidase herein may have an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of any of the transglycosidase sequences described above and possess hydrolytic activity for alpha-1,5 glycosylfructose linkages in saccharides. All of the above transglycosidases, when used in a hydrolysis reaction in the present, are preferably in a mature form that lacks an N-terminal signal peptide.

[0068] Transglycosidase enzyme in certain embodiments of the present can comprise the amino acid sequence of SEQ ID NO: 1 (transglycosidase L-2000), which is an A. niger transglycosidase (U.S. Pat. Published No. 2008/0229514 ). Alternatively, transglycosidase can comprise an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% identical to SEQ ID NO: 1 and having hydrolytic activity for alpha-1,5 glycosylfructose linkages in saccharides. Any one of SEQ ID NO: 1 or variants thereof can be produced by following the descriptions of published US Patent Application No. 2008/0229514, for example, which is incorporated herein by reference. SEQ ID NO: 1 is a mature transglycosidase that does not contain an N-terminal signal peptide. As SEQ ID NO:1 does not begin with a methionine residue, it would be understood that an initial N-terminal methionine would typically be added to SEQ ID NO:1 (directly or via an intervening heterologous amino acid sequence such as a epitope) if you express it without using a signal peptide (such as with an expression system in which the enzyme is expressed intracellularly and obtained from a cell lysate).

[0069] Glucoamylase (EC 3.2.1.3; alpha-1,4-glucan glucohydrolase) can be used in certain embodiments of the present as alpha-glucosidase to hydrolyze an alpha-1,5-glycosylfructose bond in a saccharide comprising at least one bond alpha-1,5 glycosylfructose. This class of enzymes was previously recognized as exo-acting enzymes that catalyze hydrolysis of alpha-1,4 and alpha-1,6 glycosidic linkages from non-reducing ends of di-, oligo-, and glucose-containing polysaccharides. Glucoamylases are now described at present as also exhibiting hydrolytic activity for alpha-1,5 glycosylfructose linkages. In certain embodiments, alpha-glucosidase is not glucoamylase.

[0070] Glucoamylase enzyme in this can be derived from any microbial source such as bacteria or fungi. Examples of bacterial glucoamylases include, but are not limited to, those from Bacillus species (e.g., B. alkalophilus, B. amyloliquefaciens, B. lentus, B. licheniformis, B. stearothermophilus, B. subtilis, and B. thuringiensis) and species of Streptomyces (eg S. lividans). Examples of fungal glucoamylases include, but are not limited to, those from Trichoderma species (e.g., T. reesei, T. longibrachiatum, T. strictipilis, T. asperellum, T. konilangbra, and T. hazianum), Aspergillus species (e.g., , A. niger, A. oryzae, A. terreus, A. clavatus, A. nidulans, A. kawachi and A. awamori), Rhizopus species (eg R. oryzae and R. niveus), Talaromyces species ( eg T. emersonii, T. thermophilus and T. duponti), Mucor species, Hypocrea species (e.g. H. gelatinosa, H. orientalis, H. vinosa and H. citrina), Fusarium species (e.g. eg F. oxysporum, F. roseum and F. venenatum), Neurospora species (e.g. N. crassa), Humicola species (e.g. H. grisea, H. insolens and H. lanuginose), Penicillium species (eg P. notatum and P. chrysogenum) and Saccharomycopsis species (eg S. fibuligera). Examples of such bacterial and fungal glucoamylases for use herein are described in published US Patent Application No. 2013/0102035, which is incorporated herein by reference. Other examples of useful glucoamylase enzymes in the present are described in Svensson et al (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 ); US Patent No. 5,024,941, US Patent No. 4,794,175, US Patent No. 4,247,637, US Patent No. 6,255,084, US Patent No. 6,620,924, Ashikari et al, (1986, Agric. Biol. Chem. 50: 957-964), Ashikari et al, (1989, Appl. Microbiol. Biotechnol. 32: 129-133), US Patent No. 4,863,864; US Patent No. 4,618,579, Houghton-Larsen et al (2003, Appl. Microbiol. Biotechnol. 62: 210-217) and US Patent No. 7,413,887, all of which are incorporated herein by reference . Alternatively, glucoamylase herein may have an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of any of the glucoamylase sequences described above and possess hydrolytic activity for alpha-1,5 glycosylfructose linkages in saccharides. All of the above glucoamylases, when used in a hydrolysis reaction in the present, are preferably in a mature form that lacks an N-terminal signal peptide. Commercially available glycoamylases useful herein include, for example, OPTIDEX L-400, GC 147, GC 321, G ZYME G990 4X, OPTIMAX 7525, DEXTROZYME, DISTILLASE and GLUCZYME.

[0071] Glucoamylase enzymes in certain embodiments herein may comprise the amino acid sequence of SEQ ID NO: 2 (GC 321), which is a T. reesei glucoamylase. Alternatively, glucoamylase can 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:2 and having hydrolytic activity for alpha-1,5 glycosylfructose linkages in saccharides. Any one of SEQ ID No. 2 or variants thereof can be produced by following the descriptions of US Patent No. 7,413,887 or Published US Patent Application No. 2013/0102035, for example, which are incorporated herein. as reference. SEQ ID NO: 2 is a mature glucoamylase that does not contain an N-terminal signal peptide. As SEQ ID NO:2 does not begin with a methionine residue, it would be understood that an initial N-terminal methionine would typically be added to SEQ ID NO:2 (directly or via an intervening heterologous amino acid sequence as an epitope). ) if you express it without using a signal peptide (for example, with an expression system in which the enzyme is expressed intracellularly and obtained from a cell lysate).

[0072] Alpha-glucosidase enzymes herein, such as transglycosidase or glucoamylase, may be from a commercial source (eg, DuPont Industrial Biosciences/Genencor, United States; Megazyme International, Ireland; Amano Enzyme Inc., Japan). Alternatively, such an enzyme can be produced by any means known in the art, such as as described in Published U.S. Patent Application No. 2008/0229514, U.S. Patent No. 7,413,887, or U.S. Patent Application Published No. ° 2009/0102035, which are incorporated herein as a reference. Alpha-glucosidase can be produced recombinantly, for example, in a heterologous expression system, such as a heterologous microbial or fungal expression system. Examples of heterologous expression systems include bacterial (eg E. coli, Bacillus sp) and eukaryotic systems. Eukaryotic systems may, for example, employ yeast (eg Pichia sp and Saccharomyces sp) or fungal (eg Trichoderma sp such as T. reesei, Aspergillus species such as A. niger) expression systems. The transglucosidase of SEQ ID NO: 1, the glucoamylase of SEQ ID NO: 2 and variants thereof may be expressed, for example, in a T. reesei host.

[0073] Alpha-glucosidase enzymes, when used in a hydrolysis reaction in this, are preferably found in a mature form that lacks an N-terminal signal peptide. An expression system for producing a mature alpha-glucosidase enzyme at present may employ an enzyme-encoding polynucleotide further comprising a sequence encoding an N-terminal signal peptide to direct extracellular secretion. The signal peptide in these embodiments is cleaved from the enzyme during the secretion process. The signal peptide can be native or heterologous to the transglycosidase or glucoamylase. Alternatively, an alpha-glucosidase enzyme in mature form can be provided by expressing it without using a signal peptide, such as with an expression system in which the enzyme is expressed intracellularly and obtained from cell lysate. In either scenario (secretion or expressed intracellularly), a heterologous amino acid sequence such as an epitope can optionally be included at the N-terminus of the alpha-glucosidase.

[0074] An alpha-glucosidase enzyme in certain embodiments can be provided in a hydrolysis reaction in the present through direct use of cells expressing the enzyme(s). In other words, alpha-glucosidase that is brought into contact with a saccharide may be present by virtue of its expression from a cell placed under the appropriate conditions for hydrolysis. This cell can therefore be used in place of the addition of the isolated alpha-glucosidase preparation for the hydrolysis reaction. A cell for this purpose can be, for example, a bacterial, yeast or fungal cell. Examples of yeast include those from the genera Saccharomyces (eg, S. cerevisiae), Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, and Schwanniomyces. Other expression systems useful herein are described in US Patent Application Published No. 2013/0323822, which is incorporated herein by reference.

[0075] A saccharide of the present comprises at least one alpha-1,5 glycosylfructose bond. Depending, therefore, on the length of the saccharide, it may contain one, two, three, four, five, six, seven or eight alpha-1,5 glycosylfructose bonds. The saccharide preferably contains one, two or three bonds of this type.

[0076] As a saccharide herein comprises at least one alpha-1,5 glycosylfructose bond, the saccharide comprises at least one glucose unit and at least one fructose unit. In certain embodiments, the present saccharide comprises only glucose and fructose units. This composition can characterize the disaccharide and oligosaccharide by-products of a glucan synthesis reaction. Alternatively, the present saccharide may contain monosaccharides other than glucose and fructose, such as galactose, ribose and xylose.

[0077] Hydrolyzed saccharide in certain embodiments of the present invention may be an oligosaccharide. The present oligosaccharide may contain, for example, two, three, four, five, six, seven, eight or nine monosaccharide units. As will be understood in the art, an oligosaccharide herein may be indicated with respect to its degree of polymerization (DP) number, which specifies the amount of monomer units in the oligosaccharide. A DP3 oligosaccharide contains, for example, three monomer units. The oligosaccharide can therefore be, for example, a DP3, DP4, DP5, DP6, DP7, DP8 or DP9 oligosaccharide. The DP of a saccharide in certain embodiments is 3 to 7 (i.e. DP 3-7).

[0078] An oligosaccharide in the present with three or more monosaccharide units may comprise linkages other than at least one alpha-1,5 glycosylfructose linkage (note that an oligosaccharide with two monosaccharide units (i.e. disaccharide) is leucrose , whereas a saccharide in a hydrolyzation method of the present contains at least one alpha-1,5 glycosylfructose bond). There may also be, for example, alpha-1,3, alpha-1,6 and/or alpha-1,4 linkages in the oligosaccharide, which are also susceptible to hydrolysis by alpha-glucosidases as shown herein.

[0079] An oligosaccharide comprises only, in certain embodiments, glucose monomers linked by alpha-1,3 and/or alpha-1,6 glycosidic linkages. Such oligosaccharides therefore only comprise alpha-1,3-glycosylglucose and/or alpha-1,6-glycosylglucose linkages. Examples of this oligosaccharide contain only alpha-1,3 linkages or alpha-1,6 linkages. An oligosaccharide may comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of glycosyl-glucose linkages in certain embodiments. In other embodiments, there can be about 75-85% alpha-1,3 glycosylglucose linkages and about 15-25% alpha-1,6 glycosylglucose linkages in oligosaccharides of the present invention. Alternatively, the present oligosaccharides can comprise any percentage (any integer value from 1% to 99%) alpha-1,3 glycosylglucose linkages and any percentage (any integer value from 1% to 99%) alpha-1,6 linkages glycosylglucose, provided that the total of these percentages is not more than 100%. Any of these oligosaccharides can be found in a glucan synthesis reaction fraction that produces (i) an insoluble alpha-glucan (eg, poly alpha-1,3-glucan) or (ii) a soluble alpha-glucan product, for example. This binding content can characterize (i) each individual oligosaccharide or (ii) a group of oligosaccharides (ie average binding content). Oligosaccharides comprising only glucose monomers linked by alpha-1,3 and/or alpha-1,6 glycosidic linkages can be, for example, DP2-DP7 or DP3-DP7. It should be understood that the exact distribution of linkages in oligosaccharides can vary depending on the conditions of the glucan synthesis reaction (eg gtf enzyme) that generates oligosaccharide by-products. It should be further understood that exact binding distribution is not critical to the methods described herein.

[0080] The Examples herein demonstrate that alpha-glucosidases (eg, transglycosidase and glucoamylase enzymes) can hydrolyze (i) leucrose, which comprises an alpha-1,5 glycosylfructose bond; and (ii) oligosaccharides comprising only alpha-1,3 glycosylglucose and/or alpha-1,6 glycosylglucose linkages. An alpha-glucosidase can therefore be used, for example, in a hydrolysis reaction of alpha-1,5-glycosylfructose bonds, alpha-1,3-glycosylglucose bonds and/or alpha-1,6-glycosylglucose bonds.

[0081] At least one alpha-1,5 glycosylfructose linkage in a saccharide of this can be hydrolyzed by an alpha-glucosidase in this. Alternatively, it is believed, for example, that two, three, four, five or more alpha-1,5-glycosylfructose linkages in a saccharide can be hydrolyzed by an alpha-glucosidase. Hydrolysis of at least one alpha-1,5 glycosylfructose bond can occur at the non-reducing end of the saccharide in certain embodiments. When the saccharide is the disaccharide, eg leucrose, the final non-reducing glucose is split from fructose, yielding fructose and free glucose. As another example, in which the saccharide is an oligosaccharide with a non-reducing end glucose that has alpha-1,5 bond to fructose, it is believed that this glucose can be split, leaving a fructose residue at the non-reducing end of the oligosaccharide.

[0082] The amount of saccharide is reduced in the described hydrolysis method compared to the amount of saccharide that was present before the contacting step. This reduction results from the hydrolytic cleavage of at least one alpha-1,5 glycosylfructose bond in the saccharide. The amount (e.g., concentration) of saccharide after the contacting step in a hydrolyzation method of the present can be less than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or any integer value from 1% to 90%) of the amount of saccharide that was present before the contacting step (before contacting a present alpha-glucosidase with a saccharide under appropriate conditions).

[0083] Hydrolyzed saccharide in certain embodiments of the present invention is leucrose, which is disaccharide with an alpha-1,5 glycosylfructose bond. The concentration of leucrose after the contacting step in a hydrolyzation method of the present can be less than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (or any integer value from 1% to 90%) of the concentration of leucrose that was present before the contacting step (before the contact of an alpha-glucosidase of the present with leucrose under appropriate conditions). A hydrolyzation method in some aspects of the present may alternatively be called a method of reducing the amount of leucrose in compositions.

[0084] Leucrose can be contacted with transglycosidase such as that comprising SEQ ID No. 1 (Transglycosidase L-2000), for example, in a hydrolyzation method of the present. The leucrose concentration after completion of this method can be less than about 1-3% of the original leucrose concentration in certain embodiments.

[0085] The amount of saccharide is reduced in the described hydrolysis method compared to the amount of saccharide that was present before the contacting step. It will be understood that this comparison can be carried out in any number of ways. The saccharide concentration can be measured, for example, before and after carrying out the hydrolysis method. Alternatively, the comparison can be performed against a control reaction which has the same conditions, except for the absence of addition of alpha-glucosidase as described herein to the control reaction.

[0086] Alpha-glucosidase in certain embodiments of the present can be immobilized. The enzyme can be immobilized using any method and/or means known in the art, such as those described in US Patent Nos. 5,541,097 and 4,713,333, both of which are incorporated herein by reference. One or more enzymes can be immobilized, for example, by contacting the enzyme(s) with a solution of amine-reactive material (e.g., glutaraldehyde) to form an adduct (e.g., enzyme and glutaraldehyde adduct). ), after which the adduct is attached to a solid carrier which has been treated with polyamine (e.g., polyethyleneimine such as EPOMIN P-1050).

[0087] A solid carrier (solid support) to which an alpha-glucosidase enzyme can be immobilized in certain embodiments can be an organic or inorganic material. Such materials include, for example, gamma-alumina, titania, granular activated carbon, granular diatomaceous earth, viro beads, porous glass, pumice, silica gel, metal oxide and aluminum oxide.

[0088] Polyamine may be used to treat a solid carrier such that subsequent exposure of the solid carrier to an adduct comprising an enzyme and amine reactive material generates binding of the enzyme to the solid carrier. Examples of polyamines useful herein include polyethylenediamine, polyethyleneimine (e.g., polydiethylenetriamine, polytriethylenetetramine, polypentaethylenehexamine, polyhexamethylenediamine), polymethylenedicyclohexylamine, polymethylenedianiline, polytetraethylenepentamine, polyphenylenediamine, and mixtures of two or more such polyamine compounds. Preferred polyamines are water soluble and/or have a molecular weight of about 500 to 100,000 Daltons. Polyethyleneimine such as EPOMIN P-1050 can be used in certain embodiments.

[0089] Amine reactive material useful for preparing an adduct comprising an enzyme in the present may be, for example, an aldehyde, organic halide, anhydride, azo compound, isothiocyanate and/or isocyanate. Examples of such amine-reactive materials include glutaraldehyde, succinodialdehyde, terephthaldehyde, bisdiazobenzidine-2,2'-disulfonic acid, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone, diphenyl-4,4'-dithiocyanate-2,2 acid '-Disulfonic, 3-methoxydiphenylmethane-4,4'-diisocyanate, toluene-2-isocyanate-4-isothiocyanate, toluene-2,4-diisothiocyanate, diazobenzidine, diazobenzidino-3,3'-dianisidine, N, N'-hexamethylene bisiodoacetamide, hexamethylene diisocyanate, cyanuric chloride and/or 1,5-difluoro-2,4-dinitrobenzene. Preferably, the amine reactive material is an aldehyde such as glutaraldehyde.

[0090] An alpha-glucosidase enzyme in adduction with an amine-reactive compound can be placed in contact with a polyamine-treated solid vehicle, in order to immobilize the enzyme on the solid vehicle. An enzyme immobilized herein can be employed in various reactor systems, such as a column (e.g., packed column) or stirred tank reactor, to perform hydrolysis reaction as described herein.

[0091] Appropriate conditions for contacting a saccharide in the present with an alpha-glucosidase in the present (eg, transglycosidase or glucoamylase) are those conditions which sustain the hydrolysis of one or more alpha-1,5-glycosylfructose linkages in the saccharide by the alpha-glucosidase. Examples of appropriate conditions are described in the Examples below. Conditions (e.g., temperature, pH, time) of contacting alpha-glucosidase of the present with a sugar substrate are also described in Published US Patent Application No. 2008/0229514, US Patent No. 7,413,887 and US Patent No. U.S. Published Patent Application No. 2013/0102035 (all of which are incorporated herein by reference) and may also be applicable to the described hydrolyzation method.

[0092] The disaccharides and oligosaccharides in the described hydrolyzation method are typically soluble in water or aqueous solution. Contacting the present saccharide with an alpha-glucosidase is therefore preferably carried out under appropriate conditions which are aqueous, in which the saccharide is dissolved. Aqueous conditions can characterize a solution or mixture comprising at least about 20% by weight of water. Alternatively, the aqueous conditions of the present are, for example, at least about 20, 30, 40, 50, 60, 70, 80, 85, 90 or 95% by weight water (or any integer value from 20 to 95% by weight). Aqueous conditions may further comprise a buffer, for example such as acidic, neutral or alkaline buffer, in appropriate concentration and selected on the basis of the pH range provided by the buffer. Examples of buffers/buffers include citrate, acetate (eg sodium acetate), KH2PO4, MOPS, CHES, borate, sodium carbonate and sodium bicarbonate.

[0093] The pH of a hydrolysis reaction of this may be, for example, from about 3.0 to 9.0. The hydrolysis reaction pH can be, for example, about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 , 7.5, 8.0, 8.5 or 9.0. Alternatively, the pH can be around 4-5. Methods of setting pH include the use of buffers, alkalis and/or acids, for example, and are well known in the art.

[0094] The temperature of a hydrolysis reaction of this may 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 from 20 to 80°C). A hydrolysis temperature of about 60°C, 65°C or 60-65°C is preferred in certain embodiments.

[0095] Hydrolysis reaction in this can be carried out, for example, for a period of at least about ten minutes to about ninety hours. The time of a hydrolysis reaction can be, for example, at least about 0.5, 1, 2, 3, 4, 8, 12, 16, 20, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, or 90 hours (or any integer from 0.5 to 72 hours). In certain embodiments, such as for hydrolysis of leucrose, one can perform, for example, hydrolysis reaction in less than four hours (e.g., 0.5-4 hours). The length of time required to achieve the desired level of hydrolysis will vary based on the exact conditions used and would be understood by those skilled in the art. Increasing the amount of enzyme added to a reaction or immobilized on a solid support used in a reaction, for example, will reduce the contact time.

[0096] One or more alpha-glucosidase enzymes herein may be used in a hydrolysis reaction in certain embodiments. Transglucosidase and glucoamylase, for example, can be used in reactions. The amount of alpha-glucosidase in a hydrolysis reaction of the present can be, for example, plus/minus 10% to 20% (or 5% to 10%) of any of the amounts used in the Examples below (e.g. Example 2) . Alternatively, about 0.1-0.5% by volume or 0.1-1.0% by volume of alpha-glucosidase can be used in a hydrolysis reaction. Still alternatively, alpha-glucosidase herein may be used at about, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 ppm in a hydrolysis reaction. A transglycosidase unit (TGU) can be defined, for example, as the amount of a transglycosidase enzyme that will produce one micromole of panose per minute under the test conditions below. Transglycosidase activity can be tested, for example, as follows: transglycosidase is brought up in 100 mM sodium acetate buffer, pH 4.5, containing 4 mM paranitrophenylalphaglycoside and 1 mg/ml bovine serum albumin (BSA). After thirty minutes of incubation at 30 °C, the reaction is terminated by adding an equal volume of 1 M sodium carbonate and OD405 is recorded. A glucoamylase unit (GAU) can be defined, for example, as the amount of glucoamylase enzyme that will produce 1 g of reducing sugar, calculated as glucose per hour from a soluble starch substrate (4% DS (degree of replacement)) under pH 4.2 and 60 °C.

[0097] The initial concentration of saccharide in hydrolysis reaction in certain embodiments of the present invention can be, for example, from about 1% by weight to 50% by weight. The leucrose concentration can be, for example, about 5, 10, 15, 20, 25, 30, 35 or 40% by weight (or any integer value from 5 to 40% by weight). As another example, the concentration of one or more oligosaccharides (e.g. DP2, DP3, DP4, DP2-DP7, DP3-DP7) in a hydrolysis reaction of this can be about 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% by weight. Those skilled in the art would recognize that the total concentration of sugars (which includes disaccharides and oligosaccharides) can impact the activity of alpha-glucosidase enzymes; Preferred concentrations of total sugars in a hydrolysis reaction to maximize enzyme activity can be less than 50% by weight dry solids (DS), with more preferred concentrations of 20-35% by weight DS in some respects.

[0098] Appropriate conditions in certain embodiments for contacting a saccharide with an alpha-glucosidase of the present may comprise (i) glucan synthesis reaction or (ii) a fraction obtained from a glucan synthesis reaction, in which the saccharide is a by-product of the glucan synthesis reaction. In other words, hydrolysis reaction in the present can be conducted in the context of glucan synthesis reaction or a fraction of glucan synthesis reaction, although it is typically conducted in the latter. Glucan synthesis reaction in the present can generate, for example, one or more soluble and/or insoluble alpha-glucan products. Glucan synthesis reaction can be characterized in some embodiments of the present, for example, as "alphaglucan synthesis reaction".

[0099] Glucan synthesis reaction generally designates a solution comprising at least sucrose, water, an active glycosyltransferase enzyme, and optionally other components. Other components that may be present in a glucan synthesis reaction include fructose, glucose, leucrose, soluble oligosaccharides (eg, DP2-DP7) and soluble glucan product(s). Furthermore, one or more alphaglucanhydrolase enzymes may be comprised in a glucan synthesis reaction in some aspects. It would be understood that certain glucan products, such as poly alpha-1,3-glucan with a degree of polymerization (DP) of at least 8 or 9, may be insoluble in water and therefore not dissolved in a reaction. of glucan synthesis, but they can be present out of solution. Glucan produced via glucan synthesis reaction in this can therefore be insoluble. Alphaglucosidase enzyme of the present can be added to the glucan synthesis reaction at any of its stages, such as during the initial preparation of the reaction or when the reaction is near completion (e.g., 80 to 90% complete) or at its completion, in that these last two moments are preferred.

[00100] Glucan synthesis reaction in the present can be one that, in addition to generating a glucan product, generates by-products such as leucrose and/or soluble oligosaccharides. Glucan, in some respects, is an alpha-glucan poly. Glucan synthesis reaction in the present can therefore be aimed at the production of poly alpha-1,3-glucan or mutane, for example, which are typically co-produced with at least oligosaccharide and/or leucrose by-products in a synthesis reaction of glucan.

[00101] Glucan synthesis reaction in certain embodiments comprises a glycosyltransferase enzyme that produces a polyalphaglucan such as polyalpha-1,3-glucan. Examples of such glycosyltransaferase enzymes useful herein are described in U.S. Patent No. 7,000,000 and U.S. Published Patent Applications Nos. 2013/0244288, 2013/0244287 and 2014/0087431, all of which are incorporated herein. as reference.

[00102] Glycosyltransferase enzyme in the present can be derived from any microbial source, such as bacteria or fungus. Examples of bacterial glycosyltransferase enzymes are those derived from a Streptococcus species, Leuconostoc species or Lactobacillus species. Examples of Streptococcus species include S. salivarius, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S. gallolyticus and S. sanguinis. Examples of Leuconostoc species include L. mesenteroides, L. amelibiosum, L. argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicum and L. fructosum. Examples of Lactobacillus species include L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L. reuteri.

[00103] A glycosyltransferase enzyme of the present can be primer-independent or primer-dependent. Primer-independent glycosyltransferase enzymes do not require the presence of a primer to carry out glucan synthesis. Primer-dependent glycosyltransferase enzymes require the presence of an initiator molecule in the reaction solution to act as a primer for the enzyme during glucan polymer synthesis. The term "primer" as used herein means any molecule that can act as a primer for a glycosyltransferase enzyme. Primers that can be used in certain embodiments include dextran and other carbohydrate-based primers, such as hydrolyzed glucan, for example. Published US Patent Application No. 2013/0244287, which is incorporated herein by reference, describes the preparation of hydrolyzed glucan using poly alpha-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).

[00104] A glycosyltransferase enzyme for glucan synthesis reaction in the present can be produced by any means known in the art. Glycosyltransferase enzyme can be produced recombinantly in a heterologous expression system, such as a heterologous microbial expression system. Examples of heterologous expression systems include bacterial (eg E. coli such as TOP10 or MG1655; Bacillus sp) and eukaryotic (eg yeast such as Pichia sp and Saccharomyces sp) expression systems.

[00105] A glycosyltransferase enzyme described herein can be used in any purification state (eg pure or non-pure). A glycosyltransferase enzyme can be purified and/or isolated prior to use. Examples of glycosyltransferase enzymes that are impure include those in cell lysate form. Cell extract or lysate can be prepared from a bacterium (eg E. coli) used to express the enzyme heterologously. The bacteria can be subjected to disruption, for example using a French press cell. In alternative embodiments, bacteria can be homogenized with a homogenizer (eg, APV, Rannie, Gaulin). A glycosyltransferase enzyme is typically soluble in these types of preparations. A lysate, extract or homogenate of bacterial cells in the present can be used at about 0.15-0.3% (v/v), for example, in a reaction solution to produce polyalphaglucan such as polyalpha-1, 3-glucan from sucrose.

[00106] The reaction temperature of glucan synthesis in this can be controlled if desired. In certain embodiments, the reaction temperature is from about 5°C to about 50°C. The temperature in some other embodiments is from about 20°C to about 40°C.

[00107] The initial concentration of sucrose in a glucan synthesis reaction in the present can be, for example, from about 20 g/l to about 400 g/l. Alternatively, the initial sucrose concentration can be from about 75 g/l to about 175 g/l or about 150 g/l. Still alternatively, the initial sucrose concentration can be, for example, about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 g/l (or any whole number from 40 to 160 g/l). “Initial sucrose concentration” means the concentration of sucrose in a gtf reaction solution shortly after addition of all reaction solution components (at least water, sucrose, and gtf enzyme).

[00108] Sucrose used in a glucan synthesis reaction in the present can have high purity (>99.5%) or any other grade or purity. Sucrose can be, for example, at least 99.0% pure or it can be reagent grade sucrose. As another example, incompletely refined sucrose can be used. Incompletely refined sucrose herein means sucrose that has not been processed into white refined sucrose. Incompletely refined sucrose can therefore be completely unrefined or partially refined. Examples of unrefined sucrose are “crude sucrose” (“raw sugar”) and solutions thereof. Examples of partially refined sucrose have not passed through one, two, three or more crystallization steps. The ICUMSA (International Commission on Uniform Methods of Sugar Analysis) of incompletely refined sucrose at present can be, for example, over 150. Sucrose at present can be derived from any renewable sugar source, such as sugar cane, beet , cassava, sweet sorghum or maize. Appropriate forms of sucrose useful herein are, for example, crystalline form or non-crystalline form (e.g., syrup, cane juice and beet juice). Additional suitable forms of incompletely refined sucrose are described in US Patent Application No. 61/969,958.

[00109] Methods for determining ICUMSA values for sucrose are well known in the art and described by the International Commission for Uniform Methods of Sugar Analysis in ICUMSA Methods of Sugar Analysis: Official and Tentative Methods Recommended by the International Commission for Uniform Methods of Sugar Analysis (ICUMSA) (Whalley Ed. H. C. S., Elsevier Pub. Co., 1964), for example, which is incorporated herein by reference. ICUMSA can be measured, for example, using the ICUMSA GS1/3-7 Method as described by R. J. McCowage, R. M. Urquhart and M. L. Burge (Determination of the Solution Color of Raw Sugars, Brown Sugars and Colored Syrups at pH 7.0 - Official, Verlag Dr. Albert Bartens, 2011 review), which is incorporated herein by reference.

[00110] The pH of a glucan synthesis reaction in certain embodiments can be from about 4.0 to about 8.0. Alternatively, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0. The pH can be adjusted or controlled by adding or incorporating an appropriate buffer, including, but not limited to: phosphate, tris, citrate or a combination thereof. Buffer concentration in a glucan synthesis reaction can be, for example, from 0 mM to about 100 mM or about 10, 20 or 50 mM.

[00111] Poly alpha-1,3-glucan produced in a glucan synthesis reaction in the present may contain at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% , 98%, 99%, or 100% (or any integer from 50% to 100%) of glycosidic linkages that are alpha-1,3. Accordingly, in these embodiments, poly alpha-1,3-glucan contains less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% or 0% (or any integer from 0% to 50%) of glycosidic linkages that are not alpha-1,3.

[00112] Poly alpha-1,3-glucan at present preferably has a backbone that is straight/unbranched. In certain embodiments, the poly alpha-1,3-glucan has no branch points or has less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% branch points as a percentage of glycosidic bonds in the polymer. Examples of branch points include alpha-1,6 branch points.

[00113] The molecular weight of poly alpha-1,3-glucan produced in a glucan synthesis reaction in the present can be represented as number average molecular weight (Mn) or weight average molecular weight (Mw). Alternatively, molecular weight can be measured in Daltons or grams/mol. It may also be useful to refer to the DPw (weight average degree of polymerization) or DPn (number average degree of polymerization) of the poly alpha-1,3-glucan polymer.

[00114] The Mn or Mw of poly alpha-1,3-glucan in the present can be at least about 1000. Alternatively, Mn or Mw can be, for example, from at least about 1000 to about 600,000 (or any integer from 1000 to 600,000). Still alternatively, poly alpha-1,3-glucan can have molecular weight in DPn or DPw of at least about 100 or at least about 100 to 1000 (or any integer from 100 to 1000).

[00115] A glucan synthesis reaction fraction may constitute appropriate conditions for contacting a saccharide with an alpha-glucosidase as described herein. A fraction can be part or all of the liquid solution of a glucan synthesis reaction. Typically, a fraction was separated from soluble or insoluble glucan product(s) synthesized in the reaction. A fraction can be separated, for example, from one or more glucan products that are insoluble in water (for example, poly alpha-1,3-glucan) that come out of solution during its synthesis. A fraction in certain preferred embodiments of the present invention is from a poly alpha-1,3-glucan synthesis reaction.

[00116] The fraction volume (before optional dilution or fraction concentration, see below) in certain embodiments may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80% or 90% (or any integer from 10% to 90%) of the volume of the glucan synthesis reaction from which it is obtained. Typically, in glucan synthesis reactions producing insoluble glucan (such as poly alpha-1,3-glucan), the fraction will be a part (not all) of the liquid solution component of the reaction. A fraction can be obtained at any step in a glucan synthesis reaction, but is preferably obtained near completion (eg, greater than 80% or 90% complete) or after completion of the reaction.

[00117] Examples of a glucan synthesis reaction fraction in certain embodiments include filtrates and supernatants. In embodiments in which an insoluble glucan product is synthesized, therefore, a fraction according to the present can be obtained (separated) from a glucan synthesis reaction using a funnel, filter (e.g., filter press), centrifuge or any other method or equipment known in the art that allows removal of some or all of the liquids from the solids. Filtration can be carried out, for example, by means of gravity, vacuum or pressure filtration. Filtering preferably removes all or most of an insoluble glucan; any filter material (eg, filter paper) with an average pore size (eg, about 40 to 50 microns) sufficient to remove solids from liquids can be used. A fraction typically retains, in whole or for the most part, its dissolved components, such as by-products of the glucan synthesis reaction. Leucrose is a preferred saccharide in a filtrate at present.

[00118] A fraction according to the present can be optionally diluted or concentrated, if desired. Concentration of a fraction can be carried out using any other method or equipment known in the art suitable for concentrating solutions. A fraction can be concentrated by means of evaporation, for example, such as with a rotary evaporator (eg, set at about 40-50°C). A fraction in some aspects of the present can be concentrated to a volume that is about 75%, 80%, 85%, 90% or 95% of the original fraction volume. A concentrated fraction (eg concentrated filtrate) may optionally be called a syrup.

[00119] A fraction, in some aspects, may comprise water that replaces the water that was present in the composition from which the fraction was obtained. Saccharide by-product(s) of a glucan synthesis reaction can be separated, for example, in certain chromatographic methods in which the original solvent is replaced by another solvent (saccharide by-products that are attached to a column (removed from the original solvent), for example, can be eluted in a new solvent).

[00120] A fraction, in some aspects, may be treated to have any of the appropriate conditions (eg, temperature, pH, and time) described above for contacting a saccharide with an alpha-glucosidase. A fraction can be modified, for example, to have a pH of about 4 to 5 before alpha-glucosidase is added to the fraction. As another example, the temperature of a fractional hydrolysis reaction can be about 55-65°C (e.g. about 60°C). A fraction that has been concentrated to syrup can be used in a hydrolysis reaction in yet another example.

[00121] A fraction in certain preferred embodiments of the present is from a poly alpha-1,3-glucan synthesis reaction; this fraction is preferably a filtrate. A poly alpha-1,3-glucan synthesis reaction fraction herein comprises at least water, fructose and one or more types of saccharide (leucrose and/or oligosaccharides such as DP2-DP7). Other components that may be found in this type of fraction include, for example, sucrose (i.e. residual sucrose not consumed in the gtf reaction), one or more gtf enzymes, glucose, buffer, salts, FermaSure®, borates, sodium hydroxide , hydrochloric acid, cell lysate components, proteins and/or nucleic acids. At a minimum, the components of a poly alpha-1,3-glucan synthesis reaction fraction include, for example, water, fructose, glucose, one or more types of saccharide (leucrose and/or oligosaccharides such as DP2-DP7 ) and, optionally, sucrose. It would be understood that the composition of a fraction depends, in part, on the conditions of the glucan synthesis reaction from which the fraction is obtained. In those fractions that contain one or more gtf enzymes, it is preferred that those one or more gtf enzymes are deactivated (eg, hot-deactivated) before using the fraction in a hydrolysis reaction according to the present invention.

[00122] It should be understood that the exact distribution of sugar by-products generated through the polymerization of sucrose in a glucan synthesis reaction may vary based on the reaction conditions and gtf enzyme used, especially on the temperature and the sucrose concentration. It should also be understood that the exact composition of sugars in a glucan synthesis reaction fraction is not critical to the described hydrolysis process. Generally, as the amount of sucrose increases, the selectivity of the reaction for leucrose and oligosaccharides will increase. On the other hand, as the temperature increases, the selectivity of the reaction for leucrose tends to drop, while the selectivity for oligosaccharides is largely unaffected. It should also be understood that the ratio of sugars to water, i.e. the percentage by weight of dry solids (DS), which is calculated by dividing the mass of sugar by the total weight of the solution, can be adjusted by means of of water evaporation, preferably under temperatures below 50°C under vacuum, or addition of water, without significant impact to the relative distribution of sugars in a fraction of a glucan synthesis reaction. It is also possible to increase the percentage of sucrose in a fraction by stopping the gtf reaction before reaching complete conversion (to glucan), either by reducing the pH below the active range for the gtf enzyme or by thermally deactivating the gtf enzyme. gtf enzyme.

[00123] In certain embodiments, the glucan synthesis reaction in this can generate one or more soluble alpha-glucan products. Soluble alpha-glucan product (alternatively, “soluble fiber”) can be (i) a direct product of glycosyltransferase or (ii) a product of the concerted action of glycosyltransferase and alpha-glucan hydrolase capable of hydrolyzing glucan polymers that contain one or more alpha bonds -1,3-glycosidic or one or more alpha-1,6-glycosidic linkages.

[00124] Soluble alpha-glucan in the present may comprise, for example: a. at least 75% alpha-1,3-glycosidic linkages; B. less than 25% alpha-1,6-glycosidic linkages; w. less than 10% alpha-1,3,6-glycosidic linkages; d. Mw of less than 5000 Daltons; It is. viscosity of less than 0.25 Pascal second (Pa^s) at 12% by weight in water at 20°C; f. dextrose equivalence (DE) in the range of 4 to 40; g. digestibility of less than 10% as measured using Association of Analytical Communities (AOAC) method 2009.01; H. solubility of at least 20% (w/w) in water at 25 °C under pH 7; Hey. polydispersion index (PDI) of less than 5.

[00125] This soluble alpha-glucan can be produced as described in US Application No. 62/004,290.

[00126] As an example, a soluble alpha-glucan fiber composition may comprise at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and more preferably at least 95% alpha-(1,3) glycosidic linkages.

[00127] As another example, in addition to the alpha-(1,3) glycosidic linkage embodiments described above, a soluble alpha-glucan fiber composition may additionally comprise less than 25%, preferably less than 10%, most preferably 5% or less, and even more preferably less than 1% alpha-(1,6) glycosidic linkages.

[00128] As another example, in addition to the alpha-(1,3) and alpha-(1,6) glycosidic bond content embodiments described above, a soluble alpha-glucan fiber composition may additionally comprise less than 10%, preferably less less than 5% and preferably greater than 2.5% alpha-(1,3,6) glycosidic linkages.

[00129] As another example, soluble alpha-glucan fiber compositions can comprise 93 to 97% alpha-(1,3) glycosidic linkages and less than 3% alpha-(1,6) glycosidic linkages and have weight average molecular weight corresponding to a blend having a DP of 3 to 7. In a further embodiment, soluble alpha-glucan fiber compositions can comprise about 95% alpha-(1,3) glycosidic linkages and about 1% alpha-(1,3) linkages. 6) glycosidic and have a weight average molecular weight corresponding to a blend with a SD of 3 to 7. In a further aspect of the above embodiment, a soluble alpha-glucan fiber composition may additionally comprise 1 to 3% alpha-(1,3) linkages. ,6) or, preferably, about 2% alpha-(1,3,6) bonds.

[00130] As another example, in addition to the glycosidic bond content embodiments mentioned above, a soluble alpha-glucan fiber composition may additionally comprise less than 5%, preferably less than 1%, and more preferably less than 0.5% of alpha-(1,4) glycosidic linkages.

[00131] As another example, in addition to the glycosidic bond content embodiments mentioned above, a soluble alpha-glucan fiber composition may comprise weight average molecular weight (Mw) of less than 5000 Daltons, preferably less than 2500 Daltons, more preferably of 500 to 2500 Daltons, and preferably greater from about 500 to about 2000 Daltons.

[00132] As another example, in addition to any of the above characteristics, soluble alpha-glucan fiber compositions may comprise viscosity of less than 250 centipoise (0.25 Pa^s), preferably less than 10 cP (0.01 Pa^s) more preferably less than 7 cP (0.007 Pa^s), more preferably less than 5 cP (0.005 Pa^s), more preferably less than 4 cP (0.004 Pa^s) and more preferably less than 3 cP (0.003 Pa 2 s) to 12% by weight in water at 20°C.

[00133] A soluble alpha-glucan fiber composition may have, in certain embodiments, digestibility of less than 10% or, preferably, less than 9%, 8%, 7%, 6%, 5%, 4%, 3 %, 2%, or 1% as measured using the Association of Analytical Communities (AOAC) method 2009.01. In another aspect, the relative level of digestibility can alternatively be determined using AOAC 2011.25 (Integrated Total Dietary Fiber Test) ( McCleary et al, 2012, J. AOAC Int., 95(3), 824-844 ).

[00134] In addition to any of the above embodiments, soluble alpha-glucan fiber compositions may have a solubility of at least 20% (w/w), preferably at least 30%, 40%, 50%, 60% or 70% in water at 25 °C under pH 7.

[00135] In one embodiment, a soluble alpha-glucan fiber composition may comprise reducing sugar content of less than 10% by weight, preferably less than 5% by weight, and preferably greater than 1% by weight or less.

[00136] In one embodiment, soluble alpha-glucan fiber compositions may comprise caloric content of less than 4 kcal/g, preferably less than 3 kcal/g, more preferably less than 2.5 kcal/g, and preferably greater than about 2 kcal/g or less.

[00137] As another example, soluble alpha glucan in the present may comprise: a. 10% to 30% alpha-1,3-glycosidic linkages; B. 65% to 87% alpha-1,6-glycosidic linkages; w. less than 5% alpha-1,3,6-glycosidic linkages; d. weight average molecular weight (Mw) of less than 5000 Daltons; It is. viscosity of less than 0.25 Pascal second (Pa^s) at 12% by weight in water at 20°C; f. dextrose equivalence (DE) in the range of 4 to 40, preferably 10 to 40; g. digestibility of less than 10% as measured using Association of Analytical Communities (AOAC) method 2009.01; H. solubility of at least 20% (w/w) in water at 25 °C under pH 7; Hey. polydispersion index (PDI) of less than 5.

[00138] This soluble alpha-glucan can be produced as described in US Patent Application No. 62/004,308.

[00139] As another example, soluble alpha glucan in the present may comprise: a. 25-35 alpha-1,3-glycosidic bonds; B. 55-75% alpha-1,6-glycosidic linkages; w. 5-15% alpha-1,3,6-glycosidic linkages; d. weight average molecular weight of less than 5000 Daltons; It is. viscosity of less than 0.25 Pascal second (Pa^s) at 12% by weight in water at 20°C; f. dextrose equivalence (DE) in the range of 4 to 40; g. digestibility of less than 10% as measured using Association of Analytical Communities (AOAC) method 2009.01; H. solubility of at least 20% (w/w) in water at 25 °C; Hey. polydispersion index of less than 5.

[00140] This soluble alpha-glucan can be produced as described in US Patent Application No. 62/004,312.

[00141] As another example, soluble alpha glucan in the present may comprise: a. at least 95% alpha-1,6-glycosidic linkages; B. 1% or less alpha-1,3-glycosidic linkages; w. less than 2% alpha-1,3,6-glycosidic linkages; d. less than 1.5% alpha-1,4-glycosidic linkages; It is. weight average molecular weight of less than 20,000 Daltons; f. viscosity of less than 0.25 Pascal second (Pa^s) at 12% by weight in water at 20°C; g. dextrose equivalence (DE) in the range of 1 to 30; H. digestibility of less than 10% as measured using Association of Analytical Communities (AOAC) method 2009.01; i. solubility of at least 20% (w/w) in water at 25 °C under pH 7; and j. polydispersion index of less than 5.

[00142] This soluble alpha-glucan can be produced as described in US Patent Application No. 62/004,314.

[00143] As another example, soluble alpha glucan in the present may comprise: a. a range of: i. 1% to 50% alpha-1,3-glycosidic linkages; or ii. more than 10% but less than 40% alpha-1,4-glycosidic linkages; or iii. any combination of (i) and (ii); B. 1 to 50% alpha-1,2-glycosidic linkages; w. 0-25% alpha-1,3,6-glycosidic linkages; d. less than 98% alpha-1,6-glycosidic linkages; It is. weight average molecular weight of less than 300 kDa; f. viscosity of less than 0.25 Pascal second (Pa^s) at 12% by weight in water at 20°C; g. digestibility of less than 20% as measured using Association of Analytical Communities (AOAC) method 2009.01; H. solubility of at least 20% (w/w) in water at 25 °C under pH 7; Hey. polydispersion index of less than 26, preferably less than 5.

[00144] This soluble alpha-glucan can be produced as described in US Patent Application No. 62/004,305.

[00145] In certain embodiments, soluble alpha-glucan is a direct product of glycosyltransferase. Such glycosyltransferase and conditions of its use in an appropriate glucan synthesis reaction may be as described herein, or as described, for example, in any of US Patent Applications Nos. 62/004290, 62/004308, 62/ 004312, 62/004314 and/or 62/004305.

[00146] Soluble alpha-glucan may alternatively be a product, for example, of the concerted action of glycosyltransferase and alpha-glucan hydrolase capable of hydrolyzing glucan polymers that contain one or more alpha-1,3-glycosidic linkages or one or more alpha-1 linkages ,6-glycosides. In some aspects, a glucan synthesis reaction to generate a soluble alpha-glucan product can comprise at least one glycosyltransferase and at least one alpha-glucan hydrolase. In other aspects, a glucan synthesis reaction may initially comprise one or more glycosyltransferases as the only enzyme component(s). This reaction generates a first alpha-glucan product that has not yet undergone modification by an alpha-glucan hydrolase. Then, at least one alpha-glucan hydrolase is added to the reaction for an adequate period of time to allow modification of the first product into a soluble alpha-glucan product. There are, therefore, different ways of synthesizing a soluble alpha-glucan product through the concerted action of glycosyltransferase and alpha-glucanhydrolase. Conditions for carrying out a glucan synthesis reaction in which one or more alpha-glucan hydrolase enzymes are included during the glucan synthesis reaction and/or after the glucan synthesis may be as described herein or as described, for example, in any of US Patent Applications Nos. 62/004,290, 62/004,308, 62/004,312, 62/004,314 and/or 62/004,305.

[00147] Alphaglucanhydrolase herein may be, for example, dextranase (capable of hydrolyzing alpha-1,6-linked glycosidic bonds; E.C. 3.2.1.11), mutanase (capable of hydrolyzing alpha-1,3-linked glycosidic bonds; E.C. 3.2.1.59), mycodextranase (capable of endo-hydrolyzing (1-4)-alpha-D-glycosidic linkages in alpha-D-glucans containing (1-3) and (1-4) linkages; EC 3.2.1.61) ; glucan 1,6-alphaglucosidase (EC 3.2.1.70) and alternanase (capable of alternating endohydrolytically splitting; EC 3.2.1; see US Patent No. 5,786,196).

[00148] Mutanase comprising SEQ ID NO: 47 may be used in certain aspects. Alternatively, mutanase can comprise an amino acid sequence that is, for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 47 and possess mutanase activity.

[00149] Glucan synthesis reaction as described herein to generate one or more soluble alpha-glucan products can directly serve as appropriate conditions under which a hydrolysis reaction is carried out herein, in which an alpha-glucosidase is used to hydrolyze an alpha-bond 1.5 glucosylfructose. Such hydrolysis may be carried out following any of the conditions described above in connection with, for example, the hydrolytic treatment of a glucan synthesis reaction which produces polyalpha-1,3-glucan. Alternatively, a fraction (e.g., chromatographic fraction) of a glucan synthesis reaction to generate one or more soluble alpha-glucan products can be used as appropriate conditions under which alpha-glucosidase-mediated hydrolysis of alpha-1,5 glucosylfructose linkages is performed. .

[00150] A fraction in certain embodiments of the present may be a chromatographic fraction from a glucan synthesis reaction. A fraction can be, for example, a chromatographic fraction from a glucan synthesis reaction that generates one or more soluble alpha-glucan products as described herein. Such a reaction may optionally include one or more alpha-glucan hydrolases during glucan synthesis and/or after completion of glucan synthesis. A fraction in any of these types of embodiments has typically been obtained for the purpose of separating all or most (e.g., at least about 60%, 70%, 80%, 90%, or 95%) of an alpha-glucan product. soluble part of a reaction composition from which it was produced. Once separated from a soluble alpha-glucan product, in whole or in major part, a fraction may be subjected to any of the alpha-1,5-glycosylfructose hydrolysis processes described herein, using one or more alpha-glucanases.

[00151] A chromatographic fraction of this can typically be obtained using an appropriate 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.

[00152] The present invention also relates to a composition produced by contacting a saccharide with an alpha-glucosidase enzyme (for example, transglycosidase or glucoamylase), wherein (i) the saccharide is a disaccharide or oligosaccharide comprising at least one alpha-1,5-glycosylfructose linkage and (ii) the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5-glycosylfructose linkage of the saccharide. The composition produced in this way comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to contacting. Examples of the composition include any of those described herein, such as a hydrolyzed filtrate from a glucan synthesis reaction or a hydrolyzed fraction from a glucan synthesis reaction used to produce soluble alpha-glucan. Any of the features described above and in the Examples with respect to a hydrolyzation method and its products can characterize the composition. The following composition features are examples.

[00153] An alpha-glucosidase enzyme, in certain embodiments of the composition, 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. 5, 6, 8, 9, 11, 12, 14, 15, 17, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or that of DIAZYME RDF ULTRA (DuPont Industrial Biosciences). Transglycosidase in certain embodiments of the composition may comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 1. Glycoamylase in certain embodiments of the composition may comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 2. Alternatively, any of the alpha-glucosidase enzymes described herein can be used to produce the described composition.

[00154] A composition produced by means of a hydrolyzation method according to the present may contain, for example, concentration of saccharide such as leucrose that is less than 50% of the concentration of leucrose that was present before contact of the saccharide with alphaglucosidase.

[00155] A composition produced by means of a hydrolyzation method in certain embodiments of the present may be a glucan synthesis reaction or one of its fractions, wherein a saccharide by-product of the glucan synthesis reaction is contacted with alpha-glucosidase . Fraction, in this embodiment, can be a glucan synthesis reaction filtrate or a glucan synthesis reaction fraction, for example, used to produce soluble alpha-glucan. A saccharide in this embodiment can be, for example, leucrose.

[00156] Those skilled in the art would understand that the embodiments described herein are useful, in part, for saccharifying disaccharides and oligosaccharides that otherwise can hardly be decomposed. This feature can be used, for example, to perform amplified methods of (i) fructose enrichment and (ii) fermentation.

[00157] Example 6 below demonstrates that enrichment of fructose by means of chromatography is increased when using a glucan filtrate hydrolyzed by alpha-glucosidase (transglycosidase) compared to using a filtrate that has not been hydrolyzed.

[00158] The present invention further relates, therefore, to a method of enriching fructose that is present in a fraction of a glucan synthesis reaction. This method comprises (a) contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme (e.g., transglucosidase or glucoamylase) under appropriate conditions, whereby the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5-glycosylfructose bond of a disaccharide or oligosaccharide comprised in the fraction; and (b) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition that contains a higher concentration of fructose compared to the fructose concentration of the fraction of step (a).

[00159] The characteristics of the fructose enrichment method described with respect to alpha-glucosidase enzymes (for example, transglucosidase or glucoamylase) and fractions of a glucan synthesis reaction, for example, may be in accordance with any of the descriptions provided in this with respect to each of these characteristics.

[00160] The fructose separation step (b) can be carried out by any means known in the art. Chromatography may be employed, for example, as described in the Examples below or following the description of published European Patent No. EP2292803B1, which is hereby incorporated by reference.

[00161] A composition (for example, fructose solution or fructose syrup) that contains higher concentration of fructose resulting from the described enrichment method may contain at least about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% by weight of fructose.

[00162] A fructose enrichment method in accordance with the present can perform better than one using a filtrate that has not been hydrolyzed with alpha-glucosidase as described in the present. This improved performance can be measured in terms of a fructose recovery percentage of at least 40%, 45% or 50%.

[00163] The present invention also relates to a fermentation method comprising (a) contacting a fraction obtained from a glucan synthesis reaction with an alphaglucosidase enzyme (for example, transglycosidase or glucoamylase) under appropriate conditions, in which the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose linkage of a disaccharide or oligosaccharide comprised in the fraction; (b) fermenting the fraction from step (a) with a microbe to generate a product; and (c) optionally isolating the product of (b). The fermentation step of (b) can be carried out after step (a) or simultaneously with step (a). Significantly, this method can be used to produce ethanol, for example, via fermentation of a filtrate isolated from a glucan synthesis reaction. The ethanol production through this process is higher than the ethanol production obtained by fermenting a glucan filtrate that has not been hydrolyzed.

[00164] The characteristics of the fermentation method described in relation to alpha-glucosidase enzymes (for example, transglucosidase or glucoamylase), disaccharides and oligosaccharides, fractions of a glucan synthesis reaction and appropriate contact conditions, for example, can refer to, according to any of the descriptions provided herein, to each of these characteristics.

[00165] Microbes for use in fermentation methods according to the present can be, for example, bacteria, yeast or fungi. Examples of bacteria useful herein include Lactobacillus species, Streptococcus species, Bifidobacterium species, Leuconostoc species, Escherichia species (e.g., E. coli) and Bacillus species. Examples of yeasts useful herein include Saccharomyces species, such as S. cerevisiae and S. bayanus.

[00166] Fermentation methods according to the present invention can generate a product such as ethanol or acid (eg lactic acid). It is believed, however, that other products can be generated if desired. Those skilled in the art would understand that the elaboration of certain products using a fermentation method as described would depend on various conditions, such as the microbe(s) used in the fermentation. The present fermentation conditions may be as described in the Examples below or as described in El-Mansi et al (2006, Fermentation Microbiology and Biotechnology, second edition, CRC Press) and Stanbury et al (1999, Principles of Fermentation Technology, second edition , Butterworth-Heinemann), for example, both incorporated into the present as a reference.

[00167] The yield of a product in certain embodiments of a fermentation method in accordance with the present is higher than the yield of product obtained by fermenting a glucan filtrate that has not been hydrolyzed with an alpha-glucosidase in accordance with the gift. This comparison can be performed with respect to the control fermentation, for example, which used the non-hydrolyzed fraction of a glucan synthesis reaction. The yield of fermentation product herein may be increased, for example, by at least about 10%, 20%, 40%, 60%, 80%, or 100% (or any integer from 10% to 100% ). Furthermore, the rate of product formation through fermentation according to the present invention can be increased.

[00168] Example 7 below demonstrates that leucrose can be fermented into ethanol by yeast to provide a feed comprising glucan filtrate that has not been hydrolyzed. Further described herein, therefore, is a method of using microbes to ferment leucrose into a product (eg, ethanol). Such a method may comprise fermenting a glucan filtrate that (i) has or (ii) has not been hydrolyzed with an alpha-glucosidase as described herein. Regardless of whether the leucrose is supplied in a glucan filtrate or another form (e.g., enriched or semi-purified form), methods of fermenting leucrose can comprise adapting microbes (e.g., yeast such as S. cerevisiae) using leucrose. This adaptation may comprise growing the microbe in the presence of leucrose and optionally other sugars, over at least two or three growth cycles, for example, after which the microbe uses more leucrose to ferment a product. In certain embodiments, microbes may be (i) grown on a first feed comprising leucrosis (one complete cycle), (ii) removed from the first feed, (iii) grown on a second feed comprising leucrosis (two full cycles), ( iv) optionally removed from second feeding and (v) optionally grown on third feeding (three full cycles). Microbes adapted in this way may have increased leucrose fermentation capacity in certain embodiments.

[00169] Example 9 below demonstrates that nearly all (e.g., >98% or >99%) leucrose present in a glucan filtrate can be utilized for yeast fermentation when the glucan filtrate is hydrolyzed with a transglycosidase while, at the same time it is fermented with yeast. Improved leucrose fermentation methods according to the present can therefore comprise hydrolyzing leucrose with an alpha-glucosidase (e.g., transglycosidase or glucoamylase), simultaneously fermenting the leucrose with microbes.

[00170] Non-limiting examples of compositions and methods described herein include: 1. Method of hydrolyzing an alpha-1,5 glycosylfructose bond in a saccharide comprising at least one alpha-1,5 glycosylfructose bond, wherein the saccharide is a disaccharide or oligosaccharide and the method comprises: - contacting the saccharide with an alpha-glucosidase enzyme under appropriate conditions, whereby the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose bond of the saccharide; and wherein the amount of saccharide is reduced compared to the amount of saccharide that was present prior to contacting; 2. Method according to embodiment 1, wherein alpha-glucosidase enzyme is immobilized; 3. Method according to either of Embodiments 1 or 2, wherein the saccharide is leucrose; 4. The method according to embodiment 3, wherein the leucrose concentration after the contacting step is less than 50% of the leucrose concentration that was present before the contacting; 5. A method according to any of Embodiments 1, 2, 3 or 4, wherein appropriate conditions comprise: 1. a glycan synthesis reaction; or 11. a fraction obtained from the glucan synthesis reaction; 12. that saccharide is a by-product of the glucan synthesis reaction; 13. Method according to embodiment 5, wherein the glucan synthesis reaction generates at least one insoluble alpha-glucan product; 14. Method according to embodiment 6, wherein the fraction is a filtrate from the glucan synthesis reaction; 15. Method according to embodiment 5, wherein the glucan synthesis reaction generates at least one soluble alpha-glucan product that is: i. a glycosyltransferase product; or ii. a product of the combined action of a glycosyltransferase and an alphaglucanhydrolase capable of hydrolyzing glucan polymers that contain one or more alpha-1,3-glycosidic linkages or one or more alpha-1,6-glycosidic linkages. 9. Method according to embodiment 8, wherein the fraction is a chromatographic fraction of the glucan synthesis reaction; 10. Method according to any one of embodiments 1 to 9, wherein the alpha-glucosidase enzyme is a transglycosidase or glucoamylase; 11. Composition produced by contacting saccharide with an alpha-glucosidase enzyme; wherein the saccharide is a disaccharide or oligosaccharide and comprises at least one alpha-1,5 glycosylfructose bond; wherein the enzyme hydrolyses at least one alpha-1,5 glycosylfructose linkage of the saccharide; and wherein the composition comprises a reduced amount of the saccharide compared to the amount of the saccharide that was present prior to contacting; 12. Composition according to embodiment 11, wherein the saccharide is leucrose; 13. Composition according to any of Embodiments 11 or 12, wherein the saccharide is in (i) a glucan synthesis reaction; or (ii) a fraction obtained from the glucan synthesis reaction; wherein the saccharide is a by-product of the glucan synthesis reaction; 14. Method of enriching fructose present in a glucan synthesis reaction fraction, comprising: a. contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose bond of a disaccharide or oligosaccharide comprised in 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); 15. Fermentation method comprising: a. contacting a fraction obtained from a glucan synthesis reaction with an alpha-glucosidase enzyme under appropriate conditions, wherein the alpha-glucosidase enzyme hydrolyses at least one alpha-1,5 glycosylfructose bond of a disaccharide or oligosaccharide comprised in the fraction; and b. fermenting the fraction of step (a) with a microbe to generate a product, wherein the fermentation is carried out after step (a) or simultaneously with step (a); and c. optional isolation of the product of (b); wherein the yield of product from (b) increases compared to the yield of fermentation product from a fraction of the glucan synthesis reaction which has not been contacted with the alpha-glucosidase enzyme.

EXAMPLES

[00171] The present invention is further defined in the Examples below. It is to be understood that these Examples, while indicating certain preferred aspects of the present invention, are provided by way of illustration only. From the above discussion and these Examples, those skilled in the art can determine the essential features of the present invention and, without departing from its spirit and scope, make various changes and modifications of the present invention to adapt it to various uses and conditions.

ABBREVIATIONS

[00172] The meaning of some of the abbreviations used herein is as follows: “g” indicates gram(s), “h” indicates hour(s), “ml” indicates milliliter(s), “psi” indicates pound(s) ) per square inch, "% by weight" indicates percent by weight, "μm" indicates micrometer(s), "%" indicates percent, "°C" indicates degrees Celsius, "mg" indicates milligram(s), "mm" " indicates millimeter(s), "ml/min" indicates milliliters per minute, "m" indicates meter(s), "μl" indicates microliter(s), "mmol" indicates millimol(s), "min" indicates minute( s), “% mol” indicates mole percent, “M” indicates moles, “mg/g” indicates milligrams per gram, “rpm” indicates revolutions per minute, and “MPa” indicates megaPascals.

GENERAL METHODS

[00173] All reagents were obtained from Sigma-Aldrich (St. Louis MO) unless otherwise noted. Sucrose was obtained using VWR (Radnor PA).

PREPARATION OF CRUDE EXTRACTS OF GLYCOSYLTRANSFERASE (GTF) ENZYMES

[00174] The gtfJ enzyme from Streptococcus salivarius (SEQ ID NO: 3) was expressed in E. coli DH10B strain using an expression system induced by isopropyl beta-D-1-thiogalactopyranoside (IPTG). SEQ ID NO: 3 has a 42-residue N-terminal deletion compared to the S. salivarius gtfJ amino acid sequence in GENBANK ID No. 47527, but includes an initial methionine. Briefly, E. coli DH10B cells were transformed to express SEQ ID NO: 3 of a codon-optimized DNA sequence for expressing the gtfJ enzyme in E. coli. This DNA sequence was contained in the expression vector, pJexpress404® (DNA 2.0, Menlo Park CA). Transformed cells were inoculated to an initial optical density (OD at 600nm) of 0.025 in LB medium (10 g/l of Tryptone; 5 g/l of yeast extract, 10 g/l of NaCl) and maintained in growth at 37° C in an incubator while shaking at 250 rpm. Cultures were induced by adding 1 mM IPTG when they reached OD600 of 0.8-1.0. Induced cultures were maintained on the shaker and harvested three hours after induction.

[00175] Enzyme GtfJ (SEQ ID No. 3) was harvested by centrifuging cultured cells (25 °C, 16000 rpm) in an Eppendorf® centrifuge, resuspension of cells in 5.0 mM phosphate buffer (pH 7.0) and cooling to 4 °C on ice. Cells were disrupted using a bead beater with 0.1 mm silica beads and then centrifuged at 16,000 rpm at 4°C to pellet unruptured cells and cell debris. The crude extract (which contains soluble GtfJ enzyme, SEQ ID NO: 3) was separated from the pellet and analyzed using the Bradford protein assay to determine protein concentration (mg/ml).

[00176] Streptococcus sp C150 gtf-S enzyme (SEQ ID No. 40) was prepared as follows. SG1184 is a Bacillus subtilis expression line that expresses a truncated version of the Gtf-S glycosyltransferase ("GTF0459") from Streptococcus sp C150 (GENBANK® GI: 321278321). The gene (SEQ ID NO: 41) encoding an N-terminally truncated protein GTF0459 (SEQ ID NO: 42) from the E. coli expression plasmid pMP79 was cloned into the NheI and HindIII sites of the full-length Bacillus subtilis expression plasmid p4JH under the aprE promoter and fused to the B. subtilis AprE signal peptide on the vector. The construct was first transformed into E. coli DH10B and selected on LB with ampicillin plates (100 µg/ml). The pDCQ984 construct confirmed to express GTF0459 was then transformed into B. subtilis BG6006 which contains nine protease deletions (amyE::xylRPxylAcomK-ermC, degUHy32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δ vpr, ΔwprA, Δmpr-ybfJ, ΔnprB) and selected on LB plates with chloramphenicol (5 μg/ml). Colonies grown on LB plates with 5 μg/ml chloramphenicol were streaked several times onto LB plates with 25 μg/ml chloramphenicol. The resulting B. subtilis expression line, SG1184, was first grown in LB medium with 25 µg/ml chloramphenicol and then subcultured in Grants II medium containing 25 µg/ml chloramphenicol grown at 30°C for two to three days. The cultures were centrifuged at 15000 g for thirty minutes at 4 °C and the supernatant was filtered through 0.22 μm filters. The filtered supernatant was sliced and frozen at -80 °C.

[00177] B. subtilis strain SG1184, which expresses GTF0459 (SEQ ID NO: 42), was cultured under submerged aerobic condition using conventional batch-fed fermentation. A nutrient medium containing 0-0.25% milocin solids (Roquette), 5-25 g/l of sodium potassium phosphate, a 0.3-0.6 M solution of ferrous sulfate, manganese chloride and calcium chloride, 0.5-4 g/l of magnesium sulfate and a solution of 0.01-3.7 g/l of zinc sulfate, cuprous sulfate, boric acid and citric acid. An antifoaming agent, FOAMBLAST 882, was added at 2-4 ml/l to control foaming. Ten liter fermentation was fed 50% (w/w) glucose feed when initial batch glucose was not detectable. The glucose feeding rate increased over several hours. Fermentation was controlled at 30°C and 20% DO and under initial stirring at 750 rpm. The pH was controlled at 7.2 using 50% (v/v) ammonium hydroxide. Fermentation parameters such as pH, temperature, air flow and % DO were monitored throughout the entire two-day fermentation run. The culture broth was collected at the end of the conduction and centrifuged to obtain a supernatant. The supernatant containing GTF0459 (SEQ ID NO:42) was then stored frozen at -80°C.

[00178] The gtf-C enzyme from S. mutans MT-4239 (SEQ ID NO: 43) was prepared as follows. A gene encoding a truncated version of a glycosyltransferase (gtf) enzyme identified in GENBANK® as GI: 3130088 (SEQ ID NO: 43; gtf-C from S. mutans MT-4239) was synthesized using codons optimized for expression in Bacillus subtilis and synthesized using GenScript. The gene (SEQ ID NO: 44) encoding GTF0088BsT1 with N-terminal truncation and T1 C-terminal truncation (SEQ ID NO: 45) was amplified from the GENSCRIPT plasmid and cloned into the NheI and HindIII sites of the full-length expression plasmid of Bacillus subtilis p4JH under the aprE promoter and fused with the B. subtilis AprE signal peptide on the vector. The construct was first transformed into E. coli DH10B and selected on LB with ampicillin plates (100 µg/ml). Confirmed construct pDCQ1021 expressing GTF0088BsT1 was then transformed into B. subtilis BG6006 containing nine protease deletions (amyE::xylRPxylAcomK-ermC, degUHy32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δb pr, Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB) and selected on LB plates with chloramphenicol (5 μg/ml). Colonies grown on LB plates with 5 μg/ml chloramphenicol were streaked several times onto LB plates with 25 μg/ml chloramphenicol. The resulting B. subtilis expression line, SG1221, was first grown in LB medium with 25 µg/ml chloramphenicol and then subcultured in Grants II medium containing 25 µg/ml chloramphenicol grown at 30°C for two to three days. The cultures were centrifuged at 15000 g for thirty minutes at 4 °C and the supernatant was filtered through 0.22 μm filters. The filtered supernatant was sliced and frozen at -80 °C.

[00179] B. subtilis strain SG1221, which expresses GTF0088BsT1 (SEQ ID NO: 45), was cultured under submerged aerobic condition using conventional batch-fed fermentation. A nutrient medium containing 0-0.25% milocin solids (Roquette), 525 g/l of sodium potassium phosphate, a 0.3-0.6 M solution of ferrous sulfate, manganese and calcium chloride, 0.5-4 g/l of magnesium sulfate and a solution of 0.01-3.7 g/l of zinc sulfate, cuprous sulfate, boric acid and citric acid. An antifoaming agent, FOAMBLAST 882, was added at 2-4 ml/l to control foaming. Two liter fermentation was fed 50% (w/w) glucose feed when initial batch glucose was not detectable. The glucose feeding rate increased over several hours. Fermentation was controlled at 30°C and 20% DO and under initial stirring at 400 rpm. The pH was controlled at 7.2 using 50% (v/v) ammonium hydroxide. Fermentation parameters such as pH, temperature, air flow and % DO were monitored throughout the entire two-day fermentation run. The culture broth was collected at the end of the conduction and centrifuged to obtain a supernatant. The supernatant containing GTF088BsT1 (SEQ ID NO: 45) was then stored frozen at -80°C.

DETERMINATION OF GLYCOSYLTRANSFERASE GTF0459 AND GTF088BST1 ACTIVITY

[00180] Testing of glycosyltransferase activity was performed by incubating 1-10% (v/v) crude protein extract containing GTF enzyme with 200 g/l sucrose in 25 mM or 50 mM sodium acetate buffer under pH 5.5 in the presence or absence of 25 g/l dextran (molecular weight about 1500, Sigma-Aldrich, Cat. No. 31394) at 37 °C and orbital stirring at 125 rpm. A portion of the reaction mixture was taken after one hour, two hours and three hours and heated at 90 °C for five minutes to deactivate the GTF. Insoluble material was removed by centrifugation at 13,000xg for five minutes, followed by filtration through a 0.2 μm RC (regenerated cellulose) membrane. The resulting filtrate was analyzed by HPLC using two series of AMINEX HPX-87C columns at 85°C (Bio-Rad, Hercules CA) to quantify sucrose concentration. The sucrose concentration at each time point was plotted against the reaction time and the initial reaction velocity was determined from the slope of the linear plot. A unit of GTF activity was defined as the amount of enzyme required to consume one micromole of sucrose in one minute under the test conditions.

PREPARATION OF CRUDE EXTRACT OF ALPHA-(1,3)-GLUCANE HYDROLASE (MUTANASE)

[00181] A gene encoding the Penicillium marneffei ATCC® 18224® mutanase identified in GENBANK® as GI: 212533325 was synthesized by GenScript (Piscataway NJ). The nucleotide sequence (SEQ ID NO: 46) encoding the protein sequence (MUT3325; SEQ ID NO: 47) was subcloned into the pTrex3 plasmid at SacII and AscI restriction sites, a vector designed to express the gene of interest in Trichoderma reesei , under CBHI terminal and promoter control, with Aspergillus niger acetamidase for selection. The resulting plasmid was transformed into T. reesei by biolistic injection. The detailed method of biolistic transformation is described in published PCT International Patent Application WO 2009/126773 A1, which is incorporated herein by reference. A 1 cm 2 agar plug with spores from a stable clone, TRM05-3, was used to inoculate the production media (described below). The culture was grown in shake flasks for four to five days at 28 °C and 220 rpm. To harvest the secreted proteins, the cell mass was first removed by centrifugation at 4000 g for ten minutes and the supernatant was filtered through 0.2 µm sterile filters. Expression of mutanase MUT3325 (SEQ ID NO: 47) was confirmed by means of SDS-PAGE.

[00182] The means of production component is listed below. DEFINED BY LACTOSE RICH IN NREL-T TRACE ELEMENTS OF T. REESEI

[00183] Fermentation seed culture was prepared by inoculating 0.5 l of minimal medium into a two liter flask with membrane with 1.0 ml of frozen spore suspension of the MUT3325 expression line TRM05-3 ( the minimal medium was composed of 5 g/l of ammonium sulfate, 4.5 g/l of monobasic potassium phosphate, 1.0 g/l of magnesium sulfate heptahydrate, 14.4 g/l of citric acid anhydrous, 1 g/l calcium chloride dihydrate, 25 g/l glucose and trace elements including 0.4375 g/l citric acid, 0.5 g/l ferrous sulfate heptahydrate, 0 0.04 g/l of zinc sulfate heptahydrate, 0.008 g/l of cupric sulfate pentahydrate, 0.0035 g/l of manganese sulfate monohydrate and 0.002 g/l of boric acid; the pH was 5 ,5). The culture was grown at 32 °C and 170 rpm for 48 hours before being transferred to 8 L of production medium in a 14 liter fermenter. The production medium was composed of 75 g/l of sucrose, 4.5 g/l of monobasic potassium phosphate, 0.6 g/l of calcium chloride dehydrate, 1.0 g/l of magnesium, 7.0 g/l ammonium sulfate, 0.5 g/l anhydrous citric acid, 0.5 g/l ferrous sulfate heptahydrate, 0.04 g/l zinc sulfate heptahydrate , 0.00175 g/l of cupric sulfate pentahydrate, 0.0035 g/l of manganese sulfate monohydrate, 0.002 g/l of boric acid and 0.3 ml/l of FOAMBLAST 882.

[00184] The fermentation was conducted first with batch growth on glucose at 34 °C, 500 rpm for 24 hours. At the end of 24 hours, the temperature was reduced to 28 °C and the stirring speed increased to 1000 rpm. The fermenter was then fed with a mixture of glucose and sophorose (62% w/w) at a specific feed rate of 0.030 g glucose-sophorose solids/g biomass/h. At the end of the conduction, the biomass was removed by means of centrifugation and the supernatant containing the mutanase MUT3325 (SEQ ID N° 47) was concentrated about ten times by means of ultrafiltration using a Cut-Off ultrafiltration cartridge with a molecular weight of 10 kD (UFP-10-E-35; GE Healthcare, Little Chalfont, Buckinghamshire, Great Britain). The concentrated protein was stored at -80 °C.

DETERMINATION OF ALPHA-GLUCANE-HYDROLASE (MUTANASE) ACTIVITY

[00185] Insoluble mutane polymers needed to determine mutanase activity were prepared using secreted enzymes produced by Streptococcus sobrinus ATCC® 33478®. Specifically, a set of S. sobrinus ATCC® 33478® glycerol standard was streaked onto a BHI agar plate (Heart and Brain Infusion agar, Teknova, Hollister CA) and the plate was incubated at 37°C for two days. Some colonies were picked using a circuit to inoculate 2X 100 ml BHI liquid medium in the original Teknova medium bottle and the culture was incubated at 37°C and kept static for 24 hours. The resulting cells were removed by centrifugation and the resulting supernatant was filtered through a sterile 0.2 µm filter; 2X 101 ml of filtrate was collected. To the filtrate, 2X 11.2 ml of 200 g/l sucrose was added (20 g/l sucrose final). The reaction was incubated at 37°C without agitation for 67 hours. The resulting polysaccharide polymers were collected by centrifugation at 5000xg for ten minutes. The supernatant was carefully decanted. Insoluble polymers were washed four times with 40 ml of sterile water. The resulting mutan polymers were lyophilized for 48 hours. Mutant polymer (390 mg) was suspended in 39 ml of sterile water to make a 10 mg/ml suspension. The mutan suspension was homogenized by sonication (40% amplitude until large clumps disappeared, about ten minutes total). The homogenized suspension was split and stored at 4°C.

[00186] The mutanase test was initiated by incubating an appropriate amount of enzyme with 0.5 mg/ml mutane polymer (prepared as described above) in 25 mM KOAc buffer under pH 5.5 and at 37 °C . At various times, a portion of the reaction mixture was taken out and cooled with an equal volume of 100 mM glycine buffer (pH 10). Insoluble material in each cooled sample was removed by centrifugation at 14000xg for five minutes. The oligosaccharide and polysaccharide polymer reducing ends produced at each time point were quantified by p-hydroxybenzoic acid hydrazide solution (PAHBAH) test (Lever, M., Anal. Biochem. (1972), 47: 273-279 ) and the initial velocity was determined from the slope of the linear plot of the first three or four moments of the course. The PAHBAH test was performed by adding 10 μl of reaction sample supernatant to 100 μl of PAHBAH working solution and heated at 95 °C for five minutes. The working solution was prepared by mixing one part of reagent A (0.05 g/ml p-hydroxybenzoic acid hydrazide and 5% by volume of concentrated hydrochloric acid) and four parts of reagent B (0. 05 g/ml NaOH, 0.2 g/ml potassium sodium tartrate). The absorption at 410 nm was recorded and the concentration of the reducing ends was calculated by subtracting the appropriate background absorption and using a standard curve generated with various glucose concentrations as standards.

ANALYSIS OF REACTION PROFILES USING HPLC

[00187] Periodic samples of reactions were taken and analyzed using HPLC Agilent® 1260 equipped with a refractive index detector. An Aminex® HP-87C column (BioRad, Hercules CA) containing deionized water at a flow rate of 0.6 ml/min and 85 °C was used to quantify the level of sucrose, glucose, leucrose and fructose in gtf reactions. An Aminex® HP-42A column (BioRad) containing deionized water at a flow rate of 0.6 ml/min and 85 °C was used to quantify soluble oligosaccharide by-products (DP2-DP7) in gtf reactions.

[00188] HPLC Dionex® UltiMate® 3000 (Thermo Scientific) equipped with a refractive index detector was used for samples involving immobilized enzymes (Example 4). A Phenomenex® Rezex® calcium monosaccharide column containing deionized water at a flow rate of 0.3 ml/min and 85 °C was used to analyze the sugars.

BINDING ANALYSIS OF OLIGOSACCHARIDES BY MEANS OF NMR

[00189] NMR data were obtained on an Agilent DD2 spectrometer operating at 500 MHz for 1H using a pulsed field gradient (PFG) probe with 5 mm cryogenic triple resonance. Water suppression was achieved by carefully placing the observed transmitter frequency under resonance for the residual water signal in a presat experiment and then using the first slice of a NOESY experiment with a full phase cycle (multiple of 32) and mixing time of 10 ms. One-dimensional 1H spectra were obtained with a spectrum width of 6410 Hz, acquisition time of 5.1 s, 65536 data points, pre-saturation of 4 s and pulse at 90 degrees of 5.85 μs. The sample temperature was maintained at 25 °C. Samples were prepared by adding 50 μl to a 5 mm NMR tube together with 450 μl D2O and 60 μl D2O containing 12.4 mM internal reference DSS (4,4-dimethyl acid sodium salt -4-silapentane-1-sulfonic) with methyl resonance defined at 0 ppm. Chemical change assignments for different anomeric bonds are taken from: Goffin et al (2009, Bull Korean Chem. Soc. 30: 2535-2541. Peak assignments were 5.35 ppm for alpha(1,3), 5,1 bonds ppm for leucrosis and 4.95 for alpha bonds(1,6).Reducing ends (RE) were assigned as 5.2 for alpha RE and 4.65 for beta RE.

EXAMPLE 1 PRODUCTION OF SUGAR SYRUP THROUGH SUCROSE POLYMERIZATION

[00190] This example describes the general way in which a mixture of soluble sugars was produced by polymerizing sucrose with a gtf enzyme in a glucan synthesis reaction. Specifically, a glucan synthesis reaction filtrate was prepared, which was then concentrated to a syrup.

[00191] Sucrose (3000 g) was added to a clean 19 liter polyethylene bucket. Water (18.1 L) and Fermasure® (10 ml) were added to the bucket and the pH was adjusted to 7.0 by adding 5% by volume of NaOH and 5% by volume of H2SO4. 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 adding 0.3% by volume of crude gtf enzyme extract (SEQ ID NO: 3) prepared as described in the General Methods chapter. This extract contained approximately 2.9 mg/ml of protein. Agitation was provided to the reaction solution using an overhead mechanical motor equipped with a glass rod and PTFE paddle.

[00192] After 48 hours, HPLC analysis revealed that 96% of the sucrose had been consumed and the reaction was considered complete. The insoluble polyalpha-1,3-glucan product of the reaction was removed by filtration through a Buchner filter funnel using 325 mesh steel sieve and 40 micron filter paper. The (filtered) mother liquor was then concentrated using a rotary evaporator (40-50 °C bath temperature) to a total sugar concentration of about 320 g/l of sugars. The composition of the concentrated filtrate is given in Table 2. TABLE 2 COMPOSITION OF GLUCANE SYNTHESIS REACTION CONCENTRATE FILTERATE

[00193] Table 2 indicates that the concentrated filtrate from the glucan synthesis reaction contains sucrose, fructose, glucose, leucrose and DP2-DP7 oligosaccharides.

EXAMPLE 2 EFFECT OF ENZYMES ON THE HYDROLYSIS OF SUGARS IN GLUCANE SYNTHESIS REACTION FITTRATE

[00194] This example measures the activity of several enzymes glucoamylase (EC 3.2.1.3), transglucosidase (EC 2.4.1.24), betaglucosidase (EC 3.2.1.21), alpha-amylase (EC 3.2.1.1) and glycosidase (EC 3.2. 1) for the purpose of reducing the concentration of leucrose and/or oligosaccharide by-products in a concentrated filtrate from a glucan synthesis reaction. Certain enzymes such as DIAZYME RDF ULTRA, transglycosidase (EC 2.4.1.24) and glucoamylase (EC 3.2.1.3), which are all alpha-glucosidase, have been found to be particularly effective in reducing the amount of these by-products, resulting in a corresponding increase in monosaccharides (glucose and fructose ) in the treated filtrate.

[00195] A glucan synthesis reaction filtrate was first prepared and concentrated into a syrup according to the procedure described in Example 1. The composition of this concentrated filtrate is given in Table 3. NMR analysis revealed that the ratio between alpha(1,3) and alpha(1,6) bonds present in the syrup was 78:22. TABLE 3 COMPOSITION OF GLUCANE SYNTHESIS REACTION CONCENTRATE FILTRATE

[00196] The syrup from Table 3 was used to test the hydrolytic activity of various enzymes against leucrose by-products and oligosaccharides from the glucan synthesis reaction. It was not obvious at the beginning of these experiments which enzyme could be used to hydrolyze these two by-products, considering that leucrose contains a common bond [alpha(1,5)-glycosyl fructose] and that oligosaccharides mainly comprise alpha(1,3) and alpha(1,6) glycosylglucose. Enzymes with various activities were selected for this analysis (Table 4). TABLE 4 ENZYMES EVALUATED FOR DETERMINING LEUCROSE AND OLIGOSACCHARIDE HYDROLYSIS to DuPont Industrial Biosciences

[00197] The treatment conditions of the syrup from Table 3 with each of the above enzymes are given in Table 5 (enzyme load, time, temperature, pH and sugar concentration). The syrup was diluted with water to reach the sugar concentration used in each hydrolysis reaction. Table 5 further provides the percent hydrolysis of leucrose and DP3+ oligosaccharides (at least DP3-DP7) by each enzyme. Percent DP3+ hydrolysis was calculated as (1 - (wt% DP3+ oligosaccharides in final syrup) / (wt% DP3+ oligosaccharides in starting syrup)). Similarly, the percent leucrose hydrolysis was calculated as (1 - (wt% leucrose in final syrup) / (wt% leucrose in starting syrup)). TABLE 5 HYDROLYSIS OF LEUCROSE AND OLIGOSACCHARIDES IN CONCENTRATED FILTRATE BY VARIOUS ENZYMES a Sugar concentration (total concentration of sucrose, glucose, fructose, leucrose and oligosaccharides) measured by means of HPLC; reported values are rounded to the nearest 10 g/l. b DP3+ contains DP3-DP7, but may also contain larger soluble oligosaccharides that have a high ratio of alpha-1,6 linkages to alpha-1,3 linkages when produced using certain gtf enzymes.

[00198] Table 5 indicates that 1,4-alpha-glucosidase and 1,6-alpha-glucosidase exhibited some (Example 2.1) or very little (Example 2.2) leucrose hydrolysis, but released some glucose from the oligosaccharides. The use of alpha-amylase (Example 2.3 and Example 2.4) exhibited very little activity against the compounds of interest. Similarly, the use of pullulanase (Example 2.5) exhibited very little activity.

[00199] Cellulases (Examples 2.14 and 2.15) were largely ineffective in hydrolyzing leucrose, but hydrolyzed some of the oligosaccharides.

[00200] Although the oligosaccharides did not contain beta linkages, surprisingly, beta-glucosidase enzymes also exhibited a hydrolytic conversion range from very low (ACCELERASE BG, Example 2.9) to very high (NOVO 188, Examples 2.10 and 2.11). The relative effectiveness of these enzymes varied quite dramatically. In some cases, the amount of oligosaccharide that was hydrolyzed greatly exceeded (Example 2.11) or was close (Example 2.12) to the percentage of leucrose that was hydrolyzed. In other cases, leucrose was highly hydrolyzed by beta-glucosidase, while oligosaccharides were moderately hydrolyzed (Example 2.13). The high disparity between the results observed with betaglucosidase suggests that the presence of other enzymes in the tested betaglucosidase formulations, such as glucoamylase or another type of alphaglucosidase, could be responsible for the observed activity.

[00201] On the other hand, the results of Table 5 indicate that transglycosidase (TG L-2000, Example 2.6) exhibited very high activity in the hydrolysis of oligosaccharides and leucrosis. Hydrolysis of leucrose by transglycosidase appeared quantitative under certain circumstances and over 95% of the DP3+ material was hydrolyzed to glucose and DP2 at high enzyme loads (Example 2.7). Use of a purified version of transglycosidase revealed similar activity (Example 2.8), which indicates that the observed hydrolysis is due to the transglycosidase enzyme and not background activity.

[00202] Glycoamylases (Examples 2.16-2.18) exhibited a range of activity against leucrosis and oligosaccharides. Only one tested glucoamylase (Example 2.18) generated less than 30% hydrolysis of leucrose and oligosaccharides.

[00203] The results in Table 5 indicate that alpha-glucosidases such as DIAZYME RDF ULTRA, glucoamylase and transglycosidase can hydrolyze the leucrose by-product present in a glucan reaction filtrate. The ability of alpha-glucosidases to hydrolyze leucrose indicates that these enzymes can hydrolyze alpha-1,5-glycosylfructose linkages. Although this activity was shown above using leucrose as a substrate, it is believed that this activity could also be extended to oligosaccharides comprising alpha-1,5 glycosylfructose linkages.

[00204] The results in Table 5 indicate that alpha-glucosidases such as glucoamylase and transglucosidase can hydrolyze oligosaccharide by-products present in a glucan reaction filtrate. Since these oligosaccharides are primarily composed of glucose monomer units linked by alpha-1,3 and/or alpha-1,6 linkages (Example 3), the data in Table 5 indicate that alpha-glucosidase enzymes can hydrolyze alpha-1,3 glycosylglucose and/or alpha-1,6 glycosylglucose linkages.

[00205] As alpha-glucosidase enzymes have generally been effective in hydrolyzing the leucrose and/or oligosaccharide by-products of a glucan synthesis reaction, these enzymes can be used alone or in combination to reduce the processing time required to generate high sugar syrup purity of a glucan reaction filtrate that contains higher amount of monosaccharides and reduced amount of sugar by-products. An example of an effective enzyme combination would be a transglycosidase such as TG L-2000, for hydrolysis of leucrose, and a glucoamylase enzyme (eg, GC 321) which efficiently hydrolyzes oligosaccharide by-products.

[00206] Alpha-glucosidase enzymes can therefore individually hydrolyze (i) alpha-1,5 glycosylfructose linkages and (ii) alpha-1,3 and alpha-1,6 glycosylglucose linkages in certain saccharides.

EXAMPLE 3 COMPARISON OF BINDING DISTRIBUTIONS OF GLUCANE REACTION FILTERATE COMPONENTS BEFORE AND AFTER ENZYME HYDROLYSIS

[00207] This example measures the hydrolytic activity of transglucosidase (EC 2.4.1.24) and betaglucosidase (EC 3.2.1.21) enzymes against leucrose by-products and oligosaccharides present in a concentrated filtrate from a glucan synthesis reaction. It was concluded that transglycosidase reduces the amount of these by-products, which results in a corresponding increase in monosaccharides (glucose and fructose) in the treated filtrate.

[00208] The oligosaccharide by-products present in the filtrate of the above glucan synthesis reaction comprise more than 90% of glucose-glucose linkages, as determined by means of NMR (General Methods). Of the glucose-glucose linkages, about 78% represent alpha-1,3 linkages and about 22% represent alpha-1,6 linkages.

[00209] NMR was used to determine the binding profile of material generated in Example 2.11 above after hydrolysis. As shown in Figure 1, the peak corresponding to alpha-1,3 bindings was reduced by 86%, the peak corresponding to alpha-1,6 bindings was reduced by only 2.3%, and the peak corresponding to leucrosis peaks was reduced by 21%. Although sucrose was almost quantitatively hydrolyzed by this enzyme, Novo 188 is apparently not capable of hydrolyzing alpha-1,6 bonds.

[00210] NMR was similarly used to determine the binding profile of material generated using TG L-2000 transglycosidase (SEQ ID NO: 1) (Figure 2). 210 µl concentrated filtrate of material from Table 3, 300 µl D2O and 90 µl D2O containing 12.4 mM DSS as an internal reference were mixed in an NMR tube to give a total sugar concentration of 300 g/l and heated to 60 °C. Zero time spectrum (starting material in Figure 2) was obtained after thermal equilibration at 60°C and then 0.5 vol% enzyme was added. The sample was again equilibrated in the probe at 60 °C and wedged and measurements were taken within minutes of analysis. After ten hours of treatment with TG L-2000 enzyme (material treated in Figure 2), peaks corresponding to alpha-1,3 bindings were reduced by 41%, peaks corresponding to alpha-1,6 bindings were reduced by 36% and the peak corresponding to leucrosis was reduced by more than 95% (Figure 2). An increase in peaks at the alpha reducing end and beta reducing end was observed, which corresponds to an increase in fructose and glucose (Figure 2).

[00211] These results demonstrate that transglycosidase can convert oligosaccharides containing alpha-1,3 and alpha-1,6 linkages to glucose and can convert leucrose to fructose and glucose. Transglycosidase can therefore hydrolyze (i) alpha-1,5 glycosylfructose linkages and (ii) alpha-1,3 and alpha-1,6 glycosylglucose linkages in certain saccharides.

EXAMPLE 4 HYDROLYSIS OF LEUCROSE AND OLIGOSACCHARIDES IN GLUCANE REACTION FILTERATE USING IMMOBILIZED ENZYMES

[00212] This Example describes the use of immobilized glucoamylase (EC 3.2.1.3) and transglycosidase (EC 2.4.1.24) to hydrolyse leucrose and other oligosaccharides present in filtrate obtained from a glucan synthesis reaction. Specifically, the effect of immobilized transglycosity TG L-2000 (SEQ ID NO: 1, obtained from Genencor/DuPont Industrial Biosciences) and immobilized glucoamylase GC-147 (obtained from Genencor/DuPont Industrial Biosciences) on the hydrolysis of leucrose and oligosaccharides DP2, DP3 and HS (higher sugars, DP4+) in a glucan synthesis reaction filtrate.

[00213] The immobilization of the enzymes glucoamylase and transglucosidase was conducted according to the method described in US Patent No. 5,541,097, which is hereby incorporated by reference.

[00214] In a typical process of immobilization of glucoamylase or transglucosidase, two batches of about 8.0 g/batch of porous granular diatomaceous earth (EP Minerals, Reno NV) were hydrated with distilled water and then transferred to a reactor of glass column 1.5 cm in diameter and 30 cm high. Water was pumped upstream at about 6-7 ml/min to remove fines from all three columns. Generally, within one hour, the water effluent was free of fines. Water was drained from the column to the top of the granular diatomaceous earth beds and replaced with 0.1% w/v aqueous polyethyleneimine solution (PEI, EPOMIN P-1050). 3500 ml of the PEI solution was then pumped upstream and the effluent was recycled through the beds for two hours. The granular diatomaceous earth beds were then washed upstream with distilled water for two hours to remove free PEI at room temperature. In this way, PEI vehicles and granular diatomaceous earth were obtained.

[00215] Meanwhile, 3.5 ml of GC-147 glucoamylase that has activity defined in Table 4 were added to 315 ml of 0.02 M acetate buffer (pH 4.5). 1.575 g of glutaraldehyde at 50% w/w (Protectol® GA-50) were then slowly added to the aqueous solution of glucoamylase with gentle mixing and the glutaraldehyde was maintained in reaction with the aqueous solution of glucoamylase for four hours at a temperature of 20 -25 °C with gentle agitation, which resulted in the formation of enzyme and treated glutaraldehyde adduct containing treated glucoamylase. Separately, these steps were repeated using the TG L-2000 transglycosidase that has the activity defined in Table 4 instead of the glucoamylase, so as to result in the formation of an enzyme and treated glutaraldehyde adduct that contains treated transglycosidase.

[00216] Each of the treated enzyme and glutaraldehyde adducts was then circulated for four hours (20-25 °C) in its own column prepared as above, which contains PEI carrier and granular diatomaceous earth. Excess treated adduct was then washed out of the vehicles with water. Columns with immobilized glucoamylase or transglycosidase were prepared in this way.

[00217] Glucan filtrate having the composition defined in Table 3 was diluted to 180 g/l, adjusted to pH 4.5 and passed through a column containing an immobilized enzyme. Column temperature was controlled at 60 °C. After 16 hours of column equilibration, samples were taken periodically at different flow rates. Sugar compositions of hydrolysis reaction products were determined by means of HPLC (Table 6). Each time the flow rate setting was changed, the column was allowed to re-equilibrate by at least one to two bed volumes prior to sampling. The degree of hydrolysis of leucrose and oligosaccharides was calculated using the manner described in Example 2. Three column configurations were tested: (1) immobilized glucoamylase; (2) immobilized transglycosidase; and (3) immobilized glucoamylase followed by immobilized transglycosidase. TABLE 6 APPLICATION OF IMMOBILIZED TRANSGLICOSACCHARIDE AND GLYCOAMYLASE ENZYMES TO HYDROLYZE OLIGOSACCHARIDES AND LEUCROSE:

[00218] Table 6 indicates that as the mean contact time (defined as the nominal column volume divided by the mean flow velocity) increased, the degree of hydrolysis of leucrose and oligosaccharides generally increased. The use of immobilized transglycosidase to hydrolyse leucrose was particularly effective, as no significant difference was observed, even when using the highest flow rate that was tested. While each column individually exhibited reasonable conversion, the combination of glucoamylase and transglycosidase generated higher hydrolysis of oligosaccharides.

[00219] The use of immobilized glucoamylase or transglycosidase, or both types of immobilized enzymes, therefore represents an effective method of hydrolyzing oligosaccharides that contain alpha-1,3 and alpha-1,6 glycosylglucose linkages, as well as leucrose. These results are consistent with those of Example 2. Immobilization of other alpha-glucosidase enzymes should give similar results.

EXAMPLE 5 FRUCTOSE ENRICHMENT FROM A GLUCAN REACTION FILTERATE USING CHROMATOGRAPHY

[00220] This example describes how fructose in a glucan reaction filtrate can be further enriched through chromatography.

[00221] Generally, when separating sugar molecules by chromatography, components reverse elute in molecular size so that larger molecules elute first. With respect to a filtrate from a glucan synthesis reaction, therefore, oligosaccharides elute first, followed by disaccharides and then monosaccharides. Separations using a sodium cation resin did not separate fructose and glucose well and all of leucrose, sucrose and DP2 coeluted. Preferred is the use of ion exchange resins in which the cation is calcium to separate glucose and fructose.

[00222] A glucan synthesis reaction filtrate was prepared first and concentrated into a syrup according to the procedure described in Example 1. The composition of this concentrated filtrate is given in Table 7. TABLE 7 COMPOSITION OF REACTION CONCENTRATE FILTERATE OF GLUCAN SYNTHESIS

[00223] The syrup from Table 7 was filtered and diluted to 25 g dry solids/100 g water solution with substituted ions and fed to a column containing a strong acid ion exchange resin crosslinked in the form of calcium. The physical parameters of this column appear in Table 8. Diluted syrup (15.8 l) was fed to the column, which was maintained at 65 °C, after which the column was eluted using water at a flow rate of 30 l/h. TABLE 8 PHYSICAL PARAMETERS OF THE SPINE

[00224] In this separation, leucrose remained on the column longer than sucrose, perhaps due to the formation of a leucrose complex with the calcium cation and, in fact, co-eluted with glucose. Two fractions containing fructose were isolated. Fraction 5.1 was eluted between 47 and 120 minutes and fraction 5.2 was eluted between 120 and 172 minutes. From fed fructose to chromatographic separation, 95.7% of the fructose was isolated in greater than 90% purity. The distribution of products in each fraction (5.1 and 5.2) as measured by HPLC appears in Table 9. TABLE 9 PRODUCT DISTRIBUTION OF CHROMATOGRAPHIC FRACTIONS CONTAINING SIGNIFICANT QUANTITIES OF FRUCTOSE

[00225] As the feed composition for this separation comprised 36.0% fructose, in all, 34.5% of the total flow was recovered as fructose syrup with >90% by weight DS fructose. If sucrose in the feed is disregarded, 40.7% of the sugars were recovered in the form of fructose syrup with >90% by weight DS fructose.

[00226] Fructose in a glucan reaction filtrate can therefore be further enriched by means of chromatography. Example 6 below demonstrates that this process can be improved using transglycosidase hydrolyzed glucan filtrate.

EXAMPLE 6 FRUCTOSE ENRICHMENT FROM A HYDROLYZED GLUCAN REACTION FILTERATE THROUGH CHROMATOGRAPHY

[00227] This example demonstrates that the isolation of fructose from a glucan filtrate in which oligosaccharides and leucrose have been hydrolyzed results in increased production of high purity fructose syrup compared to the isolation of fructose from an unhydrolyzed glucan filtrate .

[00228] A syrup was prepared by concentrating (vacuum at 50 °C) filtrate of glucan that had been treated with 1% vol of transglycosidase TG L-2000 (SEQ ID No. 1) for 24 hours at 60 ° C and pH 4.5. Some formation of oligosaccharides was observed during the concentration process, which was expected as transglycosidase enzymes are known to create oligosaccharides at high concentrations of monosaccharides. The syrup had the final product distribution described in Table A. TABLE THE COMPOSITION OF CONCENTRATED GLUCANE FILTERATE WHICH WAS HYDROLYZED BEFORE CONCENTRATION

[00229] The syrup described in Table A was filtered and diluted to 25.4 g DS/100 g water solution with substituted ions and fed to a column containing a strong acidic ion exchange resin crosslinked in the form of calcium. The physical parameters of the column appear in Table B. Diluted syrup (169 g) was then fed to the column, which was maintained at 65°C, after which the column was eluted using water at a flow rate of 50 ml/min. TABLE B PHYSICAL PARAMETERS OF THE SPINE

[00230] Two fractions containing fructose were isolated. Fraction 6.1 eluted between 73 and 103 minutes and fraction 6.2 eluted between 103 and 120 minutes. From the fructose fed to the chromatographic separation, 93.0% of the fructose fed to the column was isolated in fraction 6.2 with a purity of more than 90%. The distribution of products in each fraction (6.1 and 6.2) as measured by HPLC appears in Table C. TABLE C PRODUCT DISTRIBUTION OF CHROMATOGRAPHIC FRUCTIONS CONTAINING FRUCTOSE FROM A HYDROLYZED GLUCANE FILTRATE

[00231] The reduced separation efficiency in this example compared to Example 5 can be attributed to differences in column scale and the highest glucose fraction in the sample. Even so, chromatographic purification of this material resulted in increased production of high purity fructose syrup compared to that achieved in Example 5, as the syrup was chromatographically prepared from a glucan filtrate that had not been hydrolyzed by transglycosidase. As the feed composition for this separation comprised 47% fructose (Table A), 43.7% of the total flow was recovered as fructose syrup with >90% by weight DS fructose. This 43.7% recovery is significantly better than the 34.5% recovery of Example 5.

[00232] Therefore, isolation of fructose from a glucan filtrate that has been hydrolyzed with transglycosidase results in increased fructose yield compared to the isolation of fructose from a non-hydrolyzed glucan filtrate.

EXAMPLE 7 ETHANOL PRODUCTION THROUGH FILTRATE FERMENTATION OF A GLUCANE SYNTHESIS REACTION

[00233] This example describes yeast fermentation of glucan filtrate in ethanol.

[00234] Yeast cream (S. cerevisiae) (Tonon mill, Brazil) was washed by suspending the cream in tap water (2.4 l, optical density 65 to 600 nm) and then centrifuging the cream yeast for five minutes using a LEGEND XTR centrifuge (Thermo Scientific) at 4500 g. After decanting the supernatant, the yeast cells were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 2 by adding 5% by weight sulfuric acid. Optical density was measured using a GENESYS 20 4001 spectrophotometer (Thermo Scientific) and adjusted to 100 to 600 nm by adding tap water. Adjusted cream yeast (1.5 L) was added to a 7.5 L BIOFLO310 fermenter vessel (New Brunswick). The fermenter was set to maintain temperature at 30 °C and agitation at 100 rpm. Although pH was measured during fermentation, it was not controlled by adding acidic or basic solutions.

[00235] A feed solution containing yeast extract (10 g/l), peptone (20 g/l) and 200 g/l of sugars from a glucan filtrate was prepared and sterilized using the PHOENIX AV-250 PLUS autoclave at 121 °C for fifteen minutes. The feed solution was cooled to 25 °C (room temperature) before starting the fermentation. The sterilized feed solution (3.5 L) was added to the fermenter for about five hours at a rate of 684 ml/h and the fermentation was kept running for 22 hours.

[00236] Periodic samples were taken during fermentation and analyzed to determine optical density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL-3 refractometer (Atago) and sugar and ethanol concentrations using HPLC (General Methods). The results are summarized in Table 10. TABLE 10 FERMENTATION PROFILES OVER TIME AND FEEDING FOR THE FIRST ETHANOL FERMENTATION

[00237] The concentrations (g/l) of sugar and ethanol (EtOH) compounds in the feed and at different times of fermentation (0-22 hours) are related.

[00238] At the end of fermentation, the yeast cells were separated by centrifugation using a LEGEND XTR centrifuge at 4500 g for five minutes. After decanting the supernatant, the yeast was resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 2 by adding 5% by weight sulfuric acid. Optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 to 600 nm by adding tap water. Two additional fermentation cycles, each using fresh feed, were performed using yeast cells recycled from the previous fermentation following the same conditions described above. Fermentation results obtained using recycled yeast for the first and second time are provided in Tables 11 and 12, respectively. TABLE 11 PROFILES OF FERMENTATION OVER TIME AND FEEDING USING THE FIRST RECYCLING OF YEAST CELLS

[00239] The concentrations (g/l) of sugar and ethanol (EtOH) compounds in the feed and at different times of fermentation (0-21 hours) are related. TABLE 12 FERMENTATION PROFILES OVER TIME AND FEEDING USING THE SECOND YEAST CELL RECYCLING

[00240] The concentrations (g/l) of sugar and ethanol (EtOH) compounds in the feed and at different times of fermentation (0-21 hours) are related.

[00241] Very little leucrose was consumed in the first fermentation, although the yeast cells began to acclimatize and consume leucrose in the second fermentation. Ethanol fermentation titers increased from 33 g/l (Table 10, 22 hours) to 54 g/l (Table 12, 21 hours) after three cycles of fermentation with recycled yeast, although significant amounts of leucrose were present in the medium, even after the last cycle.

[00242] Therefore, glucan filtrate can be used in a fermentation process to produce ethanol.

EXAMPLE 8 ETHANOL PRODUCTION THROUGH THE FERMENTATION OF HYDROLYZED GLUCANE FILTERATE

[00243] This example demonstrates that fermentation of glucan filtrate in which the leucrose and oligosaccharide by-product components have been saccharified previously results in increased ethanol production.

[00244] Fermentations were carried out following the procedure described in Example 7, but using a glucan filtrate that was previously treated with transglycosidase (TG L-2000, SEQ ID No. 1). Hydrolyzed glucan filtrate was prepared as follows. Glucan filtrate was adjusted to 300 g of sugars/l and then the pH was adjusted to 4.0 using 1.0 M sodium hydroxide and 5 wt% sulfuric acid. The final volume of this preparation was 6.75 l. The filtrate solution was then sterilized using a PHOENIX AV-250 PLUS autoclave at 121 °C for fifteen minutes and then the temperature was adjusted to 60 °C. TG L-2000 enzyme extract as described in Table 4 (135 ml) was mixed with the sterilized filtrate and the solution was incubated in an incubator shaker (IKA KS4000) at 60 °C and 100 rpm for 72 hours. Hydrolyzed glucan filtrate was prepared in this way.

[00245] Yeast cream (S. cerevisiae) (Bom Retiro mill, Brazil) was washed by suspending the cream in tap water (2.4 l, optical density 65 to 600 nm) and then centrifugation of cream yeast for five minutes using a LEGEND XTR centrifuge at 4500 g. After decanting the supernatant, the yeast was resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 4.5 by adding 5% by weight of sulfuric acid and the optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 to 600 nm by adding water from the tap. Adjusted cream yeast (1.5 L) was added to a 7.5 L BIOFLO310 fermenter vessel. The fermenter was set to maintain temperature at 30 °C, agitation at 100 rpm and pH 4.5 using 4 M aqueous ammonium hydroxide or 5 wt% aqueous sulfuric acid.

[00246] A feed solution containing yeast extract (10 g/l), peptone (20 g/l) and 200 g/l of sugars from the hydrolyzed filtrate was prepared and sterilized using a PHOENIX AV-250 Plus autoclave at 121 ° C for fifteen minutes. The feed solution was cooled to 25 °C (room temperature) before starting the fermentation. The sterilized feed solution (3.5 L) was added to the fermenter for about five hours at a rate of 684 ml/h and the fermentation was kept running for 22 hours.

[00247] Periodic samples were taken during fermentation and analyzed to determine optical density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL-3 refractometer, and sugar and ethanol concentrations using HPLC (General Methods). The results are summarized in Table 13. TABLE 13 FERMENTATION PROFILES OVER TIME AND FEEDING FOR THE FIRST ETHANOL FERMENTATION USING HYDROLYZED GLUCANE FILTRATE

[00248] The concentrations (g/l) of sugar and ethanol (EtOH) compounds in the feed and at different times of fermentation (0-22 hours) are related.

[00249] At the end of fermentation, the yeast cells were separated by centrifugation using a LEGEND XTR centrifuge at 4500 g for five minutes. After decanting the supernatant, the yeast cells were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 2 by adding 5% by weight sulfuric acid. Optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 to 600 nm by adding tap water. Two additional fermentation cycles, each using fresh feed, were performed using yeast cells recycled from the previous fermentation following the same conditions described above. Fermentation results obtained using yeast cells recycled for the first and second time are provided in Tables 14 and 15, respectively. TABLE 14 PROFILES OF FERMENTATION OVER TIME AND FEEDING USING THE FIRST RECYCLING OF YEAST CELLS WITH HYDROLYZED GLUCAN FILTERATE

[00250] The concentrations (g/l) of sugar and ethanol (EtOH) compounds in the feed and at different times of fermentation (0-21 hours) are related. TABLE 15 PROFILES OF FERMENTATION OVER TIME AND FEEDING USING THE SECOND RECYCLING OF YEAST CELLS WITH HYDROLYZED GLUCAN FILTRATE

[00251] The concentrations (g/l) of sugar and ethanol (EtOH) compounds in the feed and at different times of fermentation (0-21 hours) are related.

[00252] All fermentations were essentially complete within about six hours of starting fermentation and resulted in ethanol titers of 57-60.0 g/l. Comparison of these fermentations with those of Example 7 demonstrates that hydrolysis of a glucan filtrate prior to its submission to fermentation results in higher and faster ethanol yields than those obtained with fermentations using unhydrolyzed glucan filtrate.

[00253] Fermentation of glucan filtrate in which the by-product components of leucrose and oligosaccharide have been saccharified therefore results in increased ethanol production at higher rates. Such saccharification can be carried out using, for example, transglycosidase.

EXAMPLE 9 SIMULTANEOUS SACHARIFYING AND FERMENTATION OF A GLUCANE FILTER SOLUTION

[00254] This example describes that simultaneous saccharification and fermentation of feed containing glucan filtrate can result in improved fermentation properties.

[00255] Yeast cream (S. cerevisiae) (Bom Retiro mill, Brazil) was washed by suspending the cream in tap water (2.4 l, optical density 65 to 600 nm) and then centrifugation of cream yeast for five minutes using a LEGEND XTR centrifuge at 4500 g. After decanting the supernatant, the yeast cells were resuspended and concentrated by centrifugation two additional times. After the third wash, the pH was adjusted to 4.5 by adding 5% by weight of sulfuric acid and the optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 to 600 nm by adding water from the tap. Adjusted cream yeast (1.5 L) was added to a 7.5 L BIOFLO310 fermenter vessel. The fermenter was set to maintain temperature at 30 °C, agitation at 100 rpm and pH 4.5 using 4 M aqueous ammonium hydroxide or 5 wt% aqueous sulfuric acid.

[00256] A feed solution containing yeast extract (10 g/l), peptone (20 g/l) and 200 g/l of sugars from the glucan filtrate was prepared and sterilized using a PHOENIX AV-250 PLUS autoclave at 121 °C for fifteen minutes. The feed solution was cooled to 25 °C (room temperature) before starting the fermentation. TG L-2000 transglucosidase enzyme extract as described in Table 4 (1% v/v) was added to the sterilized feed solution just prior to adding the solution to the fermenter. The feed solution (3.5 L) containing TG L-2000 enzyme was added to the fermenter for about five hours at 684 ml/hr and the fermentation was kept running for 48 hours.

[00257] Periodic samples were taken during fermentation and analyzed to determine optical density using a GENESYS 20 4001 spectrophotometer, Brix using a PAL-3 refractometer (Atago) and sugar and ethanol concentrations using HPLC (General Methods). These results are summarized in Table 16. TABLE 16 FERMENTATION PROFILES OVER TIME AND FEED FOR SIMULTANEOUS SACHARIFICATION AND ETHANOL FERMENTATION OF GLUCANE FILTERATE

[00258] The concentrations (g/l) of sugar and ethanol (EtOH) compounds in the feed and at different times of fermentation (0-48 hours) are related.

[00259] Fermentation nominally ended in six hours, similar to fermentations in which the filtrate was hydrolyzed before the fermentation step (Example 8), and provided slightly higher ethanol titer (62 g/l) compared to use of unhydrolyzed filtrate (Example 7). Furthermore, nearly all of the leucrose was consumed within six hours (compare Table 16 with Tables 13-15). In addition to the addition of a saccharifying enzyme, such as TG L-2000, to a feed containing glucan filtrate shortly before fermentation, similar results should be obtained if the saccharifying enzyme is added directly to the fermentation.

[00260] Simultaneous saccharification and fermentation of a feed containing glucan filtrate may therefore result in improved fermentation properties such as increased (i) consumption of glucan filtrate components (e.g. leucrose) and (ii) ethanol production and production speed.

EXAMPLE 10 PREPARATION OF VARIOUS ALPHAGLYCOSIDASES

[00261] This example describes the preparation of various alpha-glucosidases in addition to the alpha-glucosidases (transglycosidase, glucoamylase, DIAZYME RDF ULTRA) used in some of the Examples above. These additional alpha-glucosidases were tested to determine hydrolytic activity against oligosaccharides comprising alpha-1,5 glycosylfructose or alpha-1,3 and/or alpha-1,6 glycosylglucose linkages in Examples 11, 12, 15 and 16 provided below.

DISCOVERY OF ALPHAGLICOSIDASIS FROM ASPERGILLUS CLAVATUS (ACGLUI)

[00262] A strain of Aspergillus clavatus was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Aspergillus clavatus encodes an alpha-glucosidase and the sequence of this gene, named "Aclglu1", is provided in SEQ ID NO: 4. The corresponding protein encoded by SEQ ID NO: 4 is provided in SEQ ID NO: 5. Aclglu1 belongs to the family of glycosyl hydrolase 31 based on PFAM search (pfam.sanger.ac.uk). At the N-terminus, the protein (SEQ ID NO: 5) contains a signal peptide nineteen amino acids long, as predicted by SignalP version 4.0 (Nordahl Petersen et al, 2011, Nature Methods, 8: 785-786). The presence of a signal sequence suggests that Aclglu1 is a secreted enzyme. The amino acid sequence of the predicted mature form of Aclglu1 is described as SEQ ID NO:6.

ASPERGILLUS CLAVATUS ACLGLUI ALPHAGLICOSIDASE EXPRESSION

[00263] A synthetic Aclglu1 gene was cloned into pTrex3gM expression vector (described in published US Patent Application No. 2011/0136197, incorporated herein by reference) and the resulting plasmid was named pJG294. The sequence of the Aclglu1 gene was confirmed by DNA sequencing.

[00264] Plasmid pJG294 was transformed into a quad-deleted Trichoderma reesei strain (described in WO 05/001036) using a biolistic method (Te'o VS et al, J. Microbiol. Methods, 51: 393-9, 2002 ). The protein, predicted to comprise SEQ ID NO:6, was secreted into the extracellular medium and filtered culture medium was used to perform SDS-PAGE and alpha-glucosidase activity tests to confirm expression of the enzyme.

DISCOVERY OF ALPHAGLICOSIDASIS OF NEOSARTORYA FISCHERI NFIGLUI

[00265] A strain of Neosartorya fischeri was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Neosartorya fischeri encodes an alpha-glucosidase and the sequence of this gene, named “Nfiglu1”, is provided in SEQ ID NO: 7. The corresponding protein encoded by SEQ ID NO: 7 is provided in SEQ ID NO: 8. Nfligu1 belongs to the glycosyl hydrolase 31 family based on PFAM research (pfam.sanger.ac.uk). At the N-terminus, the protein (SEQ ID NO:8) contains a signal peptide nineteen amino acids long, as predicted by SignalP version 4.0 (Nordahl Petersen et al, 2011, Nature Methods, 8: 785-786). The presence of a signal sequence suggests that Nfiglu1 is a secreted enzyme. The amino acid sequence of the predicted mature form of Nfiglu1 is described as SEQ ID NO:9.

EXPRESSION OF ALPHAGLICOSIDASIS OF NEOSARTORYA FISCHERI NFIGLUI

[00266] A synthetic Nfiglu1 gene was cloned into pTrex3gM expression vector (described in published US Patent Application No. 2011/0136197) and the resulting plasmid was named pJG295. The sequence of the Nfiglu1 gene was confirmed by DNA sequencing.

[00267] Plasmid pJG295 was transformed into a quad-deleted Trichoderma reesei strain (described in WO 05/001036) using a biolistic method (Te'o VS et al, J. Microbiol. Methods, 51: 393-9, 2002 ). The protein, predicted to comprise SEQ ID NO:9, was secreted into the extracellular medium and filtered culture medium was used to perform SDS-PAGE and alpha-glucosidase activity tests to confirm expression of the enzyme.

DISCOVERY OF ALPHAGLYCOSIDASIS FROM NEUROSPORA CRASSA NCRGLUI

[00268] A strain of Neurospora crassa was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Neurospora crassa encodes an alpha-glucosidase and the sequence of this gene, named "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 31 family based on PFAM research (pfam.sanger.ac.uk). At the N-terminus, the protein (SEQ ID NO: 11) contains a signal peptide of 22 amino acids in length, as predicted by SignalP version 4.0 (Nordahl Petersen et al, 2011, Nature Methods, 8: 785-786). The presence of a signal sequence suggests that Ncrglu1 is a secreted enzyme. The amino acid sequence of the predicted mature form of Ncrglu1 is described as SEQ ID NO: 12.

EXPRESSION OF NEUROSPORA CRASSA NCRGLUI ALPHAGLICOSIDASIS

[00269] A synthetic Ncrglu1 gene was cloned into pTrex3gM expression vector (described in published US Patent Application No. 2011/0136197) and the resulting plasmid was named pJG296. The sequence of the Ncrglu1 gene was confirmed by DNA sequencing.

[00270] Plasmid pJG296 was transformed into a quad-deleted Trichoderma reesei strain (described in WO 05/001036) using a biolistic method (Te'o VS et al, J. Microbiol. Methods, 51: 393399, 2002). The protein, predicted to comprise SEQ ID NO:12, was secreted into the extracellular medium and filtered culture medium was used to perform SDS-PAGE and alpha-glucosidase activity tests to confirm expression of the enzyme.

DISCOVERY OF RASAMSONIA COMPOSTICOLA ALPHAGLICOSIDASIS TAUSEC098

[00271] A strain of Rasamsonia composticola was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Rasamsonia composticola encodes an alpha-glucosidase and the sequence of this gene, named "TauSec098", is provided in SEQ ID NO: 13. The corresponding protein encoded by SEQ ID NO: 13 is provided in SEQ ID NO: 14. TauSec098 belongs to the glycosyl hydrolase 31 family and contains an N-terminal CBM 20 domain based on PFAM research (pfam.sanger.ac.uk). At the N-terminus, the protein (SEQ ID NO: 14) contains a signal peptide of 22 amino acids in length, as predicted by SignalP version 4.0 (Nordahl Petersen et al, 2011, Nature Methods, 8: 785-786). The presence of a signal sequence suggests that TauSec098 is a secreted enzyme. The amino acid sequence of the predicted mature form of TauSec098 is described as SEQ ID NO: 15.

EXPRESSION OF RASAMSONIA COMPOSTICOLA ALPHAGLICOSIDASIS TAUSEC098

[00272] A synthetic TauSec098 gene was cloned into the Trichoderma reesei expression vector pGXT (plasmid derived from pTTT) by Generay Biotech Co. (Shanghai, China) and the resulting plasmid was named pGX256-TauSec098. The sequence of the TauSec098 gene was confirmed by DNA sequencing.

[00273] Plasmid pGX256-TauSec098 was transformed into a quad-deleted Trichoderma reesei strain (described in WO 05/001036) using protoplast transformation (Te'o et al, J. Microbiol. Methods 51: 393-399, 2002 ). Transformers were selected on a medium containing acetamide as the sole 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; agar, 20 g/l; pH 4.25). Transformed colonies (about 50-100) emerged in about a week. After growth on acetamide plates, transformer spores were collected and transferred to new acetamide agar plates. After five days of growth on acetamide plates, 1x108 spores were inoculated into 30 ml of glucose/sophorose defined media in a 250 ml shake flask. The shake flask was shaken at 28 °C for five days. Supernatants from these cultures were used to confirm expression (SDS PAGE) and activity of mature TauSec098 enzyme (SEQ ID NO: 15).

RASAMSONIA COMPOSTICOLA TAUSEC099 ALPHAGLYCOSIDASIS DISCOVERY

[00274] A strain of Rasamsonia composticola was selected as a potential source of other enzymes that may be useful in various industrial applications. One of the genes identified in Rasamsonia composticola encodes an alpha-glucosidase and the sequence of this gene, named “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 31 family based on PFAM research (pfam.sanger.ac.uk). At the N-terminus, the protein (SEQ ID NO: 17) contains a signal peptide of 17 amino acids in length, as predicted by SignalP version 4.0 (Nordahl Petersen et al, 2011, Nature Methods, 8: 785-786). The presence of a signal sequence suggests that TauSec099 is a secreted enzyme. The amino acid sequence of the predicted mature form of TauSec099 is described as SEQ ID NO: 18.

EXPRESSION OF RASAMSONIA COMPOSTICOLA ALPHAGLICOSIDASE TAUSEC099

[00275] A synthetic TauSec099 gene was cloned into the Trichoderma reesei expression vector pGXT (plasmid derived from pTTT) by Generay Biotech Co. (Shanghai, China) and the resulting plasmid was named pGX256-TauSec099. The sequence of the TauSec0998 gene was confirmed by DNA sequencing.

[00276] Plasmid pGX256-TauSec099 was transformed into a quad-deleted Trichoderma reesei strain (described in WO 05/001036) using protoplast transformation (Te'o et al, J. Microbiol. Methods 51: 393-399, 2002 ). Transformers were selected on a medium containing acetamide as the sole 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; agar, 20 g/l; pH 4.25). Transformed colonies (about 50-100) emerged in about a week. After growth on acetamide plates, transformer spores were collected and transferred to new acetamide agar plates. After five days of growth on acetamide plates, 1x108 spores were inoculated into 30 ml of glucose/sophorose defined media in a 250 ml shake flask. The shake flask was shaken at 28 °C for five days. Supernatants from these cultures were used to confirm expression (SDS PAGE) and activity of mature TauSec099 enzyme (SEQ ID NO: 18).

BIFIDOBACTERIUM LONGUM ALPHAGLICOSIDASE SEQUENCES BLOGLUI

[00277] 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 account No. NC014169.1, complementary sequence from positions 140600 to 142414) and the amino acid sequence of the hypothetical protein (SEQ ID No. 20) encoded by SEQ ID No. 19 were found in the GENBANK account No. YP_003660432.1.

EXPRESSION OF BIFIDOBACTERIUM LONGUM ALPHAGLICOSIDASE BLOGLUI

[00278] The DNA sequence encoding the complete BloGlu1 protein (SEQ ID No. 20) was optimized for expression in B. subtilis, then synthesized (generating SEQ ID No. 21) and inserted into plasmid p3JM by Generay Biotech Co. (Shanghai, China), resulting in p3JM-BloGlu1. Plasmid p3JM-BloGlu1 contains an aprE promoter to drive expression of the optimized BloGluI sequence (SEQ ID NO:21).

[00279] Plasmid p3JM-BloGlu1 was used to transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr) and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm of chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250 ml shake flask with MBD medium (defined medium based on MOPS supplemented with additional 5 mM CaCl2) to express protein BloGluI (SEQ ID NO:20).

BIFIDOBACTERIUM LONGUM ALPHAGLICOSIDASE SEQUENCES BLOGLU2

[00280] Alpha-glucosidase, BloGlu2, was identified from Bifidobacterium longum. The amino acid sequence (SEQ ID No. 22) of BloGlu2 was found in the NCBI database (GENBANK account no. WP_007054665.1).

BIFIDOBACTERIUM LONGUM ALPHAGLICOSIDASE EXPRESSION BLOGLU2

[00281] A DNA sequence encoding BloGlu2 protein was optimized for expression in B. subtilis, then synthesized (generating SEQ ID No. 23) and inserted into plasmid p3JM by Generay Biotech Co., resulting in p3JM-BloGlu2. SEQ ID NO: 23 encodes the amino acid sequence of SEQ ID NO: 24. Plasmid p3JM-BloGlu2 contains an aprE promoter to drive expression of the optimized BloGlu2 sequence (SEQ ID NO: 23).

[00282] Plasmid p3JM-BloGlu2 was used to transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr) and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm of chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250 ml shake flask with MBD medium (defined medium based on MOPS supplemented with additional 5 mM CaCl2) to express protein BloGlu2 (SEQ ID NO: 24).

BIFIDOBACTERIUM LONGUM ALPHAGLICOSIDASE SEQUENCES BLOGLU3

[00283] An alpha-glucosidase gene, “BloGlu3”, was identified from Bifidobacterium longum subsp. longum F8. The nucleic acid sequence for the BloGlu3 gene (SEQ ID No. 25, GENBANK account No. NC_021008.1, positions 2130627 to 2132441) and the amino acid sequence of the hypothetical protein (SEQ ID No. 26) encoded by SEQ ID No. 25 were found in the GENBANK account No. YP_007768249.1.

BIFIDOBACTERIUM LONGUM ALPHAGLICOSIDASE EXPRESSION BLOGLU3

[00284] The DNA sequence encoding the complete BloGlu3 protein (SEQ ID No. 26) was optimized for expression in B. subtilis, then synthesized (generating SEQ ID No. 27) and inserted into plasmid p3JM by Generay Biotech Co. , resulting in p3JM-BloGlu3. Plasmid p3JM-BloGlu3 contains an aprE promoter to drive expression of the optimized BloGlu3 sequence (SEQ ID NO:27).

[00285] Plasmid p3JM-BloGlu3 was used to transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr) and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm of chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250 ml shake flask with MBD medium (defined medium based on MOPS supplemented with additional 5 mM CaCl2) to express protein BloGlu3 (SEQ ID NO: 26).

BIFIDOBACTERIUM PSEUDOLONGUM ALPHAGLYCOSIDASE SEQUENCES BPSGLUI

[00286] Alpha-glucosidase, BpsGlu1, was identified from Bifidobacterium pseudolongum. The amino acid sequence (SEQ ID No. 28) of BpsGlul was found in the NCBI database (GENBANK account no. WP_022858408.1).

EXPRESSION OF ALPHAGLICOSIDASE OF BIFIDOBACTERIUM PSEUDOLONGUM BPSGLUI

[00287] A DNA sequence encoding BpsGlu1 protein was optimized for expression in B. subtilis, then synthesized (generating SEQ ID No. 29) and inserted into plasmid p3JM by Generay Biotech Co., resulting in p3JM-BpsGlu1. SEQ ID NO: 29 encodes the amino acid sequence of SEQ ID NO: 30. Plasmid p3JM-BpsGlu1 contains an aprE promoter to drive expression of the optimized BpsGlu1 sequence (SEQ ID NO: 29).

[00288] Plasmid p3JM-BpsGlu1 was used to transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr) and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm of chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250 ml shake flask with MBD medium (defined medium based on MOPS supplemented with additional 5 mM CaCl2) to express protein BpsGlul (SEQ ID NO: 30).

BIFIDOBACTERIUM THERMOPHILUM BTHGLUI ALPHAGLYCOSIDASE SEQUENCES

[00289] An alpha-glucosidase gene, “BthGlu1” was identified from Bifidobacterium thermophilum RBL67. The nucleic acid sequence for the BthGluI gene (SEQ ID No. 31, GENBANK account No. NC_020546.1, positions 150690 to 152495) and the amino acid sequence of the hypothetical protein (SEQ ID No. 32) encoded by SEQ ID No. 31 were found in the GENBANK account No. YP_007592840.1.

EXPRESSION OF BIFIDOBACTERIUM THERMOPHILUM ALPHAGLYCOSIDASE BTHGLUI

[00290] The DNA sequence encoding the complete BthGlu1 protein (SEQ ID No. 32) was optimized for expression in B. subtilis, then synthesized (generating SEQ ID No. 33) and inserted into plasmid p3JM by Generay Biotech Co. , resulting in p3JM-BthGlu1. Plasmid p3JM-BthGlu1 contains an aprE promoter to drive expression of the optimized BthGlul sequence (SEQ ID NO: 33).

[00291] Plasmid p3JM-BthGlu1 was used to transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr) and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm of chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250 ml shake flask with MBD medium (defined medium based on MOPS supplemented with additional 5 mM CaCl2) to express protein BthGlul (SEQ ID NO: 32).

BIFIDOBACTERIUM BREVE BBRGLU2 ALPHAGLYCOSIDASE SEQUENCES

[00292] Alpha-glucosidase, BbrGlu2, was identified from Bifidobacterium breve. The amino acid sequence (SEQ ID No. 34) of BbrGlu2 was found in the NCBI database (GENBANK account no. WP_003827971.1).

EXPRESSION OF ALPHAGLYCOSIDASE OF BIFIDOBACTERIUM BREVE BBRGLU2

[00293] A DNA sequence encoding BpsGlu2 protein was optimized for expression in B. subtilis, then synthesized (generating SEQ ID NO: 35) and inserted into plasmid p3JM by Generay Biotech Co., resulting in p3JM-BbrGlu2. SEQ ID NO: 35 encodes the amino acid sequence of SEQ ID NO: 36. Plasmid p3JM-BbrGlu2 contains an aprE promoter to drive expression of the optimized BbrGlu2 sequence (SEQ ID NO: 35).

[00294] Plasmid p3JM-BbrGlu2 was used to transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr) and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm of chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250 ml shake flask with MBD medium (defined medium based on MOPS supplemented with additional 5 mM CaCl2) to express SEQ ID No. 36.

BIFIDOBACTERIUM BREVE BBRGLU5 ALPHAGLYCOSIDASE SEQUENCES

[00295] Alpha-glucosidase gene, “BbrGlu5”, was identified from Bifidobacterium breve ACS-071-V-Sch8b. The nucleic acid sequence for the BbrGlu5 gene (SEQ ID No. 37, GENBANK account no. NC_017218.1, complementary sequence from positions 2241075 to 2242895) and the amino acid sequence of the hypothetical protein (SEQ ID No. 38) encoded by SEQ ID No. 37 were found on GENBANK account No. YP_005583701.1.

EXPRESSION OF ALPHAGLYCOSIDASE OF BIFIDOBACTERIUM BREVE BBRGLU5

[00296] A DNA sequence encoding the complete BbrGlu5 protein (SEQ ID No. 38) was optimized for expression in B. subtilis, then synthesized (generating SEQ ID No. 39) and inserted into plasmid p3JM by Generay Biotech Co. , resulting in p3JM-BbrGlu5. Plasmid p3JM-BbrGlu5 contains an aprE promoter to drive expression of the optimized BbrGlu5 sequence (SEQ ID NO: 39).

[00297] Plasmid p3JM-BbrGlu5 was used to transform B. subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr) and the transformed cells were spread on Luria Agar plates supplemented with 5 ppm of chloramphenicol. A colony with correct insertion, as confirmed by PCR and sequencing, was selected and subjected to fermentation in a 250 ml shake flask with MBD medium (defined medium based on MOPS supplemented with additional 5 mM CaCl2) to express protein BbrGlu5 (SEQ ID NO: 38).

PURIFICATION OF ALPHAGLICOSIDASES FROM ACLGLUI AND NCRGLUI EXPRESSION CULTS

[00298] AclGlu1 (SEQ ID NO: 6) and NcrGluI (SEQ ID NO: 12) alpha-glucosidases were purified using two chromatography steps. For each purification, the crude broth from the shake flask was concentrated and then ammonium sulfate was added to a final concentration of 2 M. The solution was loaded onto a 50 ml phenyl HP column previously equilibrated with 20 mM Tris , pH 8.0, 2 M ammonium sulfate. The target protein (SEQ ID NO: 6 or SEQ ID NO: 12) was eluted from the column with 1 M ammonium sulfate, 20 mM Tris, pH 8.0. Corresponding fractions were pooled, concentrated and their buffer replaced to 20 mM Tris, pH 8.0 (buffer A), using a VIVAFLOW 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to a 40 ml Q HP column previously equilibrated with Buffer A. The target protein was eluted from the column with 0.3 M NaCl in Buffer A. Fractions containing target protein were pooled and then concentrated using AMICON ULTRA-15 10K devices and stored in 40% glycerol at -20 °C until use.

NFIGLUI

[00299] NfiGlu1 alpha-glucosidase (SEQ ID NO: 9) was purified using two-step hydrophobic interaction chromatography. The crude broth from the shake flask was concentrated and then ammonium sulfate was added to a final concentration of 1 M. The solution was loaded onto a 50 ml phenyl HP column previously equilibrated with 20 mM Tris, pH 8 .0.1 M ammonium sulfate. The target protein (SEQ ID NO:9) flowed through the column. Flow-through fractions were pooled and then ammonium sulfate was added to a final concentration of 2 M. The solution was loaded onto the same phenyl HP column previously equilibrated with 20 mM Tris, pH 8.0 and 2 M ammonium sulfate. The target protein was eluted from the column with 1 M ammonium sulfate, 20 mM Tris, pH 8.0. Fractions containing target protein were pooled and then concentrated using AMICON ULTRA-15 10K devices and stored in 40% glycerol at -20°C until use.

TAUSEC098 AND TAUSEC099

[00300] Alpha-glycosidases from TauSec098 (SEQ ID NO: 15) and TauSec099 (SEQ ID NO: 18) were purified by means of hydrophobic interaction chromatography. For each purification, ammonium sulfate was added to about 180 ml of concentrated crude broth from a seven liter fermenter to a final concentration of 1 M. This solution was then loaded onto a 50 ml column of HIPREP phenyl-FF Sepharose (GE Healthcare) previously equilibrated with 20 mM sodium acetate, pH 5.0, 1 M ammonium sulfate (buffer A). After washing with the same buffer with three column volumes (CVs), the column was eluted in stages with 75%, 50% and 0% Buffer A using three CVs each, followed by two CVs of MILLIQ H2O. All fractions were analyzed using SDS-PAGE. The target protein (SEQ ID NO: 15 or SEQ ID NO: 18) was primarily present in the flow-through fraction, which was concentrated and buffer replaced to remove excess ammonium sulfate using 10 kDa AMICON ULTRA-15 devices. The final product, which was more than 90% pure, was stored in 40% glycerol at -80 °C until use.

BLOGLUI, BLOGLU2 AND BLOGLU3

[00301] Alpha-glucosidases from BloGlu1 (SEQ ID NO: 20), BloGlu2 (SEQ ID NO: 24) and BloGlu3 (SEQ ID NO: 26) were all purified in three steps. For each purification, crude broth from a 1 L DASGIP fermenter was concentrated and then ammonium sulfate was added to 60% saturation. The solution was stirred at 4 °C for one hour and then centrifuged at 8000 x g for thirty minutes. The resulting pellet was resuspended in 20 mM Tris, pH 8.0 (buffer A). Ammonium sulfate was added to the resulting solution to a final concentration of 1 M; this preparation was then equilibrated on a 40 ml HiPrep® Phenyl FF column previously equilibrated with 20 mM Tris, pH 8.0, 1 M ammonium sulfate (buffer B). After washing, the column was eluted in stages with 75%, 50% and 0% Buffer B and H2O in three column volumes each. All fractions were analyzed using SDS-PAGE and activity tests. Fractions containing target protein (SEQ ID N° 20, SEQ ID N° 24 or SEQ ID N° 26) were pooled, concentrated and then loaded onto a HiLoad® 26/60 Superdex® 75 column previously equilibrated with 20 mM sodium phosphate. sodium, pH 7.0, 0.15 M NaCl. Flow-through fractions containing the target protein were pooled and then concentrated using AMICON ULTRA-15 10K devices and stored in 40% glycerol at -20°C until use.

BPSGLUI AND BTHGLUI

[00302] BpsGlu1 (SEQ ID NO: 30) and BthGlu1 (SEQ ID NO: 32) alpha-glucosidases were purified in two steps. For each purification, crude broth from a 1 L DASGIP fermenter was concentrated and then ammonium sulfate was added to 60% saturation. The solution was stirred at 4 °C for one hour and then centrifuged at 8000 x g for thirty minutes. The resulting pellet was resuspended in 20 mM Tris, pH 8.0 (buffer A). Ammonium sulfate was added to the resulting solution to a final concentration of 1M; this preparation was then equilibrated on a 40 ml HiPrep® Phenyl FF column previously equilibrated with 20 mM Tris, pH 8.0, 1 M ammonium sulfate (buffer B). After washing, the column was eluted in stages with 75%, 50% and 0% Buffer B and H2O in three column volumes each. All fractions were analyzed using SDS-PAGE and activity tests. The target protein (SEQ ID NO: 30 or SEQ ID NO: 32) was present in the eluate from the 0% Buffer B elution step; this eluate was pooled and concentrated using AMICON ULTRA-15 10K devices. The final product, which was more than 95% pure, was stored in 40% glycerol at -20 °C until use.

BBRGLU2 AND BBRGLU5

[00303] Alpha-glycosidases from BbrGlu2 (SEQ ID NO: 36) and BbrGlu5 (SEQ ID NO: 38) were purified in four steps. For each purification, crude broth from a 1 L DASGIP fermenter was concentrated and then ammonium sulfate was added to 60% saturation. The solution was stirred at 4 °C for one hour and then centrifuged at 8000 x g for thirty 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; this preparation was then loaded onto a column of HiPrep® Phenyl FF previously equilibrated with 20 mM HEPES, pH 7.0, 1 M ammonium sulfate. The target protein (SEQ ID NO: 36 or SEQ ID NO: 38) was eluted from the column with 0.5 M ammonium sulfate. Corresponding fractions were pooled, concentrated and their buffer replaced with Buffer A using a VIVAFLOW 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to a HiPrep® Q FF 16/10 column previously equilibrated with buffer A. The target protein was eluted from the column with a linear gradient of 00.5 M NaCl in buffer A. Fractions containing target protein were pooled, concentrated and then loaded onto a HiLoad® 26/60 Superdex® 75 column previously equilibrated with 20 mM HEPES, pH 7.0, 0.15 M NaCl. Fractions containing target protein were pooled and then concentrated using AMICON ULTRA-15 10K devices and stored in 40% glycerol at -20°C until use.

[00304] Several additional alpha-glucosidases have therefore been expressed and purified. These alpha-glucosidases were tested to determine hydrolytic activity against alpha-1,5 glycosylfructose or alpha-1,3 and/or alpha-1,6 glycosylglucose linkages in Examples 11, 12, 15 and 16 provided below.

EXAMPLE 11 ALPHAGLICOSIDASIS TEST TO DETERMINE HYDROLYTIC ACTIVITY AGAINST VARIOUS GLYCOSID LINKS

[00305] This example describes testing whether alpha-glucosidases possess hydrolytic activity beyond that previously associated with this class of enzymes (EC 3.2.1.20). The alpha-glucosidases of Example 10 were shown to have hydrolytic activity against alpha-1,5 glycosylfructose linkages and alpha-1,3 and alpha-1,6 glycosylglucose linkages.

SUBSTRATE SPECIFICITY OF ALPHAGLYCOSIDASES

[00306] The substrate specificity of each alpha-glucosidase described in Example 10 was tested based on the release of glucose from a specific substrate (isomaltose, maltose, panose, leucrose or nigerosis) when the substrate was incubated with alpha-glucosidase. The rate of glucose release was measured using a coupled glucose oxidase/peroxidase (GOX/HRP) method (1980, Anal. Biochem. 105: 389-397). Glucose release was quantified as the rate of oxidation of 2,2'-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) by peroxide that was generated from coupled GOX/HRP enzymes that reacted with glucose.

[00307] Individual substrate solutions were prepared by mixing a solution of 9 ml of substrate (1% in water, w/v) with 1 ml of 0.05 M sodium acetate buffer, pH 5.0, and 40 µl of 0.5 M calcium chloride in a 15 ml conical tube. A solution of enzymes coupled (GOX/HRP) with ABTS was prepared in 50 mM sodium acetate buffer (pH 5.0), with final concentrations of 2.74 mg/ml ABTS, 0.1 U/ml HRP and 1 U/ml of GOX. Serial dilutions of individual alpha-glucosidase samples and glucose standard were prepared in MILLIQ water. For nigerosis, alpha-glucosidase samples were tested with only one dosage at 10 ppm due to the limited stock of substrate solutions. Each alpha-glucosidase sample (10 μl) was transferred to a new microtiter plate (Corning 3641) containing 90 μl of substrate solution previously incubated at 50 °C for five minutes at 600 rpm. The reactions were carried out at 50 °C for ten minutes (for isomaltose, maltose, panose and nigerose substrates) or for sixty minutes (for leucrose substrate) with stirring (600 rpm) in THERMOMIXER (Eppendorf). 10 μl of each reaction mix, as well as 10 μl of glucose standard serial dilutions, were then rapidly transferred to new microtiter plates (Corning 3641), respectively, to which 90 μl of ABTS/GOX solution was suitably added /HRP. Microtiter plates containing reaction mixtures were immediately measured at 405 nm at eleven second intervals for five minutes using a SOFTMAX PRO plate reader (Molecular Devices). The output was the reaction rate, Vo, for each enzyme concentration. Linear regression was used to determine the slope of the Vo x enzyme dose plot. The specific activity of each alpha-glucosidase was calculated based on the standard glucose curve using Equation 1: specific activity (unit/mg) = slope (enzyme) / slope (standard) x 1000 (1), where 1 unit = 1 μmol of glucose/min.

[00308] For nigerosis, the reaction rate value with enzyme dosage at 10 ppm was used directly to indicate the enzyme activity.

[00309] Using the above method, the specificity of each alpha-glucosidase was determined against each substrate. The activities of oligo-1,6-glucosidase (purchased from Megazyme, see Table 4) and a transglycosidase (TG L-2000, see Table 4) against each substrate were also measured. The results of this analysis are provided in Table 17. TABLE 17 ACTIVITY OF VARIOUS ALPHAGLYCOSIDASES AGAINST DIFFERENT SUBSTRATES a Each enzyme was used in one dosage (10 ppm) against nigerosis.

[00310] Interestingly, it has been found that alpha-glucosidases, in addition to exhibiting hydrolytic activity against alpha-1,4 glycosylglucose (maltose) binding, also exhibit hydrolytic activity against alpha-1,6 glycosylglucose (isomaltose) binding, alpha-1 binding ,3 glycosylglucose (nigerose) and alpha-1,5 glycosylfructose binding (leucrose) (Table 17).

[00311] Alpha-glucosidases therefore possess hydrolytic activity beyond that previously associated with EC 3.2.1.20 enzymes. Specifically, alpha-glucosidases have hydrolytic activity against alpha-1,5 glycosylfructose linkages and alpha-1,3 and alpha-1,6 glycosylglucose linkages.

EXAMPLE 12 HYDROLYSIS OF LEUCROSE AND OLIGOSACCHARIDES IN GLUCANE REACTION FILTRATE USING ALPHAGLYCOSIDASE

[00312] This Example describes the use of alpha-glucosidase to hydrolyze leucrose and other oligosaccharides present in filtrate obtained from a glucan synthesis reaction. Specifically, the effect of alpha-glucosidases described in Example 10 on the hydrolysis of leucrose and oligosaccharides DP2, DP3 and HS (higher sugars, DP4+) in an insoluble glucan filtrate synthesis reaction (poly alpha-1,3-glucan ).

ISOLATION AND ANALYSIS OF OLIGOSACCHARIDES FOR ALPHAGLICOSIDASE ACTIVITY TEST

[00313] First, a concentrated filtrate from a glucan synthesis reaction was prepared as described in Example 1.

[00314] Briefly, oligosaccharides were isolated from the concentrated filtrate by means of chromatographic separation and analyzed to determine the glycosidic binding profile. Chromatographic separation employing a strong acid cation exchange resin was used to isolate the oligosaccharide fraction from the concentrated filtrate. The physical parameters of the column used for this separation were the following: FINEX CS11GC, resin n° 227; forms Na+ ions; 5% divinyl benzene (crosslink); particle size of 0.34 mm; bed length of 1.64 m; column diameter of 0.093 m.

[00315] In more detail, the concentrated sugar solution (ie concentrated filtrate) described in Table 3 was filtered and diluted to 25 g dry solids/100 g solution using tap water. Prior to the addition of this sugar solution to the column resin, the resin was washed with six bed volumes (BV) of sodium chloride solution (three BV in 10% by weight sodium chloride followed by three BV in sodium chloride solution). 5% by weight sodium) to convert the resin to the sodium form. The sugar solution (0.6 l) was then fed to the column and then the column was eluted using water at a flow rate of 50 ml/min. The conditions for conducting the chromatographic separation are summarized below: 0.6 L feed size, 25 g dry solids/100 g solution, column temperature 65 °C and flow rate 50 ml/min . An oligosaccharide solution was eluted between 11 and 21 minutes. A small amount of salts (indicated by increased conductivity) eluted at the same time. The oligosaccharide fraction prepared in this way was analyzed by means of HPLC to determine its product distribution. Altogether, the fraction contained more than 89% oligosaccharides containing three or more hexose units and less than 1.5% identifiable mono- and disaccharides. This fraction was concentrated to a total dry weight of 317 g/l using a thin film evaporator (LCI Corporation, Charlotte NC) followed by rotary evaporation with ROTAVAPOR (R-151; Buchi, New Castle DE). The product distribution of the concentrated fraction as measured by HPLC appears in Table 18. TABLE 18 PRODUCT DISTRIBUTION OF CONCENTRATED OLIGOSACCHARIDE FRACTION

PRIMARY SELECTION OF ALPHAGLICOSIDASES ON GLUCANE OLIGOMER HYDROLYSIS

[00316] The activities of eleven different alpha-glucosidases (Example 10), as well as the activities of two important enzymes, oligo-1,6-glucosidase (purchased from Megazyme) and transglycosidase (TG L-2000), were evaluated individually against the fraction of purified oligosaccharide prepared above (Table 18). Each alpha-glucosidase (dosed at 1 mg/ml) was incubated in a solution containing oligosaccharide substrates (2.9% dry solids) and 2 mM calcium chloride under pH 5.0 at 60 °C. Each reaction was cooled after 24 hours of incubation by adding 50 μl of 0.5 M NaOH.

[00317] The oligosaccharide and monosaccharide contents of the cooled reactions were determined as follows. A sample of each reaction was diluted five times in water for HPLC analysis. HPLC separation was performed using an Agilent 1200 series HPLC system with an AMINEX HPX-42A column (300 mm x 7.8 m) at 85 °C. The sample (10 μl) was applied to the HPLC column and separated with an isocratic gradient of MILLI-Q water as the mobile phase at a flow rate of 0.6 ml/min. Oligosaccharide products were detected using a refractor index detector. The numbers given in Table 19 below reflect the average of the peak area percentages (from each sample doubling) of each DPn as a fraction of the total from DP1 to DP7. TABLE 19 OLIGOSACCHARIDE ANALYSIS OF GLUCAN FILTERATE AFTER TREATMENT WITH ALPHAGLYCOSIDASE

[00318] As indicated with shading in Table 19, the oligosaccharide content of the reactions generally changed to sugars with smaller size, compared to the control reaction (“Standard”), in which there was no enzyme. These results indicate that alpha-glucosidase can be used to hydrolyze oligosaccharides comprised in a synthesis reaction of glucan and one of its fractions. Furthermore, by configuring the binding profile of oligosaccharides (Examples 3 and 4) and alpha-glucosidase activity against various glycosidic linkages in addition to alpha-1,4 linkages (Example 11), it is evident that alpha-glucosidase can be used to decompose oligosaccharides with alpha-1,5 glycosylfructose and/or alpha-1,3 and alpha-1,6 glycosylglucose linkages. The results provided in Table 19 also suggest that fungal alpha-glucosidases have better hydrolytic activity for soluble oligosaccharides compared to bacterial alpha-glucosidases.

CONFIRMATION OF THE HYDROLYTIC ACTIVITY OF ALPHAGLICOSIDASE FOR OLIGOSACCHARIDE PRODUCTS OF GLUCAN SYNTHESIS REACTIONS

[00319] Reactions comprising one or two alpha-glucosidases and a concentrated filtrate obtained from a poly alpha-1,3-glucan reaction were prepared (Table 3). Alpha-glucosidase reactions were dosed with enzyme at 4 ppm or, for mixtures, each enzyme was used in a 1:1 ratio with a final dosage of 4 ppm. The concentrated filtrate was loaded into each reaction at 10% dry solids. Each reaction additionally comprised 2 mM calcium chloride under pH 5.0 and was run at either 60 °C or 65 °C. Reactions were cooled by adding 50 μl of 0.5 M NaOH after incubation for 23 hours.

[00320] The oligosaccharide and monosaccharide contents of the cooled reactions were determined as follows. A sample of each reaction was diluted 25 times in water for HPLC analysis. HPLC separation was performed using an Agilent 1200 series HPLC system with an AMINEX HPX-42A column (300 mm x 7.8 m) at 85 °C. The sample (10 μl) was applied to the HPLC column and separated with an isocratic gradient of MILLI-Q water as the mobile phase at a flow rate of 0.6 ml/min. Oligosaccharide products were detected using a refractor index detector. The numbers provided in Table 20 below reflect the average of the peak area percentages (from each sample doubling) of each DPn as a fraction of the total. The results given in Table 20 generally confirm the activity of certain alpha-glucosidases as discussed above with respect to the results given in Table 19.

[00321] Alpha-glucosidase can therefore be used to hydrolyze leucrose and other oligosaccharides present in a fraction (eg filtrate) obtained from a glucan synthesis reaction, such as a poly alpha-1,3- synthesis reaction glucan.

EXAMPLE 13 ISOLATION OF OLIGOMER/LEUROSE FRACTION FROM GTF-S/MUT3325 REACTION

[00322] Sucrose (4.50 kg) was dissolved in distilled deionized water until a final total volume of 9.5 l and the resulting solution was heated with stirring at 80 °C for five minutes and then cooled to 47 °C. With stirring, 500 grams of a crude extract containing 0.6 g/l of gtf-S enzyme (GTF0459, SEQ ID No. 42) and 15.0 ml of a crude extract containing 10 g/l of mutanase (MUT3325, SEQ ID No. 47) were added with stirring (see General Methods for Enzyme Preparations). The pH of the resulting mixture was immediately adjusted to pH 5.5 to pH 6.0 by slowly adding a 1:10 (v/v) dilution of 37 wt% HCl with stirring. The reaction temperature and pH were maintained at 47 °C and pH 5.5-6.0, respectively, until >95% sucrose conversion according to HPLC analysis, and then the reaction mixture was immediately adjusted to pH 7.0 to 7.5, heated to 90 °C for twenty minutes and then cooled to 25 °C for immediate filtering to remove particulates and precipitate. The resulting filtrate was held at 5 °C prior to IEX/SEC column chromatography using the following conditions and resin: FINEX CS 11 GC SAC in Ca2+ form, column internal diameter = 9.3 cm, height of resin bed 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, RI-DS feed = 25 .1 g/100 g, sample interval = 3 min. Column fractions collected between thirty minutes and 67 minutes were pooled, concentrated via evaporation to 66% dissolved solids, and analyzed via HPLC as described in General Methods. Table 21 indicates the oligosaccharide and monosaccharide components of the isolated fraction prepared in this way. TABLE 21 ANALYSIS OF OLIGOMER/LEUCROSIS FRACTION FROM GTF-S/MUT3325 REACTION

[00323] In this Example, a glucan synthesis reaction was used to generate at least one soluble alpha-glucan product. This soluble product resulted from the concerted action of glycosyltransferase (GTF0459, SEQ ID No. 42) and alphaglucanhydrolase (MUT3325, SEQ ID No. 47) which were present in the glycosyltransferase reaction. This Example also demonstrated the preparation of a chromatographic fraction via the glucan synthesis reaction. This fraction was used in Examples 15 and 16 below to test alpha-glucosidases activity thereon.

EXAMPLE 14 ISOLATION OF OLIGOMER/LEUCROSIS FRACTION FROM GTF-C REACTION

[00324] Sucrose (4.50 kg) was dissolved in distilled deionized water until a final total volume of 9.5 l and the resulting solution was heated with stirring at 80 °C for five minutes and then cooled to 47 °C. With stirring, 500 grams of a 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 to pH 6.0 by slowly adding a 1:10 (v/v) dilution of 37 wt% HCl with stirring. The reaction temperature and pH were maintained at 47 °C and pH 5.5-6.0, respectively, until >95% sucrose conversion via HPLC analysis, and then the reaction mixture was immediately adjusted to pH 7.0 to 7.5, heated to 90 °C for twenty minutes and then cooled to 25 °C for immediate filtering to remove particulates and precipitate. The resulting filtrate was held at 5 °C prior to IEX/SEC column chromatography using the following conditions and resin: FINEX CS 11 GC SAC in Ca2+ form, column internal diameter = 9.3 cm, height of resin bed 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, RI-DS feed = 25 .8 g/100 g, sample interval = 3 min. Column fractions collected between 34 minutes and 72 minutes were pooled, concentrated via evaporation to 67% dissolved solids, and analyzed via HPLC as described in General Methods. Table 22 indicates the oligosaccharide and monosaccharide components of the isolated fraction prepared in this way. TABLE 22 ANALYSIS OF OLIGOMER/LEUCROSIS FRACTION FROM GTF-C REACTION

[00325] In this Example, a glucan synthesis reaction was used to generate at least one soluble alpha-glucan product. This Example also demonstrated the preparation of a chromatographic fraction via the glucan synthesis reaction that generated a soluble alpha-glucan product. This fraction was used in Examples 15 and 16 below to test alpha-glucosidases activity thereon.

EXAMPLE 15 PRIMARY SELECTION OF ALPHAGLICOSIDASIS USING OLIGOMER/LEUCROSIS FRACTIONS FROM GTF-S/MUT3325 AND GTF-C REACTIONS

[00326] This Example describes the use of alpha-glucosidase to hydrolyze leucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that generated soluble alpha-glucan product. Specifically, a study was carried out on the effect of alpha-glucosidases described in Example 10 on the hydrolysis of leucrose and oligosaccharides in the fractions prepared in Examples 13 and 14.

[00327] In all, twelve alpha-glucosidases and two important enzymes (oligo-1,6-glucosidase and TG L-2000 transglucosidase) were selected using oligomer/fructose fractions from reactions of gtf-S/MUT3325 (Example 13) and gtf- C (Example 14) as substrate material. All enzymes (alphaglucosidases and important enzymes) were measured at equal protein concentrations. Each alpha-glucosidase (dosed at 100 ppm) was incubated in a solution containing oligomer and leucrose substrates (10% dry solids) and 2 mM calcium chloride under pH 5.5 at 47°C. Each reaction was cooled after 21 hours of incubation by adding 50 μl of 0.5 M NaOH.

[00328] The oligosaccharide and monosaccharide contents of the cooled reactions were determined as follows. A sample of each reaction was centrifuged and its supernatant was diluted 25 times in water for HPLC analysis (General Methods). The percentages reported in Table 23 reflect the average of the peak area percentages (from duplicate analyzes of each sample) of each DPn as a fraction of the total. The results indicate that fungal alpha-glucosidases showed better hydrolytic activity for glucan oligomers compared to bacterial alpha-glucosidases. TABLE 23 SUGAR COMPOSITION ANALYSIS OF PRIMARY SELECTION OF ALPHAGLYCOSIDASES USING OLIGOMER/LEUCROSIS FRACTIONS FROM GTF-S/MUT3325 AND GTF-C REACTIONS

[00329] As indicated with shading in Table 23, the oligosaccharide content of the reactions generally changed to sugars with smaller size, compared to the control reactions (“Standard”), in which there was no enzyme. These results indicate that alpha-glucosidase can be used to hydrolyze oligosaccharides comprised in a glucan synthesis reaction and one of its fractions, particularly a chromatographic fraction of a glucan synthesis reaction that generated soluble alpha-glucan product. Furthermore, considering the binding profile of the oligosaccharides (Examples 13 and 14) and alpha-glucosidase activity against various glycosidic linkages in addition to alpha-1,4 linkages (Example 11), it is evident that alpha-glucosidase can be used to decompose oligosaccharides with alpha-1,5 glycosylfructose and also probably alpha-1,3 and alpha-1,6 glycosylglucose linkages. The results provided in Table 23 also suggest that fungal alpha-glucosidases have better hydrolytic activity for soluble oligosaccharides compared to bacterial alpha-glucosidases.

[00330] Alpha-glucosidase can therefore be used to hydrolyze leucrose and other oligosaccharides present in a fraction (eg, chromatographic fraction) obtained from a glucan synthesis reaction, such as a soluble alpha-glucan product synthesis reaction.

EXAMPLE 16 SELECTION OF ALPHAGLICOSIDASES USING OLIGOMER/LEUCROSIS FRACTIONS FROM GTF-S/MUT3325 AND GTF-C REACTIONS

[00331] This Example is in addition to Example 15, which describes the use of alpha-glucosidase to hydrolyze leucrose and other oligosaccharides present in chromatographic fractions obtained from glucan synthesis reactions that generated soluble alpha-glucan product.

[00332] Evaluation of alpha-glucosidases that were more active for hydrolysis of oligomer/leucrose fractions from gtf-S/MUT3325 and gtf-C reactions (Example 15) was carried out through the analysis of sugar compositions, resulting in reactions that contain enzymes dosed in equal concentrations of protein. Incubations of alpha-glucosidases (dosed at 4 ppm; for mixtures, the ratio of the two enzymes was 1:1 and the total dosage was 4 ppm) and oligomer/leucrose substrate (10% ds) were carried out under pH 5.5 in the presence of 2 mM calcium chloride at 60 °C and 65 °C, respectively. The reactions were cooled by adding 50 μl of 0.5 M NaOH after 23 hours of incubation.

[00333] The oligosaccharide and monosaccharide contents of the cooled reactions were determined as follows. A sample of each reaction was centrifuged and its supernatant was diluted 25 times in water for HPLC analysis (General Methods). The percentages reported in Table 24 (below) reflect the average of the peak area percentages (from duplicate analyzes of each sample) of each DPn as a fraction of the total. The results indicate that TauSec098 was effective for the hydrolysis of DP2 oligomers to DP7 and TauSec099 outperformed TG L-2000 for the hydrolysis of leucrose when incubated at 65 °C. Mixtures of TauSec098 with TauSec099 (or TG L-2000) were effective for hydrolysis of oligomers and leucrose for DP1 production.

[00334] Alpha-glucosidase can therefore be used to hydrolyse leucrose and other oligosaccharides present in a fraction (eg, chromatographic fraction) obtained from a glucan synthesis reaction, such as a soluble alpha-glucan synthesis reaction. TABLE 24 SUGAR COMPOSITION ANALYSIS OF ALPHAGLYCOSIDASE SELECTION USING OLIGOMER/LEUCROSIS FRACTIONS FROM GTF-S/MUT3325 AND GTF-C REACTIONS

Claims (11)

1. GLUCAN SYNTHESIS METHOD AND LEUCROSE BY-PRODUCT HYDROLYSIS, characterized by comprising: (i) carrying out a glucan synthesis reaction to generate an insoluble alpha-glucan product comprising at least 50% alpha-1,3 glycosidic bonds, in that the glucan synthesis reaction is carried out using a glycosyltransferase enzyme in a solution comprising sucrose and water; (ii) separating a fraction from the glucan synthesis reaction from the insoluble alpha-glucan product, wherein the fraction comprises leucrose; and (iii) bringing the leucrose present in the fraction defined in item (ii) into contact with an alpha-glucosidase enzyme, thereby hydrolyzing the leucrose so that the amount of leucrose in the fraction is reduced compared to the amount of leucrose that was present before said contact. 2. METHOD, according to claim 1, characterized in that the alpha-glucosidase enzyme is immobilized. 3. METHOD according to any one of claims 1 to 2, characterized in that the insoluble alpha-glucan product comprises at least 80% of alpha-1-3 glycosidic bonds. 4. METHOD according to claim 3, characterized in that the alpha-glucan product comprises at least 90% alpha-1,3 glycosidic bonds. 5. METHOD according to claim 4, characterized in that the insoluble alpha-glucan product comprises at least 95% of alpha-1,3 glycosidic bonds. 6. METHOD, according to any one of claims 1 to 5, characterized in that the fraction is a filtrate from the glucan synthesis reaction. 7. METHOD, according to any one of claims 1 to 5, characterized in that the fraction is a supernatant of the glucan synthesis reaction. 8. METHOD, according to any one of claims 1 to 7, characterized in that the alpha-glucosidase enzyme is a transglycosidase. 9. METHOD according to any one of claims 1 to 7, characterized in that the alphaglucosidase enzyme is a glucoamylase. 10. METHOD, according to any one of claims 1 to 9, characterized in that the alphaglucosidase enzyme consists of an amino acid sequence of SEQ ID NO: 1, 2, 6, 9, 12, 18, 20, 24, 26, 30, 32, 36, or 38. 11. METHOD according to any one of claims 1 to 10, characterized in that it additionally comprises separating the fructose from the fraction after hydrolysis of the leucrose.