Use of alpha-glucosidases for enzymatic hydrolysis of disaccharides and oligosaccharides

Abstract

A method for hydrolyzing an alpha-1,5 glucosyl-fructose bond in a levansucrose, e.g., a sucrose, is disclosed. The method comprises contacting the sucrose with an alpha-glucosidase, e.g., a transglucosidase or a glucoamylase, under suitable conditions, during which the enzyme hydrolyzes at least one alpha-1,5 glucosyl-fructose bond of the sucrose. The method is used, e.g., to reduce the amount of levansucrose in a filtrate isolated from a glucan synthesis reaction.

Description

[0001] This application claims the benefit of U.S. Provisional Applications 61 / 945,233 (filed February 27, 2014), 61 / 945,241 (filed February 27, 2014), 62 / 004,290 (filed May 29, 2014), 62 / 004,308 (filed May 29, 2014), 62 / 004,312 (filed May 29, 2014), 62 / 004,300 (filed May 29, 2014), 62 / 004,314 (filed May 29, 2014), and 62 / 004,305 (filed May 29, 2014), the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of enzymatic hydrolysis of smaller sugar polymers. Specifically, this invention relates to the hydrolysis of disaccharides and oligosaccharides containing one or more α-1,5-glucosyl-fructose bonds using α-glucosidase.

[0003] References to sequence lists submitted electronically

[0004] The official text of the sequence list was submitted electronically via EFS-Web as a sequence list in ASCII format. The file, named CL6115USNP_SequenceListing_ST25.txt, was created on February 10, 2015, and is 266 kilobytes in size. This file was submitted along with this specification. The sequence list contained in this ASCII format file is a part of this specification, and the entire text is incorporated herein by reference. Background Technology

[0005] Glucoamylase (EC 3.2.1.3, α-1,4-glucan-glucosidase) is an exogenous enzyme that catalyzes the hydrolysis of both the non-reducing α-1,4 and α-1,6 glycosidic bonds at the glucose-containing disaccharides, oligosaccharides, and polysaccharides, releasing one glucose unit at a time (1960, Pazur and Ando, ​​J. Biol. Chem. 235: 297-302). Cleavage occurs at the glycosidic bond connecting the anomeric carbon and oxygen (1962, Fleetwood and Weigel, Nature 196: 984). α-1,4, α-1,6, and α-1,3 bonds are the only bonds hydrolyzed by glucamylase at a significant rate (1957, Barker et al., J. Chem. Soc. 4865-4871).

[0006] Glucoamylase can also hydrolyze glycosidic bonds between two glucosyl units linked by α-1,2 (e.g., succinate) or α-1,1 (trehalose). However, this enzymatic activity occurs at a lower rate and with more diluted substrate concentrations compared to the activity of glucamylase for disaccharides with α-1,4 (maltose) or α-1,6 (isomaltose) bonds.

[0007] Glucoamylase has been widely used to produce high-glucose syrup from starch. High-glucose syrup can then be used as a raw material for producing various value-added compounds such as fuel ethanol, high-fructose corn syrup, organic acids, amino acids, and vitamins. Glucoamylase has been isolated from a variety of microorganisms, animals, and cinnamon plants, and among microorganisms, many fungi are good sources of this enzyme. Glucoamylase produced in fungal organisms such as *Aspergillus niger* is commonly used in commercial applications, such as the production of high-glucose syrup.

[0008] Transglucosidase (EC. 2.4.1.24, 1,4-α-glucan-6-α-glucosyltransferase) is a D-glucosyltransferase that catalyzes both hydrolysis and transglucosylation reactions when incubated with α-D-glucose-oligosaccharides (1951, Pazur and French, J. Amer. Chem. Soc. 73: 3536). Maltose is the preferred substrate for transglucosylation by this enzyme. Transglucosylation most commonly occurs at HO-6, resulting in the production of isomaltose from D-glucose, or panose (6-O-α-glucosylmaltose) from maltose. Transglucosidase can also transfer glucosyl residues to HO-2 or HO-3 of another D-glucosyl unit to form kilobitose or aspergillus niger. The enzyme can further transfer D-glucosyl units back to HO-4, thereby modifying maltose.

[0009] Because the transglucosylation reaction uses transglucosidase, malt-oligosaccharide residues are converted into isomalt-oligosaccharides (IMOs), which contain a high proportion of glucosyl residues linked by non-reducing α-D-1,6 glycosidic bonds. IMO sugars are used in a variety of food and beverage formulations in Asia. Brier et al. (US Patent Application Publication 2003 / 0167929) disclosed the production of IMOs from barley wort using transglucosidase.

[0010] Poulose et al. (US Patent Application Publication 2008 / 0229514) disclosed the degradation of polysaccharides such as xanthan gum and guar gum using transglucosidase. Xanthan gum comprises a cellulose backbone in which glucose is optionally 1,3-linked to side chains containing mannose and glucuronic acid. Guar gum comprises a backbone in which every other mannose residue is α-1,6-linked to a β-1,4-linked mannose residue.

[0011] Lantero et al. (US Patent 5,770,437) disclosed the degradation of sucrose, melitriose, and trehalose using transglucosidase. These sugars include glucose linked to fructose via 1,2-(sucrose), 1,3-(melitriose), or 1,1-(trehalose) bonds.

[0012] Although various hydrolytic activities of glucosylamylase and transglucosidase have been disclosed, these enzymes are generally considered α-glucosidases, which endows them with the ability to hydrolyze the α-bond between two glucosyl residues. For example, both glucosylamylase and transglucosidase are associated with maltase activity (hydrolyzing the α-1,4 glycosidic bond between two glucosyl residues of maltose), which is a type of α-glucosidase activity.

[0013] Despite the foregoing disclosures, it has now been surprisingly discovered that α-glucosidases, such as transglucosidase (EC 2.4.1.24), glucosylamylase (EC 3.2.1.3), and other α-glucosidases, can hydrolyze the α-1,5 glycosidic bond of glucosyl-fructose. This document discloses the use of α-glucosidases for the degradation of disaccharides and oligosaccharides containing glucosyl-α-1,5-fructose. Summary of the Invention

[0014] In one embodiment, the present invention relates to a method for hydrolyzing α-1,5-glucosyl-fructose bonds in a sugar comprising at least one α-1,5-glucosyl-fructose bond, wherein the sugar is a disaccharide or oligosaccharide, and wherein the method comprises: contacting the sugar with an α-glucosidase under suitable conditions, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of the sugar, and wherein the amount of sugar is reduced compared to the amount of sugar present prior to the contacting step.

[0015] In another embodiment, the α-glucosidase in the hydrolysis method is immobilized.

[0016] In another embodiment, the sugar used in the hydrolysis method is Leuconostoc disaccharide. In yet another embodiment, the concentration of Leuconostoc disaccharide after the contact step is less than 50% of the concentration of Leuconostoc disaccharide present before the contact step.

[0017] In another embodiment, suitable conditions for the hydrolysis method include: (i) a dextran synthesis reaction, or (ii) a fraction obtained from a dextran synthesis reaction; wherein the sugar is a byproduct of the dextran synthesis reaction. In another embodiment, the dextran synthesis reaction produces at least one insoluble α-glucan product. In another embodiment, the fraction is a filtrate from the dextran synthesis reaction. In another embodiment, the dextran synthesis reaction produces at least one soluble α-glucan product, which is: (i) a product of a glucosyltransferase, or (ii) a product of the synergistic action of a glucosyltransferase and an α-glucan hydrolase capable of hydrolyzing dextran polymers having one or more α-1,3-glycosidic bonds or one or more α-1,6-glycosidic bonds. In another embodiment, the fraction is a chromatographic fraction from the dextran synthesis reaction, wherein the dextran synthesis reaction produces at least one soluble α-glucan product.

[0018] In another embodiment, the α-glucosidase is a transglucosidase or a glucosylamylase. In another embodiment, (i) the transglucosidase contains at least 90% of the same amino acid sequence as SEQ ID NO: 1; or (ii) the glucosylamylase contains at least 90% of the same amino acid sequence as SEQ ID NO: 2.

[0019] In another embodiment, the present invention relates to a composition produced by contacting a sugar with an α-glucosidase, wherein the sugar is a disaccharide or oligosaccharide and contains at least one α-1,5-glucosyl-fructose bond, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of the sugar, and wherein the composition contains a reduced amount of sugar compared to the amount of sugar present before the contacting step.

[0020] In another embodiment, the sugar in the composition is Leuconostoc disaccharide. For example, the concentration of Leuconostoc disaccharide in the composition is less than 50% of the concentration of Leuconostoc disaccharide present prior to contact.

[0021] In another embodiment, the sugar in the composition is in (i) a dextran synthesis reaction, or (ii) a fraction obtained from a dextran synthesis reaction; wherein the sugar is a byproduct of the dextran synthesis reaction. In another embodiment, the fraction is a filtrate from a dextran synthesis reaction or a chromatographic fraction from a dextran synthesis reaction.

[0022] In another embodiment, the present invention relates to a method for enriching fructose present in fractions of a dextran synthesis reaction, the method comprising: (a) contacting a fraction obtained from a dextran synthesis reaction with an α-glucosidase under suitable conditions, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of a disaccharide or oligosaccharide contained in the fraction; and (b) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition with a higher fructose concentration than that of the fraction of step (a).

[0023] In another thirteenth embodiment, the present invention relates to a fermentation method comprising: (a) contacting a fraction obtained from a dextran synthesis reaction with an α-glucosidase under suitable conditions, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of a disaccharide or oligosaccharide contained in the fraction; (b) fermenting the fraction of step (a) with microorganisms to obtain a product, wherein fermentation may be carried out after or simultaneously with step (a); and (c) optionally, separating the product of (b); wherein the product yield of (b) is increased compared to the product yield of fermenting a fraction of a dextran synthesis reaction that has not been contacted with an α-glucosidase.

[0024] Figures and Sequence Summary

[0025] Figure 1 : Dextran reaction filtrate materials before (starting material) and after (treated material) hydrolysis with NOVO 188 enzyme 1 1H NMR spectra (see Examples 2-3).

[0026] Figure 2 : Dextran reaction filtrate materials before (starting material) and after (treated material) hydrolysis with TG L-2000 transglucosidase 1 1H NMR spectra (see Examples 2-3).

[0027] Table 1 Overview of Nucleic Acid and Protein Sequence Identifiers

[0028]

[0029]

[0030] Detailed Implementation

[0031] All cited patent and non-patent literature publications are incorporated herein by reference.

[0032] As used herein, the terms “invention” or “disclosed herein” are not intended to be limiting but generally apply to any invention as defined in the claims or described herein. These terms are used interchangeably herein.

[0033] Unless otherwise specified, the terms “sugar,” “sugar molecule,” and “carbohydrate” are used interchangeably herein and refer to disaccharides or oligosaccharides. A “disaccharide” herein refers to a carbohydrate having two monosaccharides linked by a glycosidic bond. An “oligosaccharide” herein refers to a carbohydrate consisting of, for example, 2 to 9 monosaccharides linked by a glycosidic bond. Oligosaccharides may also be referred to herein as “oligomers.” Monosaccharides contained within a disaccharide or oligosaccharide may be referred to, for example, as “monosaccharide units” or “monomer units.” Preferred monosaccharides herein are fructose and glucose.

[0034] The terms “glycosidic bond” and “glycosidic bond” are used interchangeably in this article and refer to a type of covalent bond that links one carbohydrate molecule to another.

[0035] The terms “α-1,3-glucosyl-glucose bond,” “α-1,3-glucose-glucose bond,” and “glucose-α-1,3-glucose” refer to an α-1,3-glycosidic bond between two α-D-glucose molecules. The terms “α-1,6-glucosyl-glucose bond,” “α-1,6-glucose-glucose bond,” and “glucose-α-1,6-glucose” refer to an α-1,6-glycosidic bond between two α-D-glucose molecules. In some embodiments, one or more α-1,3-glucosyl-glucose bonds and / or α-1,6-glucosyl-glucose bonds are contained within a disaccharide or oligosaccharide.

[0036] The terms “α-1,5-glucosyl-fructose bond,” “α-1,5-glucose-fructose bond,” and “glucose-α-1,5-fructose” in this document refer to the α-1,5-glycosidic bond between an α-D-glucose molecule and a fructose molecule. In some embodiments, the α-1,5-glucosyl-fructose bond is contained within a disaccharide or oligosaccharide.

[0037] In this article, "α-D-glucose" can also be referred to as "glucose".

[0038] The disaccharide containing an α-1,5-glucosyl-fructose bond is referred to herein as Leuconostoc disaccharide. The terms “Leuconostoc disaccharide” and “D-glucanopyranosyl-α(1-5)-D-frucopyranosyl” are used interchangeably herein. Leuconostoc disaccharide has the following structure:

[0039]

[0040] The terms “α-glucosidase,” “α-1,4-glucosidase,” and “α-D-glucosidase” are used interchangeably herein. α-glucosidase (EC 3.2.1.20) (“EC” refers to the enzyme identification number) has previously been identified as an enzyme that catalyzes the hydrolysis of terminally non-reduced (1,4)-linked α-D-glucose residues in oligosaccharide (e.g., disaccharide) and polysaccharide substrates. The α-glucosidases now disclosed herein also exhibit hydrolytic activity against α-1,5-glucosyl-fructose bonds, and against α-1,3 and α-1,6-glucosyl-fructose bonds. Transglucosidase and glucosylamylase are examples of α-glucosidases with this type of activity.

[0041] The terms “transglucosidase” (TG), “transglucosidase”, and “1,4-α-glucan-6-α-glucosyltransferase” are used interchangeably herein. Transglucosidase (EC 2.4.1.24) has previously been identified as a D-glucosyltransferase that catalyzes both hydrolysis and transfer reactions when incubated with certain α-D-glucose-oligosaccharides. The transglucosidase now disclosed herein also exhibits hydrolytic activity against α-1,5-glucosyl-fructose bonds, and hydrolytic activity against α-1,3 and α-1,6-glucosyl-fructose bonds.

[0042] The terms “glucosylamylase” (GA), “glucosylamylase”, and “α-1,4-glucan-glucose hydrolase” are used interchangeably herein. Glucoamylase (EC 3.2.1.3) has previously been identified as an extrinsic enzyme that catalyzes the hydrolysis of both α-1,4 and α-1,6 glycosidic bonds at the non-reducing ends of glucose-containing disaccharides, oligosaccharides, and polysaccharides. The glucosylamylase now disclosed herein also exhibits hydrolytic activity against α-1,5-glucosyl-fructose bonds.

[0043] Enzymatic hydrolysis is a process in which an enzyme promotes the breaking of bonds in a molecule in the presence of elemental water. The terms “hydrolysis,” “hydrolyzed,” or “with respect to its hydrolytic activity” for α-1,5-glucosyl-fructose bonds refer to the enzymatic hydrolysis of the α-1,5-glycosidic bond between glucose and fructose by an α-glucosidase, such as glucosylamylase or transglucosidase. Such hydrolysis occurs when a disaccharide or oligosaccharide containing an α-1,5-glucosyl-fructose bond is contacted with the α-glucosidase described herein under suitable conditions. Therefore, a “hydrolysis reaction” as described herein comprises at least: (i) a disaccharide or oligosaccharide containing an α-1,5-glucosyl-fructose bond, and (ii) an α-glucosidase.

[0044] In this article, "saccharification" refers to the process of breaking down sugars (disaccharides or oligosaccharides) into their monosaccharide components. Saccharification can be achieved during the hydrolysis reaction described in this article.

[0045] "Suitable conditions" for contacting a sugar (disaccharide or oligosaccharide) containing at least one α-1,5-glucosyl-fructose bond with the α-glucosidase described herein refer to those conditions (e.g., temperature, pH, time) that support the hydrolysis of one or more α-1,5-glucosyl-fructose bonds by the α-glucosidase. Suitable conditions may include "aqueous conditions," such as comprising at least 20% by weight of water. Aqueous conditions may be in the form of a solution or mixture. The solution or mixture in which the sugar containing at least one α-1,5-glucosyl-fructose bond is contacted with the α-glucosidase may be referred to as an α-glucosidase reaction (e.g., transglucosidase or glucosylamylase reaction).

[0046] In this document, "immobilized" enzymes refer to enzymes that bind to inert, insoluble materials. For example, U.S. Patent Publication 5,541,097 discloses a method for preparing immobilized enzymes, the contents of which are incorporated herein by reference.

[0047] The terms “glucan” and “glucan polymer” are used interchangeably herein and refer to polysaccharides of glucose monomers linked by glycosidic bonds. “α-glucan” herein refers to a glucan polymer containing 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% α-glycosidic bonds.

[0048] The term "insoluble dextran" as used herein refers to a dextran polymer that is insoluble in aqueous conditions. An example of an insoluble dextran described herein is a poly-α-1,3-glucan with a DP of at least 8 or 9. In some embodiments, a glucosyltransferase reaction, as currently disclosed, produces at least one insoluble dextran product.

[0049] The terms “soluble dextran,” “soluble α-glucan,” “soluble fiber,” “soluble dextran fiber,” “soluble dietary fiber,” etc., are used interchangeably herein to refer to dextran polymers soluble in aqueous conditions. Examples of soluble dextrans herein include specific oligosaccharides, such as poly-α-1,3-glucan with a DP less than 8, and specific oligosaccharides disclosed in the examples provided below. In some embodiments, a glucosyltransferase reaction, as currently disclosed, produces at least one soluble dextran product. In some embodiments, another set of characteristics characterizing the soluble α-glucan compounds herein is that they are (i) water-soluble glucose oligomers with a degree of polymerization of 3 or greater, (ii) digestively resistant (i.e., exhibiting very slow digestibility or no digestibility), poorly or non-absorbable in the human small intestine, and (iii) at least partially fermentable in the lower gastrointestinal tract. The digestibility of soluble dextran fiber compositions can be determined, for example, by AOAC method 2009.01.

[0050] The terms “poly-α-1,3-glucan” and “α-1,3-glucan polymer” are used interchangeably herein. Poly-α-1,3-glucan is a polymer comprising glucose monomer units linked together by glycosidic bonds, wherein at least about 50% of the glycosidic bonds are α-1,3-glycosidic bonds. As used herein, the term “α-1,3-glycosidic bond” refers to a type of covalent bond that links α-D-glucose molecules together through carbons 1 and 3 of adjacent α-D-glucose rings.

[0051] The "molecular weight" of dextran in this article can be expressed as the number-average molecular weight (M). n or weight-average molecular weight (M) w Alternatively, molecular weight can be expressed in Daltons, grams per mole, or DP. w (weight-average degree of polymerization) or DP n (Number-average degree of polymerization). Various methods for calculating these molecular weight determinations are known in the art, such as high-performance liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).

[0052] The terms “glucosyltransferase,” “gtf enzyme,” “gtf enzyme catalyst,” “gtf,” and “glucan sucrase” are used interchangeably in this paper. The glucosyltransferase active in this paper catalyzes the reaction of sucrose substrates to produce the products glucan and fructose. Other products (byproducts) of the glucosyltransferase reaction may include glucose (obtained during the hydrolysis of glucose from the glucosyl-gtf enzyme intermediate complex), various soluble oligosaccharides (e.g., DP2-DP7), and Leuconostoc disaccharide (obtained when glucose and fructose are linked in the glucosyl-gtf enzyme intermediate complex). Wild-type glucosyltransferases typically contain a signal peptide (N-terminal to C-terminal), a variable domain, a catalytic domain, and a glucan-binding domain. According to the CAZy (carbohydrate-active enzyme) database, the glucosyltransferases in this paper are classified as glycoside hydrolases family 70 (GH70) (Cantarel et al., Nucleic Acids Res. 37: D233-238, 2009).

[0053] The term "sucrose" in this article refers to a non-reducing disaccharide composed of α-D-glucose and β-D-fructose molecules linked by α-1,2-glycosidic bonds. Sucrose is commonly known as table sugar.

[0054] The terms “glucan synthesis reaction,” “glucan reaction,” and “gtf reaction” are used interchangeably herein and refer to reactions carried out by glucosyltransferases. As used herein, a glucan synthesis reaction typically involves a solution containing at least one active glucosyltransferase in a solution comprising sucrose and water, and optionally other components. Other components that may be present in the glucan synthesis reaction herein include, for example, fructose, glucose, Leuconostoc disaccharide, soluble oligosaccharides (e.g., DP2-DP7), and one or more soluble glucan products. Additionally, in some aspects, the glucan synthesis reaction may include one or more α-glucan hydrolases. It should be understood that certain glucan products, such as poly-α-1,3-glucan with a degree of polymerization (DP) of at least 8 or 9, are water-insoluble and therefore do not dissolve in the glucan synthesis reaction, but may precipitate out of the solution.

[0055] The terms “α-glucan hydrolase” and “glucan hydrolase” are used interchangeably herein and refer to enzymes capable of hydrolyzing α-glucan oligomers. α-glucan hydrolases are defined by their endolytic activity toward specific α-D-glycosidic bonds. Examples of α-glucan hydrolases described herein include glucanase (EC 3.2.1.11; capable of endolytic hydrolysis of α-1,6-linked glycosidic bonds), mutant enzyme (EC 3.2.1.59; capable of endolytic hydrolysis of α-1,3-linked glycosidic bonds), and alternanases (EC 3.2.1.-; capable of endolytic hydrolysis of alternans). Various factors, including but not limited to the degree of branching, branching type, and relative branch length within certain α-glucans, can adversely affect the ability of α-glucan hydrolases to endolytically hydrolyze some glycosidic bonds.

[0056] The "dry solids percentage" of a dextran synthesis reaction refers to the weight percentage of all sugars used in the reaction. For example, the dry solids percentage of a GTF reaction can be calculated based on the amount of sucrose used to prepare the reaction product.

[0057] The term "fraction" in this document refers to the liquid solution portion of a dextran synthesis reaction. A fraction can be a partial or complete liquid solution derived from the dextran synthesis reaction and is separated from the soluble or insoluble dextran product synthesized in the reaction. In some embodiments, the fraction may optionally be referred to as a "mother liquor," wherein the product is an insoluble (solid) dextran product. An example of a fraction is the filtrate from a dextran synthesis reaction. Because a fraction may contain dissolved sugars such as sucrose, fructose, glucose, Leuconostoc disaccharide, and soluble oligosaccharides (e.g., DP2-DP7), it may also be referred to as a "mixed sugar solution" derived from a dextran synthesis reaction. The term "hydrolyzed fraction" in this document refers to a fraction that has been treated with the α-glucosidase described herein to hydrolyze the Leuconostoc disaccharide and / or oligosaccharides present in the fraction.

[0058] The terms “filtrate,” “dextran reaction filtrate,” and “dextran filtrate” are used interchangeably in this document and refer to the fraction filtered from the solid dextran product synthesized in the dextran synthesis reaction. “Hydrolyzed filtrate” in this document refers to filtrate that has been treated with the α-glucosidase described herein to hydrolyze the Leuconostoc disaccharide and / or oligosaccharides present in the filtrate.

[0059] The terms "percentage by volume," "volume percentage," "volume %," and "v / v %" are used interchangeably in this document. The volume percentage of solute in a solution can be determined using the following formula: [(volume of solute) / (v volume of solution)] × 100%.

[0060] The terms “weight %”, “weight percentage (weight%)”, “weight-weight percentage (weight / weight%)”, etc., are used interchangeably herein. Weight % refers to the percentage of a substance by mass when it is contained in a composition, mixture, or solution. Unless otherwise specified, all percentages herein are weight percentages.

[0061] As used in this article, terms such as "polydispersity index," "PDI," "uniformity index," and "dispersion" refer to the molecular weight distribution in a measured sample of a given polymer (e.g., glucose oligomers, such as soluble α-glucan), and can be calculated by dividing the weight-average molecular weight by the number-average molecular weight (PDI = M). w / M n ).

[0062] The terms “increased,” “enhanced,” and “improved” are used interchangeably herein. These terms refer to a greater amount or activity, such as an amount or activity slightly greater than the initial amount or activity, or an amount or activity significantly exceeding the initial amount or activity, and include all amounts or activities in between. Alternatively, these terms may refer, for example, that the amount or activity is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% higher than the amount or activity compared to it.

[0063] As used herein, for polynucleotide or polypeptide sequences, the term "sequence identity" or "identity" refers to the presence of identical nucleic acid bases or amino acid residues in two sequences when aligned within a specified comparison window to obtain the maximum correspondence. Therefore, the "sequence identity percentage" or "identity percentage" refers to a value measured by comparing two optimally aligned sequences within a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) to achieve optimal alignment when compared to a reference sequence (which does not contain additions or deletions). This percentage is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues occur to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the sequence identity percentage.

[0064] The Basic Local Alignment Search Tool (BLAST) algorithm, available online at the National Center for Biotechnology Information (NCBI) website, can be used, for example, to calculate the percentage identity between two or more polynucleotide sequences (BLASTN algorithm) or peptide sequences (BLASTP algorithm) disclosed herein. Alternatively, the percentage identity between sequences can be calculated using the Clustal algorithm (e.g., ClustalW or ClustalV). For multiple alignments using the Clustal alignment method, the default values ​​can be equivalent to GAP PENALTY = 10 and GAP LENGTHPENALTY = 10. The default parameters for pairwise alignment and percentage identity calculation of protein sequences using the Clustal method can be KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5, and DIAGONALS SAVED = 5. For nucleic acids, these parameters can be KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4, and DIAGONALS SAVED = 4. Alternatively, the percentage of identity between sequences can be determined using the EMBOSS algorithm (e.g., the needle algorithm), with parameters such as GAP OPEN=10, GAPEXTEND=0.5, END GAP PENALTY=false, END GAP OPEN=10, END GAP EXTEND=0.5, using a BLOSUM matrix (e.g., BLOSUM62).

[0065] This document discloses various polypeptide amino acid sequences as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90-95% identical to the sequences disclosed herein may be used. Alternatively, the variant amino acid sequences may have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences disclosed herein. The variant amino acid sequences described herein have the same function / activity as the published sequences, or have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function / activity of the published sequences.

[0066] As used in some embodiments, the term "isolated" refers to any cellular component (e.g., isolated polynucleotide or polypeptide molecule) that is completely isolated from its natural source. In some cases, the isolated polynucleotide or polypeptide molecule is part of a larger composition, buffer system, or reagent mixture. For example, the isolated polynucleotide or polypeptide molecule may be contained heterologously within a cell or organism. Another example is isolated α-glucosidase (e.g., glucosylamylase, transglucosidase), or glucosyltransferase. The enzymatic reactions disclosed herein (e.g., α-glucosidase reactions, glucosyltransferase reactions) are synthetic, non-naturally occurring processes.

[0067] Some embodiments disclosed in this invention relate to a method for hydrolyzing the α-1,5-glucosyl-fructose bonds of a sugar containing at least one α-1,5-glucosyl-fructose bond. The sugar is a disaccharide or oligosaccharide. The method includes contacting the sugar with an α-glucosidase under suitable conditions. In the contacting step, the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of the sugar. Due to this hydrolysis, the amount of sugar is reduced compared to the amount of sugar present before the contacting step. Therefore, this hydrolysis method can also be referred to as a method for reducing the sugar content in a composition.

[0068] Significantly, it is believed that α-glucosidase can hydrolyze α-1,5-glucosyl-fructose bonds, which was previously unknown. The α-glucosidase reaction according to this hydrolysis method can therefore be used to remove Leuconostoc disaccharides containing α-1,5-glucosyl-fructose bonds and other oligosaccharide byproducts from dextran synthesis reactions and / or from the fractions obtained therefrom. Such removal represents an improvement over byproduct removal chemical processes (e.g., acid hydrolysis) that can lead to the degradation of dextran products. Ultimately, dextran reaction fractions treated according to the above hydrolysis method are more suitable for downstream applications such as fermentation, for example, because the content of glucose and fructose monosaccharides increases in the fraction. For downstream processes, monosaccharides are generally easier to handle than Leuconostoc disaccharides and oligosaccharide byproducts.

[0069] The α-glucosidase (EC 3.2.1.20) used in the embodiments herein hydrolyzes the α-1,5-glucosyl-fructose bonds of sugars containing at least one α-1,5-glucosyl-fructose bond. α-glucosidases have previously been identified for catalytic hydrolysis that releases terminally unreduced (1,4)-linked α-D-glucose residues from oligosaccharide (e.g., disaccharide) and polysaccharide substrates. These enzymes, now disclosed herein, also possess hydrolytic activity against, for example, α-1,5-glucosyl-fructose bonds.

[0070] Alpha-glucosidases can be obtained from any source, such as plants, animals, microorganisms, like bacteria or fungi / yeasts, including, for example, the transglucosidases and / or glucosylamylases disclosed below. For instance, an alpha-glucosidase can be a fungal alpha-glucosidase. Other examples of suitable alpha-glucosidases herein include those disclosed in U.S. Patents 6,355,467, 5,922,580, 5,795,766, 5,763,252, and 8,633,006, all of which are incorporated herein by reference.

[0071] In some embodiments, the α-glucosidase may include 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 the amino acid sequence of DIAZYME RDF ULTRA (DuPont Industrial Biosciences). Alternatively, the α-glucosidase may comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence 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 DIAZYME RDF ULTRA, and has hydrolytic activity against the α-1,5-glucosyl-fructose bond of sugars. Several of the aforementioned sequences are, for example, mature α-glucosidases lacking an N-terminal signal peptide. For such sequences, it should be understood that if they are expressed without a signal peptide (e.g., using an expression system in which the enzyme is expressed intracellularly and obtained from cell lysates), an N-terminal initiating methionine (if necessary) is typically added (directly or via the insertion of a heterologous amino acid sequence, such as an epitope).

[0072] Transglucosidase (EC 2.4.1.24; 1,4-α-glucan-6-α-glucosyltransferase) can be used as an α-glucosidase in some embodiments herein to hydrolyze the α-1,5-glucosyl-fructose bonds of sugars containing at least one α-1,5-glucosyl-fructose bond. Such enzymes have previously been identified as D-glucosyltransferases that catalyze both hydrolysis and transfer reactions when incubated with specific α-D-glucose-oligosaccharides. Transglucosidases as now disclosed herein also possess hydrolytic activity against α-1,5-glucosyl-fructose bonds.

[0073] The transglucosidases described in this article can be derived from any microbial source, such as bacteria or fungi. Examples of fungal transglucosidases include, but are not limited to, those from the genus *Trichoderma* (e.g., *Trichoderma reesei*), *Aspergillus*, and *Neosartorya* (e.g., *N. fischeri*). Examples of *Aspergillus* species from which transglucosidases can be derived include, but are not limited to, *Aspergillus niger*, *Aspergillus awamori*, *Aspergillus oryzae*, *Aspergillus terreus*, *Aspergillus clavatus*, *Aspergillus fumigatus*, and *Aspergillus nidulans*. Other examples of transglucosidases that can be used herein are described in Barker et al. (1953, J. Chem. Soc. 3588-3593); Pazur et al. (1986, Carbohydr. Res. 149: 137-147); Nakamura et al. (1997, J. Biotechnol. 53: 75-84); and U.S. Patent Application Publication 2008 / 0229514, all of which are incorporated herein by reference. Other examples of transglucosidases that can be used herein are those that are thermostable; U.S. Patent 4,689,296 discloses a method for preparing thermostable transglucosidases, the disclosure of which is incorporated herein by reference. Further examples of transglucosidases that can be used in this article are any of those in the GENBANK database (NCBI), such as accession number: D45356 (GID: 2645159, Aspergillus niger), BAD06006.1 (GID: 4031328, Aspergillus awamori), BAA08125.1 (GID: 1054565, Aspergillus oryzae), XP_001210809.1 (GID: 115492363, Aspergillus terreus), XP_001216899.1 (GID: 115433524, Aspergillus terreus). The following transglucosidases are incorporated herein by reference: XP_001271891.1 (GID: 121707620, Aspergillus lanceolata), XP_751811.1 (GID: 70993928, Aspergillus fumigatus), XP_659621.1 (GID: 67523121, Aspergillus nidus), XP_001266999.1 (GID: 119500484, N. fischeri), and XP_001258585.1 (GID: 119473371, N. fischeri). Alternatively, the transglucosidases described herein may have an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of any of the aforementioned disclosed transglucosidase sequences, and be hydrolyzed on the α-1,5-glucosyl-fructose bond of sugars.When all of the aforementioned transglucosidases are used in the hydrolysis reaction described herein, the mature form lacking the N-terminal signal peptide is preferred.

[0074] In some embodiments herein, the transglucosidase may comprise the amino acid sequence of SEQ ID NO: 1 (transglucosidase L-2000), which is an Aspergillus niger transglucosidase (U.S. Patent Application Publication 2008 / 0229514). Alternatively, the transglucosidase may comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 1, and be hydrolyzed on the α-1,5-glucosyl-fructose bond of sugars. Any SEQ ID NO: 1 or a variant thereof may be prepared, for example, according to the disclosure of U.S. Patent Application Publication 2008 / 0229514, which is incorporated herein by reference. SEQ ID NO: 1 is a mature transglucosidase lacking an N-terminal signal peptide. Because SEQ ID NO:1 does not begin with a methionine residue, it should be understood that if it is expressed without using a signal peptide (e.g., using an expression system in which the enzyme is expressed in the cell and obtained from cell lysates), an N-terminal starting methionine is usually added to SEQ ID NO:1 (directly or via the insertion of a heterologous amino acid sequence such as an epitope).

[0075] Glucoamylase (EC 3.2.1.3; α-1,4-glucan glucosylhydrolase) may be used as an α-glucosidase in some embodiments herein to hydrolyze the α-1,5-glucosyl-fructose bonds of sugars containing at least one α-1,5-glucosyl-fructose bond. This class of enzymes has previously been identified as extrinsic enzymes catalyzing the hydrolysis of both α-1,4 and α-1,6 glycosidic bonds at the non-reducing ends of disaccharides, oligosaccharides, and polysaccharides containing glucose. Glucoamylases as now disclosed herein also possess hydrolytic activity against α-1,5-glucosyl-fructose bonds. In some embodiments, the α-glucosidase is not a glucosidase.

[0076] The glucosylamylases described in this article can be derived from any microbial source, such as bacteria or fungi. Examples of bacterial glucosylamylases include, but are not limited to, those from the genus Bacillus (e.g., B. alkalophilus, B. amyloliquefaciens, B. lentus, B. licheniformis, B. stearothermophilus, B. subtilis, B. thuringiensis) and the genus Streptomyces (e.g., S. lividans). Examples of fungal glucosylamylases include, but are not limited to, species of the genus *Trichoderma* (e.g., *Trichoderma longibrachiatum*, *Trichoderma strictipilis*, *Trichoderma asperellum*, *Trichoderma konilangbra*, *Trichoderma hazianum*), species of the genus *Aspergillus* (e.g., *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus terreus*, *Aspergillus lanceolata*, *Aspergillus nidus*, *Aspergillus nidus*, *Aspergillus leucosus*, *Aspergillus amblympica*), species of the genus *Rhizopus* (e.g., *Rhizopus oryzae*, *Rhizopus niveus*), species of the genus *Talaromyces* (e.g., *T. emersonii*, *T. thermophilus*, *T. duponti*), species of the genus *Mucor*, and species of the genus *Hypocrea* (e.g., *H. g.*). Those species of the genera *Elatinosa*, *H. orientalis*, *H. vinosa*, *H. citrina*, *Fusarium* (e.g., *F. oxysporum*, *F. roseum*, *F. venenatum*), *Neurospora* (e.g., *N. crassa*), *Humicola* (e.g., *H. grisea*, *H. insolens*, *H. lanuginose*), *Penicillium* (e.g., *P. notatum*, *P. chrysogenum*), and *Saccharomycopsis* (e.g., *S. fibuligera*). Examples of these bacterial and fungal glucosylamylases used herein are disclosed in U.S. Patent Application Publication 2013 / 0102035, which is incorporated herein by reference.Other examples of glucosylamylases that can be used in this paper are Svensson et al. (1983, Carlsberg Res. Commun. 48: 529-544), Boel et al. (1984, EMBO) The patents described herein are found in J.3:1097-1102, Havashida et al. (1989, Agric. Biol. Chem. 53:923-929); U.S. Patents 5,024,941, 4,794,175, 4,247,637, 6,255,084, 6,620,924, Ashikari et al. (1986, Agric. Biol. Chem. 50:957-964), Ashikari et al. (1989, Appl. Microbiol. Biotechnol. 32:129-133), U.S. Patent 4,863,864, U.S. Patent 4,618,579, Houghton-Larsen et al. (2003, Appl. Microbiol. Biotechnol. 62:210-217), and U.S. Patent 7,413,887, all of which are incorporated herein by reference. Alternatively, the glucosylamylase described herein may have an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence of any of the aforementioned disclosed glucosylamylase sequences, and be hydrolyzed on the α-1,5-glucosyl-fructose bond of sugars. When all the aforementioned glucosylamylases are used in the hydrolytic reactions described herein, the mature form lacking the N-terminal signal peptide is preferred. Commercially available glucosylamylases that can be used herein include, for example, OPTIDEX L-400, GC 147, GC 321, GZYME G990 4X, OPTIMAX 7525, DEXTROZYME, DISTILLASE, and GLUCZYME.

[0077] In some embodiments herein, the glucosylamylase may comprise the amino acid sequence of SEQ ID NO: 2 (GC 321), which is *Trichoderma reesei* glucosylamylase. Alternatively, the glucosylamylase may comprise at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 2, and have hydrolytic activity against the α-1,5-glucosyl-fructose bond of sugars. Any SEQ ID NO: 2 or a variant thereof may be prepared, for example, according to the disclosure of U.S. Patent 7,413,887 or U.S. Patent Application Publication 2013 / 0102035, which are incorporated herein by reference. SEQ ID NO: 2 is a mature glucosylamylase lacking the N-terminal signal peptide. Because SEQ ID NO:2 does not begin with a methionine residue, it should be understood that if it is expressed without using a signal peptide (e.g., using an expression system in which the enzyme is expressed in the cell and obtained from cell lysates), an N-terminal starting methionine is usually added to SEQ ID NO:2 (directly or via the insertion of a heterologous amino acid sequence such as an epitope).

[0078] The α-glucosidases described herein, such as transglucosidases or glucosylamylases, are available from commercial sources (e.g., DuPont Industrial Biosciences / Genencor, USA; Megazyme International, Ireland; AmanoEnzyme Inc., Japan). Alternatively, such enzymes can be prepared by any method known in the art, such as that described in U.S. Patent Application Publication 2008 / 0229514, U.S. Patent 7413887, or U.S. Patent Application Publication 2013 / 0102035, which are incorporated herein by reference. For example, α-glucosidases can be recombinantly generated in heterologous expression systems, such as microbial or fungal heterologous expression systems. Examples of heterologous expression systems include bacterial (e.g., E. coli, Bacillus sp.) and eukaryotic systems. Eukaryotic systems can be expressed using, for example, yeast (e.g., *Pichiasp.*, *Saccharomyces sp.*) or fungal (e.g., *Trichoderma* species, such as *Trichoderma reesei*; *Aspergillus* species, such as *Aspergillus niger*) expression systems. The transglucosidase of SEQ ID NO: 1 and the glucosylamylase of SEQ ID NO: 2, as well as their variants, can be expressed, for example, in *Trichoderma reesei* hosts.

[0079] When α-glucosidase is used in the hydrolysis reaction described herein, a mature form lacking an N-terminal signal peptide is preferred. The expression system used to produce the mature α-glucosidase described herein may employ a polynucleotide encoding the enzyme, which also contains a sequence encoding an N-terminal signal peptide to direct extracellular secretion. In such embodiments, the signal peptide is cleaved from the enzyme during the secretion process. The signal peptide may be native or heterologous to transglucosidase or glucosylamylase. Alternatively, the mature form of α-glucosidase may be provided by expression (without using a signal peptide), for example, using an expression system in which the enzyme is expressed intracellularly and obtained from cell lysates. In either case (secretion or intracellular expression), a heterologous amino acid sequence, such as an epitope, may optionally be included at the N-terminus of the α-glucosidase.

[0080] In some embodiments, α-glucosidase can be provided in the hydrolysis reaction described herein by directly using cells expressing one or more enzymes. In other words, the α-glucosidase in contact with the sugar can be present due to its expression by cells placed under suitable conditions for hydrolysis. Such cells can therefore be used instead of isolated α-glucosidase preparations added to the hydrolysis reaction. Cells used for this purpose can be, for example, bacterial, yeast, or fungal cells. Examples of yeasts include those derived from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Kluyveromyces*, *Candida*, *Pichia*, *Schizosaccharomyces*, *Hansenula*, *Kloeckera*, and *Schwanniomyces*. Other expression systems that can be used herein are disclosed in U.S. Patent Application Publication 2013 / 0323822, which is incorporated herein by reference.

[0081] The sugars described herein contain at least one α-1,5-glucosyl-fructose bond. Therefore, depending on the length of the sugar, it may contain, for example, 1, 2, 3, 4, 5, 6, 7, or 8 α-1,5-glucosyl-fructose bonds. Sugars preferably contain 1, 2, or 3 such bonds.

[0082] Because the sugars described herein contain at least one α-1,5-glucosyl-fructose bond, the sugars contain at least one glucose unit and at least one fructose unit. In some embodiments, the sugars described herein contain only glucose and fructose units. Such compositions can act as disaccharide and oligosaccharide byproducts of dextran synthesis reactions. Alternatively, in addition to glucose and fructose, the sugars described herein may also contain other monosaccharides, such as galactose, ribose, and xylose.

[0083] In some embodiments of this invention, the hydrolyzed sugar may be an oligosaccharide. The oligosaccharide herein may have, for example, 2, 3, 4, 5, 6, 7, 8, or 9 monosaccharide units. As understood in the art, the oligosaccharide herein may be referred to by its degree of polymerization (DP) number—which specifies the number of monomer units in the oligosaccharide. For example, DP3 oligosaccharide has 3 monomer units. Therefore, the oligosaccharide may be, for example, DP3, DP4, DP5, DP6, DP7, DP8, or DP9 oligosaccharides. In some embodiments, the DP of the sugar is 3 to 7 (i.e., DP 3-7).

[0084] In addition to at least one α-1,5-glucosyl-fructose bond (note that oligosaccharides with two monosaccharide units—i.e., disaccharides—are Leuconostoc disaccharides, and the sugars given in the hydrolysis method described herein have at least one α-1,5-glucosyl-fructose bond), oligosaccharides with three or more monosaccharide units may also contain other bonds. For example, α-1,3, α-1,6, and / or α-1,4 bonds may also be present in oligosaccharides, which are also readily hydrolyzed by α-glucosidases as shown herein.

[0085] In some embodiments, the sugar comprises only glucose monomers linked by α-1,3 and / or α-1,6 glycosidic bonds. Therefore, such oligosaccharides contain only α-1,3-glucosyl-glucose bonds and / or α-1,6-glucosyl-glucose bonds. Examples of such oligosaccharides contain only α-1,3 or α-1,6 bonds. In some embodiments, the oligosaccharide may contain at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% glucosyl-glucose bonds. In other embodiments, about 75-85% α-1,3-glucosyl-glucose bonds and about 15-25% α-1,6-glucosyl-glucose bonds may be present in the oligosaccharides described herein. Alternatively, the oligosaccharides described herein may contain any percentage (any integer value between 1% and 99%) of α-1,3-glucose bonds and any percentage (any integer value between 1% and 99%) of α-1,6-glucose bonds, provided that these percentages do not exceed 100%. Any such oligosaccharides may be derived from fractions of dextran synthesis reactions that produce, for example, (i) insoluble α-glucans (e.g., poly-α-1,3-glucans), or (ii) soluble α-glucan products. The bond content may characterize: (i) individual oligosaccharides, or (ii) a group of oligosaccharides (i.e., average bond content). Oligosaccharides containing only glucose monomers linked via α-1,3 and / or α-1,6 glycosidic bonds may be, for example, DP2-DP7 or DP3-DP7. It should be understood that the specific distribution of bonds in oligosaccharides can vary depending on the conditions of the dextran synthesis reaction that produces oligosaccharide byproducts (e.g., GTF enzymes). It should also be understood that the specific distribution of bonds is not critical to the currently disclosed method.

[0086] The examples in this article demonstrate that α-glucosidases (e.g., transglucosidases and glucosylamylases) can hydrolyze (i) Leuconostoc disaccharides containing α-1,5-glucosyl-fructose bonds, and (ii) oligosaccharides containing only α-1,3-glucose and / or α-1,6-glucose bonds. Therefore, α-glucosidases can be used, for example, in reactions used to hydrolyze α-1,5-glucosyl-fructose bonds, α-1,3-glucose bonds, and / or α-1,6-glucose bonds.

[0087] At least one α-1,5-glucosyl-fructose bond of the sugar described herein can be hydrolyzed by the α-glucosidase described herein. Alternatively, it is believed that 2, 3, 4, 5, or more α-1,5-glucosyl-fructose bonds of the sugar can be hydrolyzed, for example, by an α-glucosidase. In some embodiments, the hydrolysis of at least one α-1,5-glucosyl-fructose bond can occur at the non-reducing end of the sugar. For example, where the sugar is a disaccharide, namely Leuconostoc disaccharide, the non-reducing glucose is cleaved from the fructose to obtain free glucose and fructose. As another example, where the sugar is an oligosaccharide having a non-reducing glucose α-1,5-linked to fructose, it is believed that this glucose can be cleaved, leaving a fructose residue at the non-reducing end of the oligosaccharide.

[0088] In the disclosed hydrolysis method, the amount of sugar is reduced compared to the amount of sugar present before the contact step. This reduction is due to the hydrolytic breaking of at least one α-1,5-glucosyl-fructose bond in the sugar. In the hydrolysis method described herein, the amount of sugar (e.g., concentration) after the contact step may be less than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any integer value between 1% and 90%) of the amount of sugar present before the contact step (before contacting the α-glucosidase described herein with the sugar under suitable conditions).

[0089] In some embodiments of this invention, the sugar hydrolyzed is Leuconostoc disaccharide, a disaccharide having an α-1,5-glucosyl-fructose bond. In the hydrolysis method described herein, the concentration of Leuconostoc disaccharide after the contact step may be less than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any integer value between 1% and 90%) of the concentration of Leuconostoc disaccharide present before the contact step (before contacting the α-glucosidase described herein with the Leuconostoc disaccharide under suitable conditions). In some aspects herein, the hydrolysis method may alternatively be referred to as a method for reducing the amount of Leuconostoc disaccharide in a composition.

[0090] In the hydrolysis method described herein, Leuconostoc mesenteroides can be contacted, for example, with a transglucosidase, such as a transglucosidase comprising SEQ ID NO: 1 (transglucosidase L-2000). In some embodiments, the concentration of Leuconostoc mesenteroides after completing this method may be about 1-3% less than the initial concentration of Leuconostoc mesenteroides.

[0091] The amount of sugar in the disclosed hydrolysis method is reduced compared to the amount of sugar present before the contact step. It should be understood that this comparison can be made in any manner. For example, the sugar concentration can be measured both before and after the hydrolysis process. Alternatively, comparisons can be made with a control reaction under the same conditions, except that an α-glucosidase as currently disclosed is not added to the control reaction.

[0092] In some embodiments, α-glucosidase may be immobilized. Enzymes can be immobilized using any methods and / or approaches known in the art, such as those disclosed in U.S. Patents 5,541,097 and 4,713,333, both of which are incorporated herein by reference. For example, one or more enzymes can be immobilized by contacting one or more enzymes with an amine-reactive material (e.g., glutaraldehyde) to form an adduct (e.g., an enzyme-glutaraldehyde adduct), and then binding the adduct to a solid support treated with a polyamine (e.g., polyethyleneimine, such as EPOMINP-1050).

[0093] In some embodiments, the solid support (solid carrier) to which α-glucosidase can be immobilized can be an inorganic or organic material. Such materials include, for example, γ-alumina, titanium dioxide, granular activated carbon, granular diatomaceous earth, glass beads, porous glass, pumice, silica gel, metal oxides, and alumina.

[0094] Polyamines can be used to treat solid supports, thereby exposing the solid supports to adducts comprising enzymes and amine reactive materials, resulting in enzyme binding to the solid supports. Examples of polyamines that can be used herein include: polyethylene diamine, polyethyleneimine (e.g., poly(diethylenetriamine), poly(triethylenetetramine), poly(pentaethylenehexamine), poly(hexamethylenediamine), poly(methylenedicyclohexylamine), poly(methylenediphenylamine), poly(tetraethylenepentamine), poly(phenylene diamine), and blends of two or more of these polyamine compounds. Preferred polyamines are water-soluble and / or have a molecular weight of about 500 to 100,000 Daltons. Polyethyleneimine, such as EPOMIN P-1050, may be used in some embodiments.

[0095] Amine-reactive materials used to prepare adducts containing the enzymes described herein may be, for example, aldehydes, organohalides, acid anhydrides, azo compounds, isothiocyanates, and / or isocyanates. Examples of such amine-reactive materials include: glutaraldehyde, succinal, terephthalaldehyde, di-diazobenzidine-2,2'-disulfonic acid, 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone, diphenyl-4,4'-dithiocyanate-2,2'-disulfonic acid, 3-methoxydiphenylmethane-4,4'-diisocyanate, toluene-2-isocyanate-4-isothiocyanate, toluene-2,4-diisocyanate, diazobenzidine, diazobenzidine-3,3'-o-anisidine, N,N'-hexamethylenediiodoacetamide, hexamethylenediisocyanate, cyanuric chloride, and / or 1,5-difluoro-2,4-dinitrobenzene. Preferably, the amine reactive material is an aldehyde, such as glutaraldehyde.

[0096] An α-glucosidase that adducts to amine-reactive compounds can be contacted with a polyamine-treated solid support, thereby immobilizing the enzyme on the solid support. The enzymes immobilized herein can be used in various reactor systems, such as column (e.g., packed column) or stirred tank reactors, to carry out the hydrolysis reactions as disclosed herein.

[0097] Suitable conditions for contacting the sugars described herein with the α-glucosidases described herein (e.g., transglucosidase or glucosylamylase) are those conditions that support the hydrolysis of one or more α-1,5-glucosyl-fructose bonds of the sugar by the α-glucosidase. Examples of suitable conditions are disclosed in the following embodiments. Conditions (e.g., temperature, pH, time) for contacting the α-glucosidases described herein with the sugar substrate are also disclosed in U.S. Patent Application Publication 2008 / 0229514, U.S. Patent 7,413,887, and U.S. Patent Application Publication 2013 / 0102035 (all of which are incorporated herein by reference) and are also applicable to the disclosed hydrolysis methods.

[0098] In the disclosed hydrolysis methods, disaccharides and oligosaccharides are generally soluble in water or aqueous solutions. Therefore, contacting the sugars described herein with α-glucosidase is preferably carried out under suitable aqueous conditions in which the sugars are dissolved. Aqueous conditions may be a solution or mixture containing at least about 20% by weight of water. Alternatively, the aqueous conditions described herein are, for example, at least about 20, 30, 40, 50, 60, 70, 80, 85, 90, or 95% by weight of water (or any integer value between 20 and 95% by weight). Aqueous conditions may also include, for example, a buffer solution of suitable concentration, such as an acidic, neutral, or alkaline buffer solution, selected based on the pH range provided by the buffer solution. Examples of buffer solutions / buffers include citrates, acetates (e.g., sodium acetate), KH₂PO₄, MOPS, CHES, borates, sodium carbonate, and sodium bicarbonate.

[0099] The pH of the hydrolysis reaction described herein may, for example, be from about 3.0 to 9.0. The pH of the hydrolysis reaction may, for example, be 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 may be about 4-5. Techniques for setting the pH include the use of, for example, buffer solutions, bases, and / or acids, and are well known in the art.

[0100] The hydrolysis reaction temperature described herein can be, for example, from about 20°C to about 80°C. The hydrolysis reaction temperature can be, for example, about 20, 30, 40, 50, 60, 70, or 80°C (or any integer value between 20 and 80°C). In some embodiments, a hydrolysis temperature of about 60°C, 65°C, or 60-65°C is preferred.

[0101] The hydrolysis reaction described herein can proceed over a period of, for example, at least about 10 minutes to about 90 hours. The hydrolysis reaction time can be, for example, at least about 0.5, 1, 2, 3, 4, 8, 12, 16, 20, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, or 90 hours (or any integer value between 0.5 and 72 hours). In some embodiments, such as the hydrolysis of Leuconostoc disaccharide, the hydrolysis reaction can, for example, proceed for less than 4 hours (e.g., 0.5-4 hours). The time period required to achieve the desired level of hydrolysis will vary depending on the specific conditions used, and will be understood by those skilled in the art. For example, increasing the amount of enzyme added to the reaction or immobilized on a solid carrier used for the reaction will reduce the contact time.

[0102] In some embodiments, one or more α-glucosidases described herein may be used in the hydrolysis reaction. For example, both transglucosidase and glucosylamylase may be used in the reaction. In the hydrolysis reaction described herein, the amount of α-glucosidase may be increased / decreased by, for example, 10% to 20% (or 5% to 10%) compared to any amount used in the following examples (e.g., Example 2). Alternatively, about 0.1-0.5 vol% or 0.1-1.0 vol% of α-glucosidase may be used in the hydrolysis reaction. Still alternatively, the α-glucosidase described herein may be used in the hydrolysis reaction at about, or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 ppm. A transglucosidase unit (TGU) may be defined, for example, as the amount of transglucosidase that produces one micromolar of panose per minute under the conditions determined below. Transglucosidase activity can be determined, for example, as follows: Transglucosidase is introduced into a 100 mM sodium acetate buffer (pH 4.5) containing 4 mM p-nitrophenyl-α-glucosidase and 1 mg / ml bovine serum albumin (BSA). After incubation at 30°C for 30 minutes, the reaction is terminated by adding an equal volume of 1 M sodium carbonate, and the OD is recorded. 405 A glucosylamylase unit (XU) can be defined, for example, as the amount of glucosylamylase that produces 1g of reducing sugar, calculated as glucose per hour from a soluble starch substrate (4% DS [degree of substitution]) at pH 4.2 and 60°C.

[0103] In some embodiments disclosed in this invention, the initial concentration of sugars in the hydrolysis reaction may, for example, be from about 1% to 50% by weight. For example, the concentration of Leuconostoc disaccharide may be about 5, 10, 15, 20, 25, 30, 35, or 40% by weight (or any integer value between 5 and 40% by weight). Similarly, in the hydrolysis reaction described herein, the concentration of one or more oligosaccharides (e.g., DP2, DP3, DP4, DP2-DP7, DP3-DP7) may 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 will recognize that the concentration of total sugars (including disaccharides and oligosaccharides) can affect the activity of α-glucosidase; in some aspects, the preferred total sugar concentration for maximizing enzyme activity in the hydrolysis reaction may be less than 50% by weight dry solids (DS), and the most preferred concentration is 20-35% by weight DS.

[0104] In some embodiments, suitable conditions for contacting the sugar with the α-glucosidase described herein may include: (i) a dextran synthesis reaction, or (ii) a fraction obtained from a dextran synthesis reaction; wherein the sugar is a byproduct of the dextran synthesis reaction. In other words, the hydrolysis reaction described herein may be carried out within or as a part of a dextran synthesis reaction, although it is generally carried out within the latter. The dextran synthesis reaction described herein may, for example, produce one or more insoluble and / or soluble α-glucan products. Therefore, in some embodiments herein, the dextran synthesis reaction may be characterized as “α-glucan synthesis reaction”.

[0105] Dextran synthesis reactions typically involve a solution containing at least one sucrose, water, an active glucosyltransferase, and optional other components. Other components that may be present in a dextran synthesis reaction include fructose, glucose, Leuconostoc disaccharide, soluble oligosaccharides (e.g., DP2-DP7), and one or more soluble dextran products. Additionally, in some aspects, the dextran synthesis reaction may include one or more α-glucan hydrolases. It should be understood that certain dextran products, such as poly-α-1,3-glucans with a DP of at least 8 or 9, may be water-insoluble and therefore insoluble in the dextran synthesis reaction, but may precipitate out of the solution. Therefore, the dextran produced by the dextran synthesis reaction described herein may be insoluble. The α-glucosidase described herein may be added to the dextran synthesis reaction at any stage, such as during the initial preparation of the reaction or when the reaction is near completion (e.g., 80 to 90% complete) or complete, with the latter two time points being preferred.

[0106] In addition to producing the dextran product, the dextran synthesis reaction described herein may also produce byproducts such as Leuconostoc disaccharide and / or soluble oligosaccharides. In some respects, the dextran is a poly-α-glucan. Therefore, the dextran synthesis reaction described herein can, for example, be used to produce poly-α-1,3-glucan or dentin dextran, which is typically produced in conjunction with at least one Leuconostoc disaccharide and / or oligosaccharide byproduct during the dextran synthesis reaction.

[0107] In some embodiments, the dextran synthesis reaction includes a glucosyltransferase that produces polyα-glucan, such as α-1,3-glucan. Examples of such glucosyltransferases that may be used herein are disclosed in U.S. Patent 7,000,000 and U.S. Patent Application Publications 2013 / 0244288, 2013 / 0244287, and 2014 / 0087431, all of which are incorporated herein by reference.

[0108] The glucosyltransferases described in this article can be derived from any microbial source, such as bacteria or fungi. Examples of bacterial glucosyltransferases include those derived from species of the genus *Streptococcus*, *Leuconostoc*, or *Lactobacillus*. Examples of *Streptococcus* species include *Lactobacillus salivarius*, *Streptococcus sobrinus*, *Streptococcus denirousetti*, *Streptococcus downei*, *Streptococcus mutans*, *Streptococcus oralis*, *Streptococcus gallolyticus*, and *Streptococcus sanguinis*. Examples of species in the genus *Leuconostoc* include *L. mesenteroides*, *L. amelibiosum*, *L. argentinum*, *L. carnosum*, *L. citreum*, *L. cremoris*, *L. deextranicum*, and *L. fructosum*. Examples of species in the genus *Lactobacillus* 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*.

[0109] The glucosyltransferases described herein may be primer-independent or primer-dependent. Primer-independent glucosyltransferases do not require the presence of primers for glucan synthesis. During polymer synthesis, primer-dependent glucosyltransferases require the presence of a starting molecule in the reaction solution to act as an enzyme primer. As used herein, the term "primer" refers to any molecule capable of serving as an initiator for glucosyltransferases. Primers that can be used in certain embodiments include, for example, dextran and other carbohydrate-based primers, such as hydrolyzed dextran. U.S. Patent Application Publication 2013 / 0244287 (incorporated herein by reference) discloses the preparation of hydrolyzed dextran using poly-α-1,3-glucan as a starting material. The dextran used as a primer may be, for example, dextran T10 (i.e., a dextran with a molecular weight of 10 kDa).

[0110] The glucosyltransferases used in the dextran synthesis reactions described herein can be produced by any method known in the art. For example, glucosyltransferases can be recombinantly produced in heterologous expression systems, such as microbial heterologous expression systems. Examples of heterologous expression systems include bacterial (e.g., Escherichia coli, such as TOP10 or MG1655; Bacillus) and eukaryotic (e.g., yeast, such as Pichia pastoris and Saccharomyces) expression systems.

[0111] The glucosyltransferases described herein can be used in any purified state (e.g., pure or impure). For example, the glucosyltransferases may be purified and / or isolated before use. Examples of impure glucosyltransferases include those in the form of cell lysates. Cell lysates or extracts can be obtained from bacteria (e.g., *Escherichia coli*) used for heterologous expression of the enzyme. For example, the bacteria can be destroyed using a French crusher. In an alternative embodiment, the bacteria can be homogenized using a homogenizer (e.g., APV, Rannie, Gaulin). Glucosyltransferases are generally soluble in these types of formulations. The bacterial cell lysates, extracts, or homogenates described herein can, for example, be used in a reaction solution at about 0.15–0.3% (v / v) to produce polyalpha-glucan, such as polyalpha-1,3-glucan, from sucrose.

[0112] The temperature of the dextran synthesis reaction described herein can be controlled if desired. In some embodiments, the reaction temperature is from about 5°C to about 50°C. In some other embodiments, the temperature is from about 20°C to about 40°C.

[0113] In the dextran synthesis reaction described herein, the initial concentration of sucrose can be, for example, from about 20 g / L to about 400 g / L. Alternatively, the initial concentration of sucrose can be from about 75 g / L to about 175 g / L, or from about 50 g / L to about 150 g / L. Still alternatively, the initial concentration of sucrose can be, for example, from about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 g / L (or any integer value between 40 and 160 g / L). "Initial concentration of sucrose" refers to the concentration of sucrose in the GTF reaction solution only after all reaction solution components (at least water, sucrose, and GTF enzyme) have been added.

[0114] The sucrose used in the dextran synthesis reactions described herein can be of high purity (≥99.5%) or any other purity or grade. For example, the sucrose can have a purity of at least 99.0% or can be reagent-grade sucrose. Alternatively, partially refined sucrose can be used. In this context, partially refined sucrose refers to sucrose that has not been processed into refined white sucrose. Therefore, partially refined sucrose can be completely unrefined or partially refined. Examples of unrefined sucrose are “crude sucrose” (“raw sugar”) and solutions thereof. Examples of partially refined sucrose are those that have not undergone one, two, three, or more crystallization steps. The ICUMSA (International Commission for Uniform Methods of Sugar Analysis) of the partially refined sucrose described herein can, for example, be greater than 150. The sucrose described herein can be derived from any renewable sugar source, such as sugarcane, sugar beets, cassava, sweet sorghum, or corn. Suitable forms of sucrose that can be used herein are, for example, crystalline or amorphous forms (e.g., syrup, cane juice, beet juice). Another suitable form of unrefined sucrose is disclosed in U.S. Application 61 / 969,958.

[0115] Methods for determining the sucrose ICUMSA value are well known in the art and are disclosed, for example, by the International Commission for Uniform Methods of Sugar Analysis. ICUMSA Methods of Sugar Analysis:Official and Tentative Methods Recommended by the International Commission for Uniform Methods of Sugar A nalysis (ICUMSA) (HCS de Whalley, ed., Elsevier Pub. Co., 1964), this citation is incorporated herein by reference. ICUMSA can be seen, for example, from RJ McCowage, RMUrquhart, MLBurge. (Determination of the Solution Color of Raw Sugars, Brown Sugars and Colored Syrups at pH 7.0-OfficialThe GS1 / 3-7 determination of the ICUMSA method described in Verlag Dr. Albert Bartens (2011 revised edition) is incorporated herein by reference.

[0116] In some embodiments, the pH of the dextran synthesis reaction may be from about 4.0 to about 8.0. Alternatively, the pH may be from 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 a suitable buffer solution, including but not limited to phosphates, tris, citrates, or combinations thereof. The concentration of the buffer solution for the dextran synthesis reaction may be, for example, from 0 mM to about 100 mM, or from about 10, 20, or 50 mM.

[0117] The poly-α-1,3-glucan produced in the dextran synthesis reaction described herein may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any integer value between 50% and 100%) α-1,3-glycosidic bonds. In such embodiments, the poly-α-1,3-glucan has less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or 0% (or any integer value between 0% and 50%) non-α-1,3-glycosidic bonds.

[0118] The poly-α-1,3-glucan described herein preferably has a linear / unbranched backbone. In some embodiments, the poly-α-1,3-glucan has no branching points or has less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branching points (as a percentage of glycosidic bonds in the polymer). Examples of branching points include α-1,6 branching points.

[0119] The molecular weight of the poly-α-1,3-glucan produced in the dextran synthesis reaction in this paper can be determined as a number-average molecular weight (M). n or weight-average molecular weight (M) w Alternatively, molecular weight can be determined in Daltons or grams per mole. It can also be used to refer to the DP of poly(α-1,3-glucan) polymers. w (weight-average degree of polymerization) or DP n (Number-average degree of aggregation)

[0120] The Mn or Mw of the poly-α-1,3-glucan described herein may be at least about 1000. Alternatively, Mn or Mw may, for example, be at least about 1000 to about 600,000 (or any integer value between 1000 and 600,000). Still alternatively, the poly-α-1,3-glucan may have a DPn or DPw with a molecular weight of at least about 100, or at least about 100 to 1000 (or any integer value between 100 and 1000).

[0121] The fractions of the dextran synthesis reaction can provide suitable conditions for contacting the sugar with α-glucosidases as disclosed herein. The fractions can be partial or complete liquid solutions derived from the dextran synthesis reaction. Typically, the fractions are separated from one or more soluble or insoluble dextran products synthesized in the reaction. For example, the fractions can be separated from one or more water-insoluble dextran products (e.g., poly-α-1,3-glucan) that precipitate from the solution during their synthesis. In certain preferred embodiments of this disclosure, the fractions are derived from the poly-α-1,3-glucan synthesis reaction.

[0122] In some embodiments, the volume of the fraction (see below before optionally diluting or concentrating the fraction) may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (or any integer value between 10% and 90%) of the volume of the dextran synthesis reaction from which the fraction is obtained. Typically, in dextran synthesis reactions that produce insoluble dextran (e.g., poly-α-1,3-glucan), the fraction will be a portion (not all) of the liquid solution component of the reaction. The fraction can be obtained at any stage of the dextran synthesis reaction, but is preferably obtained near completion (e.g., greater than 80% or 90% of completion) or after completion.

[0123] In some embodiments, examples of fractions from the dextran synthesis reaction include filtrate and supernatant. Therefore, in those embodiments where insoluble dextran products are synthesized, the fractions described herein can be obtained (separated) from the dextran synthesis reaction using funnels, filters (e.g., filter presses), centrifuges, or any other methods or apparatus known in the art that allow the removal of some or all of the liquid from the solids. Filtration can be carried out, for example, by gravity, vacuum, or pressure filtration. Filtration preferably removes all or most of the insoluble dextran; any filter material (e.g., filter paper) with an average pore size (e.g., about 40-50 micrometers) sufficient to remove solids from the liquid can be used. The fraction typically retains all or most of its dissolved components, such as byproducts of the dextran synthesis reaction. In the filtrate described herein, Leuconostoc disaccharide is a preferred sugar.

[0124] If desired, the fractions described herein may be optionally diluted or concentrated. Concentration of the fractions may be carried out using any other method or apparatus known in the art suitable for concentrating solutions. For example, the fractions may be concentrated by evaporation, such as using a rotary evaporator (e.g., set to a temperature of about 40-50°C). In some aspects of this document, the fractions may be concentrated to a volume of about 75%, 80%, 85%, 90%, or 95% of the initial fraction volume. The concentrated fraction (e.g., the concentrated filtrate) may optionally be referred to as a syrup.

[0125] The fraction may contain water in some respects, which replaces the water present in the composition from which the fraction is obtained. For example, one or more sugar byproducts can be separated from a dextran synthesis reaction in certain chromatographic methods in which the initial solvent is replaced by another solvent (e.g., sugar byproducts bound to the column [thereby being removed from the initial solvent] can be eluted into the new solvent).

[0126] In some respects, the fraction can be processed in such a way that it has any of the suitable conditions (e.g., temperature, pH, and reagents) disclosed above for contacting the sugar with α-glucosidase. For example, the fraction can be altered to have a pH of about 4 to 5 before the α-glucosidase is added to it. Also, the temperature for the hydrolysis reaction of the fraction can be about 55-65°C (e.g., about 60°C). Furthermore, the fraction already concentrated into a syrup can be used for the hydrolysis reaction.

[0127] In certain preferred embodiments described herein, fractions are obtained from the poly-α-1,3-glucan synthesis reaction; for example, the fraction is preferably a filtrate. The fractions from the poly-α-1,3-glucan synthesis reaction described herein comprise at least water, fructose, and one or more types of sugars (Leuconostoc disaccharides and / or oligosaccharides, such as DP2-DP7). Other components that may be 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 solution, salt, etc. Borate, sodium hydroxide, hydrochloric acid, cell lysate components, proteins and / or nucleic acids. Minimally, the components of fractions derived from poly-1,3-glucan synthesis reactions include, for example, water, fructose, glucose, one or more types of sugars (Leuconostoc disaccharides and / or oligosaccharides, such as DP2-DP7), and optionally sucrose. It should be understood that the composition of the fraction depends on the conditions of the glucan synthesis reaction from which the fraction is obtained. In those fractions containing one or more GTF enzymes, preferably, such one or more GTF enzymes are inactivated (e.g., heat-inactivated) prior to use in the hydrolysis reactions described herein.

[0128] It should be understood that the specific distribution of sugar byproducts generated via sucrose polymerization in the dextran synthesis reaction can vary based on the reaction conditions and GTF enzymes used, particularly temperature and sucrose concentration. It should also be understood that the specific composition of sugars in the fractions of the dextran synthesis reaction is not critical to the disclosed hydrolysis method. Generally, as the amount of sucrose increases, the selectivity of the reaction for both Leuconostoc disaccharides and oligosaccharides increases. Conversely, as the temperature increases, the selectivity for Leuconostoc disaccharides tends to decrease, while the selectivity for oligosaccharides remains largely unaffected. It should be understood that the sugar-to-water ratio, calculated by dividing the mass of sugar by the total solution weight (wt% dry solids (DS), can be adjusted by evaporating water (preferably under vacuum and at temperatures below 50°C) or by adding water, without substantially affecting the relative distribution of sugars in the fractions of the dextran synthesis reaction. The percentage of sucrose in the fraction can also be increased by terminating the GTF reaction before achieving complete conversion (to dextran), which is achieved by lowering the pH below the activity range of the GTF enzyme or by thermally inactivating the GTF enzyme.

[0129] In some embodiments, the dextran synthesis reaction described herein may produce one or more soluble α-glucan products. The soluble α-glucan products (or "soluble fibers") may be: (i) direct products of glucosyltransferases, or (ii) products of the synergistic action of both glucosyltransferases and α-glucan hydrolases, said α-glucan hydrolases being capable of hydrolyzing dextran polymers having one or more α-1,3-glycosidic bonds or one or more α-1,6-glycosidic bonds.

[0130] The soluble α-glucans described herein may include, for example:

[0131] a) At least 75% α-1,3-glycosidic bonds;

[0132] b) Less than 25% α-1,6-glycosidic bonds;

[0133] c) Less than 10% α-1,3,6-glycosidic bonds;

[0134] d) M less than 5000 Daltons w ;

[0135] e) Viscosity less than 0.25 Pa·s in water at 20°C and 12% by weight;

[0136] f) Dextran equivalents (DE) ranging from 4 to 40;

[0137] g) Digestibility less than 10%, as measured by the Association of Analytical Communities (AOAC) method 2009.01;

[0138] h) Solubility in water at pH 7 at 25°C at at least 20% (w / w); and

[0139] i) Polydispersion index (PDI) less than 5.

[0140] Such soluble α-glucans can be prepared as disclosed in US patent application 62 / 004,290.

[0141] For example, the soluble α-glucan fiber composition may contain at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% α-(1,3) glycosidic bonds.

[0142] For example, in addition to the above-described α-(1,3) glycosidic bond embodiments, the soluble α-glucan fiber composition may also contain less than 25%, preferably less than 10%, more preferably 5% or less, and even more preferably less than 1% α-(1,6) glycosidic bonds.

[0143] For example, in addition to the above-described embodiments for the content of α-(1,3) and α-(1,6) glycosidic bonds, the soluble α-glucan fiber composition may also contain less than 10%, preferably less than 5%, and most preferably less than 2.5% of α-(1,3,6) glycosidic bonds.

[0144] For example, the soluble α-glucan fiber composition may contain 93 to 97% α-(1,3) glycosidic bonds and less than 3% α-(1,6) glycosidic bonds, and has a weight-average molecular weight corresponding to a mixed DP of 3 to 7. In another embodiment, the soluble α-glucan fiber composition may contain about 95% α-(1,3) glycosidic bonds and about 1% α-(1,6) glycosidic bonds, and has a weight-average molecular weight corresponding to a mixed DP of 3 to 7. In another aspect of the above embodiments, the soluble α-glucan fiber composition may contain about 1 to 3% α-(1,3,6) bonds or preferably about 2% α-(1,3,6) bonds.

[0145] For example, in addition to the glycosidic bond content embodiments mentioned above, the soluble α-glucan fiber composition may also contain less than 5%, preferably less than 1%, and most preferably less than 0.5% α-(1,4) glycosidic bonds.

[0146] For example, in addition to the glycosidic bond content embodiments mentioned above, the soluble α-glucan fiber composition may also include a weight-average molecular weight (M) of less than 5000 Daltons, preferably less than 2500 Daltons, more preferably 500 to 2500 Daltons, and most preferably about 500 to about 2000 Daltons. w ).

[0147] For example, in addition to any of the above characteristics, the soluble α-glucan fiber composition may also include a viscosity of less than 250 centipoise (0.25 Pa·s), preferably less than 10 cP (0.01 Pa·s), preferably less than 7 cP (0.007 Pa·s), more preferably less than 5 cP (0.005 Pa·s), more preferably less than 4 cP (0.004 Pa·s), and most preferably less than 3 cP (0.003 Pa·s) at 20°C and 12 wt% in water.

[0148] In some embodiments, as determined by Association of Analytical Communities (AOAC) Method 2009.01, the soluble α-glucan fiber composition may have a digestibility of less than 10%, preferably less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. Alternatively, the relative level of digestibility may also be determined using AOAC 2011.25 (Integrated Total Dietary Fiber Assay) (McCleary et al., 2012, J. AOAC Int., 95(3), 824-844).

[0149] In addition to any of the above embodiments, the soluble α-glucan fiber composition has a solubility of at least 20% (w / w), preferably at least 30%, 40%, 50%, 60% or 70%, in water at pH 7 at 25°C.

[0150] In one embodiment, the soluble α-glucan fiber composition may contain reducing sugars in a content of less than 10% by weight, preferably less than 5% by weight, and most preferably less than 1% by weight or less.

[0151] In one embodiment, the soluble α-glucan fiber composition may include less than 4 kcal / g, preferably less than 3 kcal / g, more preferably less than 2.5 kcal / g, and most preferably about 2 kcal / g or less of calories.

[0152] For example, the soluble α-glucan in this article may include:

[0153] a) 10% to 30% α-1,3-glycosidic bonds;

[0154] b) 65% to 87% α-1,6-glycosidic bonds;

[0155] c) Less than 5% α-1,3,6-glycosidic bonds;

[0156] d) Weight-average molecular weight (Mw) less than 5000 Daltons;

[0157] e) Viscosity less than 0.25 Pa·s in water at 20°C and 12% by weight;

[0158] f) A dextran equivalent (DE) in the range of 4 to 40, preferably 10 to 40;

[0159] g) Digestibility less than 10%, as measured by the Association of Analytical Communities (AOAC) method 2009.01;

[0160] h) Solubility in water at pH 7 at 25°C at at least 20% (w / w); and

[0161] i) Polydispersion index (PDI) less than 5.

[0162] Such soluble α-glucans can be prepared as disclosed in US patent application 62 / 004,308.

[0163] For example, the soluble α-glucan in this article may include:

[0164] a) 25-35α-1,3-glycosidic bonds;

[0165] b) 55-75% α-1,6-glycosidic bonds;

[0166] c) 5-15% α-1,3,6-glycosidic bonds;

[0167] d) Weight-average molecular weight less than 5000 Daltons;

[0168] e) Viscosity less than 0.25 Pa·s in water at 20°C and 12% by weight;

[0169] f) Dextran equivalents (DE) ranging from 4 to 40;

[0170] g) Digestibility less than 10%, as measured by the Association of Analytical Communities (AOAC) method 2009.01;

[0171] h) Solubility in water of at least 20% (w / w) at 25°C; and

[0172] i) A polydispersity index less than 5.

[0173] Such soluble α-glucans can be prepared as disclosed in US patent application 62 / 004,312.

[0174] For example, the soluble α-glucan in this article may include:

[0175] a) At least 95% α-1,6-glycosidic bonds;

[0176] b) 1% or less of α-1,3-glycosidic bonds;

[0177] c) Less than 2% α-1,3,6-glycosidic bonds;

[0178] d) Less than 1.5% α-1,4-glycosidic bonds;

[0179] e) Weight-average molecular weight less than 20,000 Daltons;

[0180] f) Viscosity less than 0.25 Pa·s in water at 20°C and 12% by weight:

[0181] g) Dextran equivalents (DE) ranging from 1 to 30;

[0182] h) Digestibility less than 10%, as measured by the Association of Analytical Communities (AOAC) method 2009.01;

[0183] i) Solubility in water at pH 7 at 25°C of at least 20% (w / w); and

[0184] j) A polydispersity index less than 5.

[0185] Such soluble α-glucans can be prepared as disclosed in U.S. Patent Application 62 / 004,314.

[0186] For example, the soluble α-glucan in this article may include:

[0187] a) The scope is:

[0188] i) 1% to 50% of α-1,3-glycosidic bonds; or

[0189] ii) More than 10% but less than 40% of α-1,4-glycosidic bonds; or

[0190] iii) any combination of i) and ii);

[0191] b) 1 to 50% α-1,2-glycosidic bonds;

[0192] c) 0-25% α-1,3,6-glycosidic bonds;

[0193] d) Less than 98% of α-1,6-glycosidic bonds;

[0194] e) Weight-average molecular weight less than 300 kDa;

[0195] f) Viscosity less than 0.25 Pa·s in water at 20°C and 12% by weight;

[0196] g) Digestibility less than 20%, as measured by the Association of Analytical Communities (AOAC) method 2009.01;

[0197] h) Solubility in water at pH 7 at 25°C at at least 20% (w / w); and

[0198] i) The polydispersity index is less than 26, preferably less than 5.

[0199] Such soluble α-glucans can be prepared as disclosed in US patent application 62 / 004,305.

[0200] In some embodiments, the soluble α-glucan is a direct product of a glucosyltransferase. In suitable dextran synthesis reactions, such glucosyltransferases and the conditions used therein may be as disclosed herein, or as disclosed in any U.S. patent application, such as 62 / 004,290, 62 / 004,308, 62 / 004,312, 62 / 004,314, and / or 62 / 004,305.

[0201] Soluble α-glucan can also be a product of the synergistic action of, for example, a glucosyltransferase and an α-glucan hydrolase, wherein the α-glucan hydrolase is capable of hydrolyzing dextran polymers having one or more α-1,3-glycosidic bonds or one or more α-1,6-glycosidic bonds. In some aspects, the dextran synthesis reaction for producing a soluble α-glucan product may include both at least one glucosyltransferase and at least one α-glucan hydrolase. In other aspects, the dextran synthesis reaction may initially contain one or more glucosyltransferases as the sole enzymatic component. Such a reaction produces a first α-glucan that has not yet been modified by an α-glucan hydrolase. Then, at least one α-glucan hydrolase is added to the reaction at a suitable time to allow the first product to be modified into a soluble α-glucan product. Thus, there are different ways to synthesize a soluble α-glucan product via the synergistic action of both a glucosyltransferase and an α-glucan hydrolase. During and / or after dextran synthesis, the conditions for carrying out the dextran synthesis reaction (which includes one or more α-glucan hydrolases) may be as disclosed herein or as disclosed in any U.S. patent application such as 62 / 004,290, 62 / 004,308, 62 / 004,312, 62 / 004,314 and / or 62 / 004,305.

[0202] The α-glucan hydrolases described herein may be, for example, glucanase (capable of hydrolyzing α-1,6-linked glycosidic bonds; EC 3.2.1.11), mutant enzyme (capable of hydrolyzing α-1,3-linked glycosidic bonds; EC 3.2.1.59), mycoglucanase (capable of endo-hydrolyzing the (1-4)-α-D-glycosidic bonds of α-D-glucans containing both (1-3)- and (1-4)- bonds; EC 3.2.1.61), glucan 1,6-α-glucosidase (EC 3.2.1.70), and alternating glucanase (EC 3.2.1.-; capable of endo-hydrolyzing alternating glucans; EC 3.2.1.-; see US Patent 5786196).

[0203] The mutant enzyme including SEQ ID NO: 47 may be used in certain aspects. Alternatively, the mutant enzyme may, for example, contain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO: 47 and have mutant enzyme activity.

[0204] The dextran synthesis reactions disclosed herein for producing one or more soluble α-glucan products can be directly used as suitable conditions for carrying out the hydrolysis reactions described herein, wherein α-glucosidase is used to hydrolyze the α-1,5-glucosyl-fructose bonds. Such hydrolysis can be carried out according to any of the above-disclosed conditions relating to the hydrolysis treatment of dextran synthesis reactions for producing, for example, poly-α-1,3-glucan. Alternatively, fractions (e.g., chromatographic fractions) of dextran synthesis reactions for producing one or more soluble α-glucan products can be used as suitable conditions for α-glucosidase-mediated hydrolysis of the α-1,5-glucosyl-fructose bonds.

[0205] In some embodiments herein, the fraction may be a chromatographic fraction of a dextran synthesis reaction. For example, the fraction may be a chromatographic fraction of a dextran synthesis reaction that produces one or more soluble α-glucan products as disclosed herein. Such reactions may optionally include one or more α-glucan hydrolases during and / or after dextran synthesis. Typically, the fraction is obtained in any of these types of embodiments to separate all or most (e.g., at least about 60%, 70%, 80%, 90%, 95%) of the soluble α-glucan product from the reaction composition that produces it. Once separated from all or most of the soluble α-glucan product, the fraction may be subjected to any of the α-1,5-glucosyl-fructose hydrolysis processes disclosed herein using one or more α-glucanases.

[0206] The chromatographic fractions described in this article can typically be obtained using a suitable type of liquid chromatography. Liquid chromatography can be performed, for example, using size exclusion chromatography (SEC), column chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, affinity chromatography, ultrafiltration, microfiltration, or dialysis.

[0207] This disclosure also relates to a composition produced by contacting a sugar with an α-glucosidase (e.g., transglucosidase or glucosidase), wherein (i) the sugar is a disaccharide or oligosaccharide containing at least one α-1,5-glucosyl-fructose bond, and (ii) the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of the sugar. The composition produced in this manner contains less sugar than the amount present prior to contact. Examples of compositions include those disclosed herein, such as hydrolyzed filtrates from dextran synthesis reactions, or hydrolyzed fractions from dextran synthesis reactions used to produce soluble α-glucans. Any characteristics of the hydrolysis methods and products disclosed above and in the examples may characterize the composition. The following characteristics of the composition are examples.

[0208] In certain embodiments of the composition, the α-glucosidase may comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence 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 DIAZYME RDF ULTRA (DuPont Industrial Biosciences). In certain embodiments of the composition, the transglucosidase may comprise at least 90% of the amino acid sequence identical to SEQ ID NO: 1. In certain embodiments of the composition, the glucosylamylase may comprise at least 90% of the amino acid sequence identical to SEQ ID NO: 2. Alternatively, any α-glucosidase disclosed herein may be used to produce the disclosed compositions.

[0209] The concentration of sugars, such as Leuconostoc disaccharide, in the compositions produced by the hydrolysis method described herein may be, for example, less than 50% of the concentration of Leuconostoc disaccharide present before the sugar is contacted with α-glucosidase.

[0210] In some embodiments herein, the composition produced by the hydrolysis method may be a dextran synthesis reactant or a fraction thereof, wherein a byproduct of the dextran synthesis reaction is contacted with α-glucosidase. In this embodiment, the fraction may be, for example, a filtrate from a dextran synthesis reaction, or a fraction from a dextran synthesis reaction used to produce soluble α-glucan. In this embodiment, the sugar may be, for example, Leuconostoc disaccharide.

[0211] Those skilled in the art should understand that the currently disclosed embodiments are partly used for the saccharification of disaccharides and oligosaccharides that may be difficult to break down by other means. For example, this feature can be used to implement enhanced methods: (i) fructose enrichment and (ii) fermentation.

[0212] Example 6 below shows that, compared to using unhydrolyzed filtrate, using dextran filtrate hydrolyzed by α-glucosidase (transglucosidase) enhances fructose enrichment by chromatography.

[0213] Therefore, the disclosed invention also relates to a method for enriching fructose present in fractions of a dextran synthesis reaction. The method comprises: (a) contacting a fraction obtained from the dextran synthesis reaction with an α-glucosidase (e.g., transglucosidase or glucosylamylase) under suitable conditions, wherein the enzyme hydrolyzes at least one α-1,5-glucosyl-fructose bond of a disaccharide or oligosaccharide contained within the fraction; and (b) separating fructose from the hydrolyzed fraction of step (a) to obtain a composition with a higher fructose concentration than that of the fraction of step (a).

[0214] Features of disclosed methods for fructose enrichment, such as those relating to fractions of α-glucosidase (e.g., transglucosidase or glucosylamylase) and dextran synthesis reactions, can be found in any disclosure provided herein relating to each of these features.

[0215] Step (b) of fructose separation can be performed by any method known to those skilled in the art. For example, chromatography can be used as disclosed in the following examples or according to European Patent Publication EP2292803B1, which is incorporated herein by reference.

[0216] Compositions with higher fructose concentrations obtained from disclosed enrichment methods (e.g., fructose solutions or fructose syrups) may have at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% fructose by weight.

[0217] The fructose enrichment method described herein performs better than methods using filtrates that have not been hydrolyzed by α-glucosidases as currently disclosed. This enhanced performance can be determined by a fructose recovery percentage of at least 40%, 45%, or 50%.

[0218] This disclosure also relates to a fermentation method comprising: (a) contacting a fraction obtained from a dextran synthesis reaction with an α-glucosidase (e.g., transglucosidase or glucosylamylase) under suitable conditions, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of a disaccharide or oligosaccharide contained in the fraction; (b) fermenting the fraction of step (a) with a microorganism to obtain a product; and (c) optionally separating the product of (b). The fermentation step of step (b) may be performed after or simultaneously with step (a). Notably, ethanol can be produced using this method, for example, by fermenting the hydrolyzed filtrate of a dextran synthesis reaction. The ethanol yield obtained from this process is higher than that obtained by fermenting an unhydrolyzed dextran filtrate.

[0219] Features of a disclosed fermentation method relating to α-glucosidase (e.g., transglucosidase or glucosylamylase), disaccharides and oligosaccharides, fractionation of the glucan synthesis reaction, and suitable contact conditions may be found, for example, in any disclosure provided herein relating to each of these features.

[0220] The microorganisms used in the fermentation methods described herein may be, for example, bacteria, yeast, or fungi. Examples of bacteria that may be used herein include species of the genus *Lactobacillus*, *Streptococcus*, *Bifidobacterium*, *Leuconostoc*, *Escherichia* (e.g., *Escherichia coli*), and *Bacillus*. Examples of yeasts that may be used herein include species of the genus *Saccharomyces*, such as *Saccharomyces cerevisiae* and *Saccharomyces bayanus*.

[0221] The fermentation methods described herein can yield products such as ethanol or acids (e.g., lactic acid). However, it is believed that other products can be produced if desired. Those skilled in the art will understand that the production of a particular product using fermentation methods such as those disclosed will depend on various conditions, such as the one or more microorganisms used for fermentation. For example, the conditions for fermentation described herein may be as disclosed in the following examples, or as described by E1-Mansi et al. (2006, Fermentation Microbiology and Biotechnology (Second edition, CRC Press) and Stanbury et al. (1999, Principles of Fermentation Technology The second edition of the book, published by Butterworth-Heinemann, is cited in this article.

[0222] In some embodiments of the fermentation method described herein, the product yield is higher than that obtained by fermenting a dextran filtrate that has not been hydrolyzed by the α-glucosidase described herein. This comparison can be made with reference, for example, a control fermentation using the unhydrolyzed fraction of the dextran synthesis reaction. The product yield of the fermentation described herein can, for example, be increased by at least about 10%, 20%, 40%, 60%, 80%, or 100% (or any integer value between 10% and 100%). Furthermore, the rate of product formation by the fermentation described herein can be increased.

[0223] Example 7 below shows that Leuconostoc disaccharide can be fermented into ethanol by yeast fed with a feed containing an unhydrolyzed dextran filtrate. Therefore, a method for fermenting Leuconostoc disaccharide into a product (e.g., ethanol) using microorganisms is further disclosed herein. This method may include fermenting (i) a dextran filtrate that has been or (ii) not hydrolyzed by an α-glucosidase as disclosed herein. Whether Leuconostoc disaccharide is provided in the dextran filtrate or in another form (e.g., semi-purified or enriched form), the method for fermenting Leuconostoc disaccharide may include adapting microorganisms (e.g., yeast, such as Saccharomyces cerevisiae) to utilize Leuconostoc disaccharide. Such adaptation may include growing the microorganisms in the presence of Leuconostoc disaccharide and optionally other sugars for at least two or three growth cycles, after which the microorganisms utilize more Leuconostoc disaccharide to ferment the product. In some embodiments, the microorganism may (i) grow in a first feed containing Leuconostoc mesenteroides (one complete cycle), (ii) be removed from the first feed, (iii) grow in a second feed containing Leuconostoc mesenteroides (two complete cycles), (iv) optionally be removed from the second feed, and (v) optionally grow in a third feed (three complete cycles). In some embodiments, microorganisms adapted in this manner may have an increased ability to ferment Leuconostoc mesenteroides.

[0224] Example 9 below shows that when the dextran filtrate is fermented by yeast while being hydrolyzed by transglucosidase, almost all (e.g., >98% or >99%) of the Leuconostoc disaccharide present in the dextran filtrate can be used for yeast fermentation. Therefore, the enhanced Leuconostoc disaccharide fermentation method described herein may include hydrolyzing the Leuconostoc disaccharide with an α-glucosidase (e.g., transglucosidase or glucosylamylase) while simultaneously fermenting the Leuconostoc disaccharide with microorganisms.

[0225] Non-limiting examples of the compositions and methods disclosed herein include:

[0226] 1. A method for hydrolyzing the α-1,5-glucosyl-fructose bond in a sugar comprising at least one α-1,5-glucosyl-fructose bond, wherein the sugar is a disaccharide or oligosaccharide, and wherein the method comprises:

[0227] The sugar is brought into contact with α-glucosidase under suitable conditions, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of the sugar.

[0228] Furthermore, the sugar content is reduced compared to the sugar content present before contact.

[0229] 2. The method according to implementation scheme 1, wherein the α-glucosidase is immobilized.

[0230] 3. The method according to implementation scheme 1 or 2, wherein the sugar is Leuconostoc disaccharide.

[0231] 4. The method according to embodiment 3, wherein the concentration of Leuconostoc disaccharide after the contact step is less than 50% of the concentration of Leuconostoc disaccharide present before the contact.

[0232] 5. The method according to implementation scheme 1, 2, 3 or 4, wherein suitable conditions include:

[0233] (i) Dextran synthesis reaction, or

[0234] (ii) Fractions obtained from the dextran synthesis reaction;

[0235] Sugars are byproducts of the dextran synthesis reaction.

[0236] 6. The method according to embodiment 5, wherein the dextran synthesis reaction produces at least one insoluble α-glucan product.

[0237] 7. The method according to Implementation Scheme 6, wherein the fraction is the filtrate from the dextran synthesis reaction.

[0238] 8. The method according to embodiment 5, wherein the dextran synthesis reaction produces at least one soluble α-glucan product, which is:

[0239] (i) products of glucosyltransferase, or

[0240] (ii) A product of the synergistic action of glucosyltransferase and α-glucan hydrolase, wherein the α-glucan hydrolase is capable of hydrolyzing glucan polymers having one or more α-1,3-glycosidic bonds or one or more α-1,6-glycosidic bonds.

[0241] 9. The method according to implementation scheme 8, wherein the fraction is the chromatographic fraction of the dextran synthesis reaction.

[0242] 10. The method according to any one of embodiments 1-9, wherein the α-glucosidase is a transglucosidase or a glucosylamylase.

[0243] 11. A composition produced by contacting a sugar with an α-glucosidase,

[0244] The sugar is a disaccharide or oligosaccharide and contains at least one α-1,5-glucosyl-fructose bond.

[0245] The enzyme hydrolyzes at least one α-1,5-glucosyl-fructose bond in the sugar.

[0246] Furthermore, the composition contains less sugar than the amount of sugar present before contact.

[0247] 12. The composition according to embodiment 11, wherein the sugar is Leuconostoc disaccharide.

[0248] 13. The composition according to embodiment 11 or 12, wherein the sugar is in (i) a dextran synthesis reaction, or (ii) a fraction obtained from a dextran synthesis reaction;

[0249] Sugars are byproducts of the dextran synthesis reaction.

[0250] 14. A method for enriching fructose present in fractions of a dextran synthesis reaction, the method comprising:

[0251] (a) Contacting a fraction obtained from a dextran synthesis reaction with an α-glucosidase under suitable conditions, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of the disaccharide or oligosaccharide contained in the fraction; and

[0252] (b) Separate fructose from the hydrolyzed fraction of step (a) to obtain a composition with a higher fructose concentration than that of the fraction of step (a).

[0253] 15. A fermentation method, the method comprising:

[0254] (a) Contacting a fraction obtained from a dextran synthesis reaction with an α-glucosidase under suitable conditions, wherein the α-glucosidase hydrolyzes at least one α-1,5-glucosyl-fructose bond of a disaccharide or oligosaccharide contained in the fraction.

[0255] (b) Obtaining the product by fractionation of step (a) using microbial fermentation, wherein fermentation is carried out after or simultaneously with step (a); and

[0256] (c) Optionally, separate the product of (b);

[0257] Among them, the product yield of (b) is increased compared to the product yield of fermenting fractions of the dextran synthesis reaction that have not been contacted with α-glucosidase.

[0258] Example

[0259] The disclosed invention will be further illustrated in the following embodiments. It should be understood that although these embodiments illustrate certain preferred aspects of the invention, they are given by way of example only. Through the foregoing discussion and these embodiments, those skilled in the art can identify the essential features of the invention, and various changes and modifications can be made to the invention to suit various uses and conditions without departing from the spirit and scope of the invention.

[0260] abbreviation

[0261] The meanings of some abbreviations used in this article are as follows: "g" refers to grams, "h" refers to hours, "mL" refers to milliliters, "psi" refers to pounds per square inch, "wt%" refers to weight percentage, "μm" refers to micrometers, "%" refers to percentage, "℃" refers to degrees Celsius, "mg" refers to milligrams, "mm" refers to millimeters per minute, "mL / min" refers to millimeters per minute, "m" refers to meters, "uL" refers to microliters, "mmol" refers to millimoles, "min" refers to minutes, "mol%" refers to mol%, "M" refers to moles, "mg / g" refers to milligrams per gram, "rpm" refers to revolutions per minute, and "MPa" refers to megapascals.

[0262] General methods

[0263] Unless otherwise specified, all reagents are available from Sigma-Aldrich (St. Louis, MO). Sucrose is available from VWR (Radnor, PA).

[0264] Preparation of crude extract of glucosyltransferase (GTF)

[0265] The *Streptococcus salivarius* gtfJ enzyme (SEQ ID NO: 3) was expressed in *Escherichia coli* strain DH10B using an isopropyl β-D-1-thiogalactopyranoside (IPTG)-induced expression system. Compared to the *Streptococcus salivarius* gtfJ amino acid sequence (GENBANK identification number 47527), SEQ ID NO: 3 has a 42-residue deletion at the N-terminus but includes a starting methionine residue. In short, *E. coli* DH10B cells were transformed to express SEQ ID NO: 3 using a codon-optimized DNA sequence, thereby expressing the gtfJ enzyme in *E. coli*. This DNA sequence is included in the expression vector. (DNA 2.0, Menlo Park CA). Transformed cells were seeded at an initial optical density (OD, at 600). nm The culture medium (10 g / L tryptone; 5 g / L yeast extract, 10 g / L NaCl) with a concentration of 0.025 was prepared and incubated at 37°C with stirring at 250 rpm. When the OD value reached 0.8–1.0, the culture was cultured.600 Add 1 mM IPTG to induce the culture. Place the induced culture on a shaker and harvest after 3 hours of induction.

[0266] By making the cultured cells in The enzyme GtfJ (SEQ ID NO: 3) was harvested by centrifugation (25°C, 16000 rpm). Cells were resuspended in 5.0 mM phosphate buffer (pH 7.0) and cooled to 4°C on ice. Cells were lysed using a granulator with 0.1-mm silica beads, followed by centrifugation at 4°C and 16000 rpm to precipitate undisturbed cells and cell debris. The crude extract (containing the soluble GtfJ enzyme, SEQ ID NO: 3) was separated from the precipitate, and protein concentration (mg / mL) was determined by the Bradford protein assay.

[0267] The following describes the preparation of the Streptococcus spp. C150gtf-S enzyme (SEQ ID NO: 40). SG1184 is a Bacillus subtilis expression strain that expresses a truncated form of Streptococcus spp. C150 (SEQ ID NO: 40). The glycosyltransferase Gtf-S (“GTF0459”) from the *E. coli* expression plasmid pMP79 (SEQ ID NO: 41) was cloned into the NheI and HindIII sites of the *Bacillus subtilis* integrated expression plasmid p4JH under the action of the aprE promoter, and then fused with the *Bacillus subtilis* AprE signal peptide. The construct was first transformed into *E. coli* DH10B and selected on LB agar plates containing ampicillin (100 μg / mL). The confirmed GTF0459-expressing construct pDCQ984 was then transformed into Bacillus subtilis BG6006 containing nine protease deletions (amyE:xylRPxylAcomK-ermC, degUHy32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB) and selected on LB agar plates containing chloramphenicol (5 μg / mL). Colonies grown on LB agar plates containing 5 μg / mL chloramphenicol were streaked several times onto LB agar plates containing 25 μg / mL chloramphenicol. The resulting Bacillus subtilis expression strain SG1184 was first grown on LB medium containing 25 μg / mL chloramphenicol and then subcultured on Grants II medium containing 25 μg / mL chloramphenicol at 30°C for 2–3 days. The culture was centrifuged at 15,000 g and 4°C for 30 min, and the supernatant was filtered through a 0.22 μm filter. The filtered supernatant was aliquoted and frozen at -80°C.

[0268] Bacillus subtilis strain SG1184 expressing GTF0459 (SEQ ID NO: 42) was grown under deep-water aerobic conditions using conventional fed-batch fermentation. A nutrient medium containing 0-0.25% solid corn steep liquor (Roquette), 5-25 g / L sodium and potassium phosphate, 0.3-0.6 M ferrous sulfate, manganese chloride, and calcium chloride, 0.5-4 g / L magnesium sulfate, and 0.01-3.7 g / L zinc sulfate, cuprous sulfate, boric acid, and citric acid was used. Foaming was controlled with 2-4 mL / L of FOAMBLAST 882 antifoaming agent. When the initial glucose in the batch was undetectable, 10⁻⁶ L of 50% (w / w) glucose was added to the fermentation feed. The glucose feed rate increased over several hours. Fermentation was controlled at 30°C and 20% DO, with initial stirring at 750 rpm. The pH was maintained at 7.2 with 50% (v / v) ammonium hydroxide. During the 2-day fermentation run, fermentation parameters such as pH, temperature, air flow rate, and DO%. At the end of the run, the culture broth was harvested and centrifuged to obtain the supernatant. The supernatant containing GTF0459 (SEQ ID NO: 42) was then frozen and stored at -80°C.

[0269] The *Streptococcus mutans* MT-4239gtf-C enzyme (SEQ ID NO: 43) was prepared as follows. A truncated form of glucosyltransferase (gtf) encoded by codons optimized for expression in *Bacillus subtilis* was synthesized. The gene identified as GI: 3130088, SEQ ID NO: 43, derived from *Streptococcus mutans* MT-4239 (gtf-C) and synthesized using GenScript, was amplified by the GENSCRIPT plasmid. The gene encoding GTF0088BsT1 (SEQ ID NO: 45), with N-terminal and C-terminal T1 truncation, was cloned into the NheI and HindIII sites of the *Bacillus subtilis* integrative expression plasmid p4JH under the action of the aprE promoter and fused with the *Bacillus subtilis* AprE signal peptide on the vector. The construct was first transformed into *Escherichia coli* DH10B and selected on LB plates containing ampicillin (100 μg / mL). The confirmed GTF0088BsT1-expressing construct pDCQ1021 was then transformed into Bacillus subtilis BG6006 containing nine protease deletions (amyE: xylRPxylAcomK-ermC, degUHy32, oppA, ΔspoIIE3501, ΔaprE, ΔnprE, Δepr, ΔispA, Δbpr, Δvpr, ΔwprA, Δmpr-ybfJ, ΔnprB) and selected on LB agar plates containing chloramphenicol (5 μg / mL). Colonies grown on LB agar plates containing 5 μg / mL chloramphenicol were streaked several times onto LB agar plates containing 25 μg / mL chloramphenicol. The resulting Bacillus subtilis expression strain SG1221 was first grown on LB medium containing 25 μg / mL chloramphenicol and then subcultured on Grants II medium containing 25 μg / mL chloramphenicol at 30°C for 2–3 days. The culture was centrifuged at 15,000 g and 4°C for 30 min, and the supernatant was filtered through a 0.22 μm filter. The filtered supernatant was aliquoted and frozen at -80°C.

[0270] Bacillus subtilis strain SG1221 expressing GTF0088BsT1 (SEQ ID NO: 45) was grown under deep-water aerobic conditions using conventional fed-batch fermentation. A nutrient medium containing 0-0.25% corn steep liquor (Roquette), 5-25 g / L sodium and potassium phosphate, 0.3-0.6 M ferrous sulfate, manganese chloride, and calcium chloride, 0.5-4 g / L magnesium sulfate, and 0.01-3.7 g / L zinc sulfate, cuprous sulfate, boric acid, and citric acid was used. Foaming was controlled with 2-4 mL / L of antifoaming agent FOAMBLAST882. When the initial glucose in the batch was undetectable, 2-L of 50% (w / w) glucose was added to the fermentation feed. The glucose feed rate increased over several hours. Fermentation was controlled at 30°C and 20% DO, with initial stirring at 400 rpm. The pH was maintained at 7.2 with 50% (v / v) ammonium hydroxide. During the 2-day fermentation run, fermentation parameters such as pH, temperature, air flow rate, and DO%. At the end of the run, the culture broth was harvested and centrifuged to obtain the supernatant. The supernatant containing GTF088BsT1 (SEQ ID NO: 45) was then frozen and stored at -80°C.

[0271] Glucosyltransferase GTF0459 Determination of GTF0088BsT1 activity

[0272] The glucosyltransferase activity assay was performed as follows: A 1–10% (v / v) crude protein extract containing GTF enzyme was incubated with 25 mM or 50 mM sodium acetate buffer (pH 5.5) containing 200 g / L sucrose in the presence or absence of 25 g / L dextran (MW 1500, Sigma-Aldrich, Cat. #31394) at 37 °C and 125 rpm with rocking. Aliquots of the reaction mixture were removed at 1 h, 2 h, and 3 h and heated at 90 °C for 5 min to inactivate GTF. Insoluble material was removed by centrifugation at 13,000 x g for 5 min, followed by filtration through a 0.2 μm RC (regenerated cellulose) membrane. Sucrose concentration was quantified by HPLC analysis of the filtrate (Bio-Rad, Hercules, CA) using two tandem AMINEX HPX-87C columns at 85 °C. The sucrose concentration at each time point relative to the reaction time was plotted, and the initial reaction rate was determined by the slope of the linear curve. One GTF activity unit is defined as the amount of enzyme required to consume one micromole of sucrose per minute under the assay conditions.

[0273] Preparation of crude extract of α-(1,3)-glucan hydrolase (mutant enzyme)

[0274] The code is Penicillium marneffei. 18224 TM mutant enzyme ( The gene identified as GI: 212533325 was synthesized by GenScript (Piscataway, NJ). Under the control of the CBHI promoter and terminator, the nucleotide sequence (SEQ ID NO: 46) encoding the protein sequence (MUT3325; SEQ ID NO: 47) was subcloned into the SacII and AscI restriction sites of plasmid pTrex3, which is the vector designed to express the target gene in *Trichoderma reesei*. *Aspergillus niger* acetamase was used for selection. The resulting plasmid was transformed into *Trichoderma reesei* via gene gun injection. The specific method of gene gun transformation is described in International PCT Patent Application Publication WO2009 / 126773 A1, the disclosure of which is incorporated herein by reference. A 1-cm sample of spores from a stable clone was used… 2 The culture medium (described below) was prepared by inoculating with TRM05-3 agar sticks. The culture was grown in shake flasks at 28°C and 220 rpm for 4–5 days. To harvest the secreted protein, the cell population was first removed by centrifugation at 4000g for 10 minutes, and the supernatant was filtered through a 0.2 μm sterile filter. The expression of the mutant enzyme MUT3325 (SEQ ID NO: 47) was confirmed by SDS-PAGE.

[0275] The following lists the components for preparing the culture medium.

[0276] NREL-Trich Lactose Defined

[0277]

[0278]

[0279] Trace elements of Trichoderma reesei

[0280]

[0281] Fermentation seed cultures were prepared by inoculating 0.5 L of basal medium (basal medium consisting of 5 g / L ammonium sulfate, 4.5 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 14.4 g / L anhydrous citric acid, 1 g / L calcium chloride dihydrate, 25 g / L glucose, and trace elements including 0.4375 g / L citric acid, 0.5 g / L ferrous sulfate heptahydrate, 0.04 g / L zinc sulfate heptahydrate, 0.008 g / L copper sulfate pentahydrate, 0.0035 g / L manganese sulfate monohydrate, and 0.002 g / L boric acid, pH 5.5) into 8 L of preparation medium in a 14 L fermenter. The cultures were grown at 32 °C and 170 rpm for 48 hours before being transferred to 8 L of preparation medium in a 14 L fermenter. The culture medium consisted of 75 g / L glucose, 4.5 g / L potassium dihydrogen phosphate, 0.6 g / L calcium chloride dihydrate, 1.0 g / L magnesium sulfate heptahydrate, 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 copper sulfate pentahydrate, 0.0035 g / L manganese sulfate monohydrate, 0.002 g / L boric acid, and 0.3 mL / L FOAMBLAST 882.

[0282] For batches grown on glucose at 34°C and 500 rpm for 24 h, fermentation was run first. At the end of 24 h, the temperature was lowered to 28°C and the stirring speed was 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 run, the biomass was removed by centrifugation, and the supernatant containing the MUT3325 mutant enzyme (SEQ ID NO: 47) was concentrated approximately 10-fold by filtration using a 10-kD molecular weight cutoff ultrafiltration cartridge (UFP-10-E-35; GE Healthcare, Little Chalfont, Buckinghamshire, UK). The concentrated protein was stored at -80°C.

[0283] Determination of α-glucan hydrolase (mutant enzyme) activity

[0284] Used from distantly related Streptococcus sobrinus 33478 TM The secreted enzymes produced are used to prepare insoluble dental plaque dextran polymers required for determining the activity of the mutant enzymes. Specifically, a loopful of distantly related streptococci is inoculated onto BHI agar plates (BrainHeart Infusion agar, Teknova, Hollister, CA). 33478 TM The glycerol culture was prepared and the plates were incubated at 37°C for 2 days. Several colonies were picked by looping and inoculated into 2 x 100 mL BHI liquid medium in the initial culture flask (obtained from Teknova), and the cultures were incubated statically at 37°C for 24 h. The cells were removed by centrifugation and the resulting supernatant was filtered through a 0.2 μm sterile filter; 2 x 10¹ mL of filtrate were collected. 2 x 11.2 mL of 200 g / L sucrose (final sucrose 20 g / L) was added to the filtrate. The reaction was incubated at 37°C for 67 h without stirring. The obtained polysaccharide polymer was collected by centrifugation at 5000 x g for 10 min. The supernatant was carefully decanted. The insoluble polymer was washed four times with 40 mL of sterile water. The resulting dentin dextran polymer was lyophilized for 48 h. Dental plaque dextran polymer (390 mg) was suspended in 39 mL of sterile water to prepare a 10 mg / mL suspension. The dental plaque dextran suspension was homogenized by sonication (40% amplitude until large clumps disappeared, total ~10 min). The homogenized suspension was aliquoted and stored at 4 °C.

[0285] The mutant enzyme assay was initiated by incubating an appropriate amount of enzyme in 25 mM KOAc buffer containing 0.5 mg / mL dentin dextran polymer (prepared as described above) at pH 5.5 and 37 °C. At each time point, aliquots of the reaction mixture were removed and quenched with an equal volume of 100 mM glycine buffer (pH 10). Insoluble material in each quenched sample was removed by centrifugation at 14,000 x g for 5 min. The reducing end of the oligosaccharide and polysaccharide polymers produced at each time point was quantified by determination of p-hydroxybenzoic acid hydrazide solution (PAHBAH) (Lever M., Anal. Biochem., (1972) 47: 273-279), and the initial rate was determined by the slope of the linear curve at the first three or four time points in the time process. PAHBAH assay was performed by adding 10 μL of the reaction sample supernatant to 100 μL of PAHBAH working solution and heating at 95 °C for 5 min. The working solution was prepared by mixing one part of reagent A (0.05 g / mL p-hydroxybenzoyl hydrazide and 5% (v / v) concentrated hydrochloric acid) with four parts of reagent B (0.05 g / mL NaOH and 0.2 g / mL potassium sodium tartrate). The absorbance at 410 nm was recorded, and the concentration at the reducing end was calculated by subtracting appropriate background absorption and using standard curves generated using glucose at various concentrations (as standards).

[0286] Reaction characterization was performed using HPLC.

[0287] Periodic sampling is performed on the reaction using a device equipped with a refractive index detector. Analysis was performed using a 1260 HPLC system. The flow rate was 0.6 mL / min, and the temperature was 85 °C, using deionized water. HP-87C column (BioRad, Hercules, CA) was used to determine the content of sucrose, glucose, Leuconostose, and fructose in the GTF reaction. The reaction was carried out at a flow rate of 0.6 mL / min and 85 °C using a column equipped with deionized water. HP-42A column (BioRad) was used to quantitatively determine soluble oligosaccharide byproducts (DP2-DP7) in the GTF reaction.

[0288] Equipped with a refractive index detector UltiMate TM A 3000 HPLC (Thermo Scientific) was used for samples including immobilized enzymes (Example 4). The flow rate was 0.3 mL / min and the temperature was 85°C, using deionized water. Rezex TM Calcium monosaccharide columns were used to analyze sugars.

[0289] Oligosaccharides were analyzed by NMR.

[0290] Using a 5-mm cryogenic triple resonance pulsed field gradient (PFG) probe at 500MHz (for 1 NMR data were acquired on an Agilent DD2 spectrometer operating at H). Water suppression was achieved by carefully placing the observed emitter frequency at the resonance of the residual water signal from the Presat experiment, and then using the first data slice from the NOESY experiment with a full phase cycle (over 32) and a mixing time of 10 ms. One-dimensional 1 The H-spectroscopy was acquired with a spectral width of 6410 Hz, an acquisition time of 5.1 s, 65536 data points, a 4 s pre-saturation, and a 5.85 μs 90-degree pulse. The sample temperature was maintained at 25 °C. The sample was prepared by adding 50 μL of D2O, 450 μL of D2O, and 60 μL of D2O containing a 12.4 mM DSS (sodium 4,4-dimethyl-4-methylsilylpentane-1-sulfonate) internal standard to a 5-mm NMR tube, with the methyl resonance set to 0 ppm. The chemical shift localizations of different anomeric bonds were obtained from: Goffin et al. (2009, Bull Korean Chem. Soc. 30: 2535-2541). The peak distribution for α(1,3) bonds was 5.35 ppm, for Leuconostoc disaccharide it was 5.1 ppm, and for α(1,6) bonds it was 4.95 ppm. The peak distribution for the αRE reducing end (RE) was 5.2 ppm, and for βRE it was 4.65 ppm.

[0291] Example 1

[0292] Syrup preparation by polymerizing sucrose

[0293] This embodiment discloses a general method for producing a mixture of soluble sugars by polymerizing sucrose using a GTF enzyme in a dextran synthesis reaction. Specifically, a filtrate from the dextran synthesis reaction is prepared and then concentrated into a syrup.

[0294] Add 3000g of sucrose to a clean 5-gallon polyethylene tank. Add 18.1L of water and Fermasure. TM 10 mL was added to the container, and the pH was adjusted to 7.0 by adding 5 vol% NaOH and 5 vol% H2SO4. The final volume was approximately 20 L, and the initial sucrose concentration was 152.5 g / L as determined by HPLC. The dextran polymerization was initiated by adding 0.3 vol% of a crude GTF enzyme (SEQ ID NO: 3) extract prepared as described in the General Methods section. This extract contained approximately 2.9 mg / mL of protein. The reaction solution was stirred using an overhead mechanical motor equipped with a glass shaft and PTFE blades.

[0295] After 48 hours, HPLC analysis showed that 96% of the sucrose had been consumed, and the reaction was considered complete. The insoluble poly-α-1,3-glucan product was removed by filtration through a Buchner filter funnel using 325-mesh wire mesh and 40-micron filter paper. The mother liquor (filtrate) was then concentrated to a total sugar concentration of approximately 320 g / L using a rotary evaporator (bath temperature 40-50°C). The composition of the concentrated filtrate is provided in Table 2.

[0296] Table 2

[0297] Composition of the concentrated filtrate from the dextran synthesis reaction

[0298] sucrose Leuconostoc disaccharide glucose fructose DP2 DP3+ Total g / L 13.5 130.6 25.5 103.8 18.3 28.3 320.1 wt% 4.2 40.8 8 32.4 5.7 8.9 100

[0299] Table 2 indicates that the concentrated filtrate from the dextran synthesis reaction contains sucrose, fructose, glucose, Leuconostoc disaccharide, and oligosaccharides DP2-DP7.

[0300] Example 2

[0301] Effect of enzymes on sugar hydrolysis in the filtrate of dextran synthesis reaction

[0302] This example measured the activities of various glucosidases (EC 3.2.1.3), transglucosidases (EC 2.4.1.24), β-glucosidases (EC 3.2.1.21), α-amylases (EC 3.2.1.1), and glucosidases (EC 3.2.1) to reduce the concentration of Leuconostoc disaccharide and / or oligosaccharide byproducts in the concentrated filtrate from the dextran synthesis reaction. Certain enzymes, such as DIAZYME RDF ULTRA, transglucosidase (EC 2.4.1.24), and glucosidase (EC 3.2.1.3), which are all α-glucosidases, were found to be particularly effective in reducing the amount of these byproducts, resulting in a corresponding increase in monosaccharides (glucose and fructose) in the treated filtrate.

[0303] According to the process summary of Example 1, the filtrate from the dextran synthesis reaction was first prepared and then concentrated into a syrup. The composition of the concentrated filtrate is provided in Table 3. NMR analysis showed that the ratio of α(1,3) to (1,6) bonds present in the syrup was 78:22.

[0304] Table 3

[0305] Composition of the concentrated filtrate from the dextran synthesis reaction

[0306]

[0307]

[0308] The syrups in Table 3 were used to test the hydrolytic activity of various enzymes on the byproducts of Leuconostoc disaccharide and oligosaccharides from the dextran synthesis reaction. Assuming that Leuconostoc disaccharide contains a specific bond [α(1,5)-glucosylfructose] and that the oligosaccharides primarily contain α(1,3) and α(1,6)-glucosyl-glucose bonds, it was not immediately obvious which enzymes could be used to hydrolyze these two byproducts at the start of these experiments. For the purposes of this analysis, enzymes with different activities were selected (Table 4).

[0309] Table 4

[0310] Enzyme evaluation for the hydrolysis of Leuconostoc disaccharides and oligosaccharides.

[0311]

[0312] a DuPont Industrial Biosciences

[0313] The conditions for treating the syrups in Table 3 with the enzymes listed above are provided in Table 5 (enzyme load, time, temperature, pH, sugar concentration). The syrups were diluted with water to achieve the sugar concentrations required for each hydrolysis reaction. Table 5 also provides the percentages of Leuconostoc mesylate and DP3+ (at least DP3-DP7) oligosaccharides hydrolyzed by each enzyme. The DP3+ hydrolysis percentage was calculated as (1 - (weight % of DP3+ oligosaccharides in the final syrup) / (weight % of DP3+ oligosaccharides in the initial syrup)). Similarly, the Leuconostoc mesylate hydrolysis percentage was calculated as (1 - (weight % of Leuconostoc mesylate in the final syrup) / (weight % of Leuconostoc mesylate in the initial syrup)).

[0314] Table 5

[0315] Leuconostoc disaccharide and oligosaccharides in the concentrated filtrate were obtained through various enzymatic hydrolysis.

[0316]

[0317]

[0318] Sugar concentrations (sucrose, glucose, fructose, Leuconostoc disaccharide, and oligosaccharides) measured by HPLC; recorded values ​​were rounded to the nearest 10 g / L increment.

[0319] b DP3+ contains DP3-DP7, but may also contain larger soluble oligosaccharides, which have a high ratio of α-1,6 bonds to α-1,3 bonds when produced by certain GTF enzymes.

[0320] Table 5 shows that 1,4-α-glucosidase and 1,6-α-glucosidase exhibit partial (Example 2.1) or minimal (Example 2.2) hydrolysis of Leuconostoc disaccharide, but release some glucose from the oligosaccharide. The use of α-amylase (Examples 2.3 and 2.4) showed minimal activity against the target compound. Similarly, the use of amylopectin (Example 2.5) showed minimal activity.

[0321] Cellulases (Examples 2.14 and 2.15) were largely ineffective in hydrolyzing Leuconostoc disaccharides, but they were effective in hydrolyzing some oligosaccharides.

[0322] Although oligosaccharides contain β-bonds, surprisingly, β-glucosidases also exhibit a very low (ACCELERASE BG, Example 2.9) to a very high (NOVO 188, Examples 2.10 and 2.11) range of hydrolytic conversion. The relative efficacy of these enzymes varies considerably. In some cases, the amount of hydrolyzed oligosaccharides greatly exceeds (Example 2.11) or approaches (Example 2.12) the percentage of *Leuconostoc mesylate* hydrolyzed. In other cases, *Leuconostoc mesylate* is highly hydrolyzed by β-glucosidases, while the oligosaccharides are moderately hydrolyzed (Example 2.13). The high degree of variation observed between the β-glucosidase results suggests that other enzymes present in the tested β-glucosidase preparations, such as glucosylamylases or another class of α-glucosidases, are responsible for the observed activity.

[0323] Conversely, the results in Table 5 indicate that the transglucosidase (TG L-2000, Example 2.6) exhibits extremely high activity in hydrolyzing both oligosaccharides and Leuconostoc disaccharides. The Leuconostoc disaccharides hydrolyzed by the transglucosidase appear quantitative in some cases, and at higher enzyme loads, more than 95% of the DP3+ material is hydrolyzed to glucose and DP2 (Example 2.7). Similar activity was observed using a purified form of transglucosidase (Example 2.8), suggesting that the observed hydrolysis is attributable to the transglucosidase rather than background activity.

[0324] Glucoamylases (Examples 2.16-2.18) showed a range of activities for Leuconostoc disaccharides and oligosaccharides. Only one tested glucoamylase (Example 2.18) resulted in less than 30% hydrolysis of both Leuconostoc disaccharides and oligosaccharides.

[0325] The results in Table 5 indicate that α-glucosidases such as DIAZYME RDF ULTRA, glucosylamylase, and transglucosidase can hydrolyze Leuconostoc disaccharide byproducts present in the dextran reaction filtrate. The ability of α-glucosidases to hydrolyze Leuconostoc disaccharides suggests that these enzymes can hydrolyze α-1,5-glucosyl-fructose bonds. Given this activity demonstrated when Leuconostoc disaccharides were used as substrates, it is believed that this activity can also be extended to oligosaccharides containing α-1,5-glucosyl-fructose bonds.

[0326] The results in Table 5 further demonstrate that α-glucosidases, such as glucosylamylase and transglucosidase, can hydrolyze oligosaccharide byproducts present in the dextran reaction filtrate. Since these oligosaccharides are primarily composed of glucose monomer units linked by α-1,3 and / or α-1,6 bonds (Example 3), the data in Table 5 indicate that α-glucosidases can hydrolyze α-1,3-glucose and / or α-1,6-glucose-fructose bonds.

[0327] Because α-glucosidases typically and efficiently hydrolyze Leuconostoc disaccharides and / or oligosaccharide byproducts of dextran synthesis reactions, these enzymes can be used alone or in combination to reduce the processing time required to produce high-purity syrups from dextran reaction filtrates containing increased amounts of monosaccharides and reduced amounts of sugar byproducts. Examples of effective enzyme combinations include transglucosidases such as TG L-2000 for hydrolyzing Leuconostoc disaccharides and glucosylamylases (e.g., GC321) for efficiently hydrolyzing oligosaccharide byproducts.

[0328] Therefore, α-glucosidase can independently hydrolyze (i) α-1,5-glucosyl-fructose bonds and (ii) α-1,3 and α-1,6-glucosyl-glucose bonds in specific sugars.

[0329] Example 3

[0330] Comparison of bond distribution of dextran reaction filtrate components before and after enzymatic hydrolysis

[0331] This example determined the hydrolytic activities of transglucosidase (EC 2.4.1.24) and β-glucosidase (EC 3.2.1.21) on Leuconostoc disaccharides and oligosaccharide byproducts present in the concentrated filtrate of the dextran synthesis reaction. It was found that transglucosidase reduced the amount of these byproducts, resulting in a corresponding increase in monosaccharides (glucose and fructose) in the treated filtrate.

[0332] The oligosaccharide byproducts present in the filtrate of the above dextran synthesis reaction contain >90% glucose-glucose bonds, as determined by NMR (general method). Of these glucose-glucose bonds, approximately 78% represent α-1,3 bonds and approximately 22% represent α-1,6 bonds.

[0333] NMR was used to determine the bond characteristics of the substance produced in Example 2.11 above after hydrolysis. For example... Figure 1 As shown, the peak corresponding to the α-1,3 bond decreased by 86%, the peak corresponding to the α-1,6 bond decreased by only 2.3%, and the peak corresponding to Leuconostoc disaccharide decreased by 21%. Although sucrose is hydrolyzed almost quantitatively by this enzyme, Novo 188 does not appear to hydrolyze the α-1,6 bond.

[0334] NMR was similarly used to determine the bond characteristics of substances produced by transglucosidase using TG L-2000 (SEQ ID NO: 1). Figure 2 210 μL of concentrated filtrate (from materials in Table 3), 300 μL of D₂O, and 90 μL of D₂O containing 12.4 mM DSS (as internal standard) were mixed in an NMR tube to obtain a total sugar concentration of 300 g / L and heated to 60 °C. After thermal equilibration at 60 °C, the zero-time spectrum was obtained. Figure 2The starting material was prepared, and then 0.5% (v / v) of enzyme was added. The sample was reequilibrated in the probe at 60°C, a shim was added, and measurements were taken within minutes of analysis. The enzyme was then used with TG L-2000 enzyme (…). Figure 2 After 10 hours of treatment with the treated material, the peak corresponding to the α-1,3 bond decreased by 41%, the peak corresponding to the α-1,6 bond decreased by 36%, and the peak corresponding to Leuconostoc disaccharide decreased by >95%. Figure 2 An increase in both the α-reducing and β-reducing peaks was observed, corresponding to an increase in fructose and glucose. Figure 2 ).

[0335] These results indicate that transglucosidase can convert oligosaccharides containing α-1,3 and α-1,6 bonds into glucose and can convert Leuconostose into fructose and glucose. Therefore, transglucosidase can hydrolyze (i) α-1,5-glucosyl-fructose bonds and (ii) α-1,3 and α-1,6-glucosyl-glucose bonds in specific sugars.

[0336] Example 4

[0337] The Leuconostobacterial disaccharide and oligosaccharides in the reaction filtrate were hydrolyzed using immobilized enzymes.

[0338] This example describes the hydrolysis of Leuconostoc melanin and other oligosaccharides present in the filtrate obtained from the dextran synthesis reaction using immobilized glucosidase (EC 3.2.1.3) and transglucosidase (EC 2.4.1.24). Specifically, the effects of immobilized transglucosidase TG L-2000 (SEQ ID NO: 1, obtained from Genencor / DuPont Industrial Biosciences) and immobilized glucosidase GC-147 (obtained from Genencor / DuPont Industrial Biosciences) on the hydrolysis of Leuconostoc melanin and oligosaccharides DP2, DP3, and HS (high sugar, DP4+) in the filtrate of the dextran synthesis reaction were investigated.

[0339] Immobilization of glucosylamylase and transglucosidase is performed according to the method described in U.S. Patent 5,541,097, the disclosure of which is incorporated herein by reference.

[0340] In a typical method for immobilizing glucosylamylase and transglucosidase, two batches of porous granular diatomaceous earth (EP Minerals, Reno, NV), approximately 8.0 g / batch, are hydrated with distilled water and then transferred to a glass column reactor with a diameter of 1.5 cm and a height of 30 cm. Water is pumped upward at approximately 6-7 mL / min to remove fine particles from all three columns. Typically, the effluent is free of fine particles within one hour. Water is drained from the columns to the top of the granular diatomaceous earth bed and replaced with a 0.1% w / v polyaziridine (PEI, EPOMIN P-1050) aqueous solution. Then, 3500 mL of the PEI solution is pumped upward and the effluent is circulated through the bed for 2 hours. The granular diatomaceous earth bed is then washed with upward-flowing distilled water for 2 hours at room temperature to remove free PEI. This yields granular diatomaceous earth-PEI.

[0341] Simultaneously, 3.5 mL of glucosyl amylase GC-147 with the activities defined in Table 4 was added to 315 mL of 0.02 M acetate buffer (pH 4.5). Then, 1.575 g of 50% w / w glutaraldehyde ( GA-50 was slowly added to the glucosylamylase aqueous solution, gently mixed, and the glutaraldehyde was reacted with the glucosylamylase aqueous solution at 20-25°C for 4 hours with gentle stirring, resulting in the formation of a treated enzyme-glutaraldehyde adduct containing the treated glucosylamylase. These steps were repeated separately using transglucosidase TG L-2000 with the activities specified in Table 4, instead of glucosylamylase, thereby resulting in the formation of a treated enzyme-glutaraldehyde adduct containing the treated transglucosidase.

[0342] Then, each treated enzyme-glutaraldehyde adduct was circulated for 4 hours (20-25°C) in its own column (prepared as above) containing a granular diatomaceous earth-PEI support. Excess treated adducts were washed off the support with water. This prepared a column with immobilized glucosidase or transglucosidase.

[0343] The dextran filtrate with the composition defined in Table 3 was diluted to 180 g / L, adjusted to pH 4.5, and passed through a column containing immobilized enzyme. The column temperature was controlled at 60°C. After 16 hours of column equilibration, samples were taken at different flow rates at regular intervals. The sugar composition of the hydrolysis products was determined by HPLC (Table 6). Each time the flow rate setting was changed, the column was reequilibrated by at least 1-2 bed volumes before sampling. The degree of hydrolysis of Leuconostoc disaccharide and oligosaccharide was calculated using the method described in Example 2. Three column configurations were tested: 1) immobilized glucosylamylase, 2) immobilized transglucosidase, and 3) glucosylamylase immobilized after immobilized transglucosidase.

[0344] Table 6

[0345] Application of fixed glucosylamylase and transglucosidase in the hydrolysis of oligosaccharides and Leuconostoc disaccharides

[0346]

[0347]

[0348] Table 6 shows that the degree of hydrolysis of Leuconostoc disaccharide and oligosaccharides generally increases with increasing average contact time (defined as nominal column volume divided by average flow rate). Hydrolysis of Leuconostoc disaccharide with a fixed transglucosidase is particularly preferred, as no significant difference was observed even at the fastest flow rates measured. While individual columns show appropriate conversions, the combination of glucosylamylase and transglucosidase yielded the highest degree of oligosaccharide hydrolysis.

[0349] Therefore, the use of immobilized glucosidase or transglucosidase, or both, has demonstrated an efficient technique for hydrolyzing oligosaccharides containing α-1,3 and α-1,6 glucosyl-glucose bonds, as well as Leuconostoc disaccharides. These results are consistent with those of Example 2. Immobilization of other α-glucosidases should yield similar results.

[0350] Example 5

[0351] Fructose was enriched from the dextran reaction filtrate using chromatography.

[0352] This embodiment discloses how to further enrich fructose in dextran reaction filtrate by chromatography.

[0353] Typically, when separating sugar molecules by chromatography, the elution of components is negatively correlated with molecular size, thus the largest molecules elute first. Therefore, for the filtrate from a dextran synthesis reaction, oligosaccharides elute first, followed by disaccharides, and then monosaccharides. Separation using sodium cation exchange resins cannot adequately separate fructose and glucose, and all Leuconostoc disaccharides, sucrose, and DP2 are co-eluted. It is preferable to use ion exchange resins with calcium cations for the separation of glucose and fructose.

[0354] According to the process summary of Example 1, the filtrate from the dextran synthesis reaction was first prepared and then concentrated into a syrup. The composition of the concentrated filtrate is provided in Table 7.

[0355] Table 7

[0356] Composition of the concentrated filtrate from the dextran synthesis reaction

[0357]

[0358] The syrup from Table 7 was filtered and diluted with non-ion-exchanged water to a concentration of 25 g dry solids / 100 g solution, and then fed into a column containing a cross-linked, strongly acidic ion-exchange resin (calcium form). The physical parameters of the column are shown in Table 8. The diluted syrup (15.8 L) was fed into the column maintained at 65 °C, and then the column was eluted with water at a flow rate of 30 L / h.

[0359] Table 8

[0360] Physical parameters of the column

[0361] Resin type FINEX CS11GC Ionic form <![CDATA[Ca 2+ ]]> Crosslinking, divinylbenzene% 5.5 Particle size (mm) 0.34 Bed length (m) 5.0 Column diameter (m) 0.225

[0362] In this separation, Leuconostoc disaccharide retained longer in the column than sucrose, likely due to its complexation with calcium cations and its co-elution with glucose. Two fractions containing fructose were then separated. Fraction 5.1 eluted between 47 and 120 minutes, and fraction 5.2 eluted between 120 and 172 minutes. Of the fructose fed to the chromatographically separated fraction, 95.7% was separated with a purity >90%. The product distributions in each fraction (5.1 and 5.2), as determined by HPLC, are shown in Table 9.

[0363] Table 9

[0364] Product distribution of chromatographic fractions containing significant amounts of fructose

[0365]

[0366] Because the feed composition used for this separation contains 36.0% fructose, a total of 34.5% of the total stream is recovered as fructose syrup with >90% DS fructose. If sucrose in the feed is ignored, 40.7% of the sugar is recovered as fructose syrup with >90% DS fructose.

[0367] Therefore, fructose in the dextran reaction filtrate can be further enriched by chromatography. Example 6 below shows that the process can be enhanced using a dextran filtrate hydrolyzed by transglucosidase.

[0368] Example 6

[0369] Fructose was enriched from the filtrate of hydrolyzed dextran reaction by chromatography.

[0370] This embodiment demonstrates that separating fructose from dextran filtrate in which oligosaccharides and Leuconostoc disaccharide have been hydrolyzed results in an increased yield of high-purity fructose syrup compared to separating fructose from unhydrolyzed dextran filtrate.

[0371] The syrup was prepared by concentrating a dextran filtrate treated with 1% (v / v) transglucosidase TG L-2000 (SEQ ID NO: 1) for 24 hours at 60 °C and pH 4.5 (under vacuum at 50 °C). Some oligosaccharide formation was observed during the concentration process, which is desirable because transglucosidase is known to produce oligosaccharides at high concentrations of monosaccharides. The syrup exhibited the final product distribution described in Table A.

[0372] Table A

[0373] Composition of concentrated dextran filtrate hydrolyzed prior to concentration

[0374]

[0375] The syrup described in Table A was filtered and diluted with non-ion-exchanged water to a concentration of 25.4 g DS / 100 g, and then fed into a column containing a cross-linked, strongly acidic cation exchange resin (calcium form). The physical parameters of the column are shown in Table B. The diluted syrup (169 g) was then fed into the column maintained at 65 °C, and subsequently eluted with water at a flow rate of 50 mL / min.

[0376] Table B

[0377] Physical parameters of the column

[0378] Resin type FINEX CS11GC Ionic form <![CDATA[Ca 2+ ]]> Crosslinking, divinylbenzene% 5.5 Particle size (mm) 0.34 Bed length (m) 1.69 Column diameter (m) 0.093

[0379] The two fractions containing fructose were separated. Fraction 6.1 was eluted between 73 and 103 minutes, and fraction 6.2 was eluted between 103 and 120 minutes. Of the fructose fed to the column in the chromatographically separated fraction, 93.0% of the fructose was separated in fraction 6.2 with a purity >90%. The product distributions in each fraction (6.1 and 6.2) as determined by HPLC are shown in Table C.

[0380] Table C

[0381] Product distribution of chromatographic fractions containing fructose derived from hydrolyzed dextran filtrate

[0382]

[0383] The reduced separation efficiency of this embodiment compared to Example 5 can be attributed to the difference in column size and the higher glucose fraction in the sample. Even so, the chromatographic purification of this material resulted in an increased yield of high-purity fructose syrup compared to that obtained in Example 5, which prepared syrup from dextran filtrate without transglucosidase hydrolysis. Because the feed composition used for this separation contained 47% fructose (Table A), 43.7% of the total stream was recovered as fructose syrup with >90% wt% DS fructose. This 43.7% recovery is significantly better than the 34.5% recovery in Example 5.

[0384] Therefore, separating fructose from dextran filtrate that has been hydrolyzed by transglucosidase results in a higher fructose yield compared to separating fructose from unhydrolyzed dextran filtrate.

[0385] Example 7

[0386] Ethanol preparation from the filtrate of the fermentation dextran synthesis reaction.

[0387] This embodiment discloses the fermentation of dextran filtrate into ethanol by yeast.

[0388] The yeast (Saccharomyces cerevisiae) paste (Tonon mill, Brazil) was washed by suspending the paste in tap water (2.4 L, optical density 65 at 600 nm) and then centrifuging it at 4500 g for 5 minutes using a LEGEND XTR centrifuge (Thermo Scientific). After decanting the supernatant, the yeast cells were resuspended and concentrated by two more centrifugations. After a third wash, the pH was adjusted to 2 by adding 5% sulfuric acid. The optical density was measured using a GENESYS 204001 spectrophotometer (Thermo Scientific) and adjusted to 100 (at 600 nm) by adding tap water. The adjusted yeast paste (1.5 L) was added to a 7.5-L BIOFLO 310 fermenter vessel (New Brunswick). The fermenter was set to maintain a temperature of 30°C and stirred at 100 rpm. Although the pH was measured during fermentation, it was not controlled by adding acid or alkali solutions.

[0389] A feed solution containing yeast extract (10 g / L), peptone (20 g / L), and 200 g / L sugar derived from dextran filtrate was prepared and sterilized in a PHOENIX AV-250 PLUS autoclave at 121 °C for 15 min. The feed solution was cooled to 25 °C (room temperature) before fermentation began. The sterilized feed solution (3.5 L) was added to the fermenter at a rate of 684 mL / h for approximately 5 hours, and fermentation was allowed to proceed for 22 hours.

[0390] During fermentation, samples were taken periodically and the optical density was analyzed using a GENESYS 20 4001 spectrophotometer. Brix content was analyzed using a PAL-3 refractometer (Atago). Sugar and ethanol concentrations were analyzed using HPLC (general method). These results are summarized in Table 10.

[0391] Table 10

[0392] Feed and time-series fermentation characteristics of primary ethanol fermentation

[0393]

[0394] The concentrations (g / L) of ethanol (EtOH) and sugar compounds in the feed and at each fermentation time point (0-22 hours) are listed.

[0395] At the end of fermentation, yeast cells were separated by centrifugation at 4500g for 5 minutes using a LEGEND XTR centrifuge. After decanting the supernatant, the yeast was resuspended and concentrated by two more centrifugations. After a third wash, the pH was adjusted to 2 by adding 5% sulfuric acid. The optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 (at 600 nm) by adding tap water. Under the same conditions as above, two more fermentation cycles were performed using yeast cells recovered from the previous fermentation, each time with fresh feed. The fermentation results obtained using the yeast recovered in the first and second cycles are provided in Tables 11 and 12, respectively.

[0396] Table 11

[0397] Feed and time-series fermentation characteristics using first-recycled yeast cells

[0398]

[0399] The concentrations (g / L) of ethanol (EtOH) and sugar compounds in the feed and at each fermentation time point (0-21 hours) are listed.

[0400] Table 12

[0401] Feed and time-series fermentation characteristics using second-generation recycled yeast cells

[0402]

[0403] The concentrations (g / L) of ethanol (EtOH) and sugar compounds in the feed and at each fermentation time point (0-21 hours) are listed.

[0404] Very little Leuconostoc disaccharide was consumed in the first fermentation, but the yeast cells began to adapt to and consume Leuconostoc disaccharide after the second recovery. After three fermentation cycles using recovered yeast, the ethanol fermentation titer increased from 33 g / L (Table 10, 22 h) to 54 g / L (Table 12, 21 h), but significant amounts of Leuconostoc disaccharide were still present in the medium even after the last cycle.

[0405] Therefore, dextran filtrate can be used in fermentation processes to produce ethanol.

[0406] Example 8

[0407] Ethanol preparation from the filtrate of dextran hydrolyzed by fermentation

[0408] This example demonstrates that fermentation of the dextran filtrate, in which Leuconostoc disaccharide and oligosaccharide byproducts were previously saccharified, resulted in an increased ethanol yield.

[0409] Fermentation was carried out according to the method described in Example 7, but here a dextran filtrate previously treated with glucosidase (TG L-2000, SEQ ID NO: 1) was used. The hydrolyzed dextran filtrate was prepared as follows: The dextran filtrate was adjusted to 300 g sugar / L, and then the pH was adjusted to 4.0 using 1.0 M sodium hydroxide and 5% sulfuric acid by weight. The final volume of this preparation was 6.75 L. The filtrate was then sterilized at 121°C for 15 minutes using a PHOENIX AV-250PLUS autoclave, and the temperature was then adjusted to 60°C. The TG L-2000 enzyme extract as described in Table 4 (135 mL) was mixed with the sterilized filtrate, and the solution was incubated at 60°C and 100 rpm in a shaker (IKA KS4000) for 72 hours. This yielded the hydrolyzed dextran filtrate.

[0410] The yeast (Saccharomyces cerevisiae) extract (Bom Retiro mill, Brazil) was washed by suspending the extract in tap water (2.4 L, optical density 65 at 600 nm) and then centrifuging it at 4500 g for 5 minutes using a LEGEND XTR centrifuge. After decanting the supernatant, the yeast was resuspended and concentrated by two more centrifugations. After a third wash, the pH was adjusted to 4.5 by adding 5 wt% sulfuric acid, and the optical density was measured using a GENESYS 204001 spectrophotometer and adjusted to 100 (at 600 nm) by adding tap water. The adjusted yeast extract (1.5 L) was added to a 7.5-L BIOFLO 310 fermenter. The fermenter was set to maintain a temperature of 30°C, stirred at 100 rpm, and the pH was maintained at 4.5 using either a 4M ammonium hydroxide aqueous solution or a 5 wt% sulfuric acid aqueous solution.

[0411] A feed solution containing yeast extract (10 g / L), peptone (20 g / L), and 200 g / L sugar derived from the hydrolyzed filtrate was prepared and sterilized in a PHOENIX AV-250 Plus autoclave at 121 °C for 15 min. The feed solution was cooled to 25 °C (room temperature) before fermentation began. The sterilized feed solution (3.5 L) was added to the fermenter at a rate of 684 mL / h for approximately 5 hours, and fermentation was allowed to proceed for 22 hours.

[0412] During fermentation, samples were taken periodically and the optical density was analyzed using a GENESYS 20 4001 spectrophotometer. The Brix content was analyzed using a PAL-3 refractometer, and the sugar and ethanol concentrations were analyzed using HPLC (general method). These results are summarized in Table 13.

[0413] Table 13

[0414] Feed and time-series fermentation characteristics using primary ethanol fermentation with hydrolyzed dextran filtrate

[0415]

[0416] The concentrations (g / L) of ethanol (EtOH) and sugar compounds in the feed and at each fermentation time point (0-22 hours) are listed.

[0417] At the end of fermentation, yeast cells were separated by centrifugation at 4500g for 5 minutes using a LEGEND XTR centrifuge. After decanting the supernatant, the yeast cells were resuspended and concentrated by two more centrifugations. After a third wash, the pH was adjusted to 2 by adding 5% by weight of sulfuric acid. The optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 (at 600 nm) by adding tap water. Under the same conditions as above, two more fermentation cycles were performed using yeast cells recovered from the previous fermentation, each using fresh feed. The fermentation results obtained using the yeast cells recovered in the first and second cycles are provided in Tables 14 and 15, respectively.

[0418] Table 14

[0419] Feed and time-series fermentation characteristics using first-recovery yeast cells and hydrolyzed dextran filtrate

[0420]

[0421]

[0422] The concentrations (g / L) of ethanol (EtOH) and sugar compounds in the feed and at each fermentation time point (0-21 hours) are listed.

[0423] Table 15

[0424] Feed and time-series fermentation characteristics using second-recycled yeast cells and hydrolyzed dextran filtrate

[0425]

[0426] The concentrations (g / L) of ethanol (EtOH) and sugar compounds in the feed and at each fermentation time point (0-21 hours) are listed.

[0427] Within approximately six hours of initiating fermentation, all fermentation was substantially complete, resulting in ethanol titers of 57-60.0 g / L. Comparison of these fermentations with those of Example 7 showed that the hydrolysis of the dextran filtrate prior to fermentation, compared to those obtained using unhydrolyzed dextran filtrate, resulted in faster and greater ethanol yields.

[0428] Therefore, fermentation of the dextran filtrate, in which the Leuconostoc disaccharide and oligosaccharide byproducts have been saccharified, results in a faster increase in ethanol yield. This saccharification can be carried out using, for example, transglucosidase.

[0429] Example 9

[0430] Simultaneous saccharification and fermentation of dextran filtrate

[0431] This embodiment discloses that simultaneous saccharification and fermentation of a feed containing dextran filtrate can lead to enhanced fermentation characteristics.

[0432] The yeast (Saccharomyces cerevisiae) extract (Bom Retiro mill, Brazil) was washed by suspending the extract in tap water (2.4 L, optical density 65 at 600 nm) and then centrifuging it at 4500 g for 5 minutes using a LEGEND XTR centrifuge. After decanting the supernatant, the yeast cells were resuspended and concentrated by two more centrifugations. After a third wash, the pH was adjusted to 4.5 by adding 5 wt% sulfuric acid, and the optical density was measured using a GENESYS 20 4001 spectrophotometer and adjusted to 100 (at 600 nm) by adding tap water. The adjusted yeast extract (1.5 L) was added to a 7.5-L BIOFLO 310 fermenter. The fermenter was set to maintain a temperature of 30°C, stirred at 100 rpm, and the pH was maintained at 4.5 using either a 4M ammonium hydroxide aqueous solution or a 5 wt% sulfuric acid aqueous solution.

[0433] A feed solution containing yeast extract (10 g / L), peptone (20 g / L), and 200 g / L sugar derived from dextran filtrate was prepared and sterilized in a PHOENIX AV-250 PLUS autoclave at 121°C for 15 minutes. The feed solution was cooled to 25°C (room temperature) before fermentation began. Immediately before adding the solution to the fermenter, TG L-2000 transglucosidase (1% v / v) as described in Table 4 was added to the sterilized feed solution. The feed solution containing TG L-2000 enzyme (3.5 L) was added to the fermenter at a rate of 684 mL / h for approximately 5 hours, and fermentation was allowed to proceed for 48 hours.

[0434] During fermentation, samples were taken periodically and the optical density was analyzed using a GENESYS 20 4001 spectrophotometer. Brix content was analyzed using a PAL-3 refractometer (Atago). Sugar and ethanol concentrations were analyzed using HPLC (general method). These results are summarized in Table 16.

[0435] Table 16

[0436] Feed and time-series fermentation characteristics of simultaneous saccharification and ethanol fermentation of dextran filtrate

[0437]

[0438]

[0439] The concentrations (g / L) of ethanol (EtOH) and sugar compounds in the feed and at each fermentation time point (0-48 hours) are listed.

[0440] Fermentation was nominally completed within 6 hours, similar to fermentation where the filtrate had been hydrolyzed prior to the fermentation step (Example 8), and yielded a slightly better ethanol titer (62 g / L) compared to using unhydrolyzed filtrate (Example 7). Furthermore, almost all of the Leuconostoc disaccharide was consumed within 6 hours (compare Table 16 with Tables 13-15). Similar results should be obtained if the saccharifying enzyme is added directly to the fermentation product, except that a saccharifying enzyme such as TG L-2000 is added to the feed containing the dextran filtrate just before fermentation.

[0441] Therefore, simultaneous saccharification and fermentation of feed containing dextran filtrate can lead to enhanced fermentation characteristics, such as increased (i) consumption of dextran filtrate components (e.g., Leuconostoc disaccharide) and (ii) ethanol yield and production rate.

[0442] Example 10

[0443] Preparation of various α-glucosidases

[0444] This embodiment discloses the preparation of various α-glucosidases other than those used in some of the foregoing embodiments (transglucosidase, glucosylamylase, DIAZYME RDF ULTRA). The hydrolytic activity of these additional α-glucosidases against oligosaccharides containing α-1,5-glucosyl-fructose bonds or α-1,3 and / or α-1,6-glucosyl-glucose bonds was tested (in Examples 11, 12, 15, and 16 provided below).

[0445] Discovery of Aspergillus anthelminticus α-glucosidase (Aclglu1)

[0446] Aspergillus strains were selected as a potential source of other enzymes for various industrial applications. A gene identified in Aspergillus encodes an α-glucosidase (designated “Aclglu1”), and the sequence of this gene 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 glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO:5) has a signal peptide of 19 amino acids, as predicted by SignalP version 4.0 (Nordahl Petersen et al. 2011, Nature Methods, 8:785-786). The presence of the signal peptide indicates that Aclglu1 is a secretory enzyme. The amino acid sequence of the predicted mature form of Aclglu1 is shown in SEQ ID NO:6.

[0447] Expression of Aclglu1, an α-glucosidase from Aspergillus lanceolata.

[0448] The Aclglu1 gene was synthesized and cloned into the pTrex3gM expression vector (described in U.S. Patent Application Publication 2011 / 0136197, which is incorporated herein by reference), and the resulting plasmid was named pJG294. The sequence of the Aclglu1 gene was confirmed by DNA sequencing.

[0449] The plasmid pJG294 was transformed into a tetrad-deleted *Trichoderma reesei* strain (described in WO05 / 001036) using the gene gun method (Te′o VS et al., J Microbiol Methods, 51:393-9, 2002). The protein containing SEQ ID NO: 6 was predicted to be secreted into extracellular culture medium, and the filtered medium was used for SDS-PAGE and α-glucosidase activity assays to confirm enzyme expression.

[0450] Neosartorya The discovery of fischeri α-glucosidase Nfiglu1

[0451] Strains of *Neosatoria fischeri* were selected as a potential source of other enzymes for various industrial applications. A gene identified in *Neosatoria fischeri* encodes an α-glucosidase (designated “Nfiglu1”), and the sequence of this gene is provided in SEQ ID NO: 7. The corresponding protein encoded by SEQ ID NO: 7 is provided in SEQ ID NO: 8. Nfiglu1 belongs to the glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO: 8) has a signal peptide of 19 amino acids, as predicted by SignalP version 4.0 (Nordahl Petersen et al. 2011, *Nature Methods*, 8: 785-786). The presence of the signal peptide indicates that Nfiglu1 is a secretory enzyme. The predicted amino acid sequence of the mature form of Nfiglu1 is shown in SEQ ID NO: 9.

[0452] Expression of α-glucosidase Nfiglu1 in Neosatoria fibrillosa

[0453] The synthesized Nfiglu1 gene was cloned into the pTrex3gM expression vector (described in U.S. Patent Application Publication 2011 / 0136197), and the resulting plasmid was named pJG295. The sequence of the Nfiglu1 gene was confirmed by DNA sequencing.

[0454] The plasmid pJG295 was transformed into a tetrad-deleted *Trichoderma reesei* strain (described in WO05 / 001036) using the gene gun method (Te′o VS et al., J Microbiol Methods, 51:393-9, 2002). The protein containing SEQ ID NO: 9 was predicted to be secreted into extracellular culture medium, and the filtered medium was used for SDS-PAGE and α-glucosidase activity assays to confirm enzyme expression.

[0455] Discovery of α-glucosidase Ncrglu1 in Neurospora crassa

[0456] Strains of *Neurospora crassa* were selected as a potential source of other enzymes for various industrial applications. A gene identified in *Neurospora crassa* encodes an α-glucosidase (designated “Ncrglu1”), and the sequence of this gene is provided in SEQ ID NO: 10. The corresponding protein encoded by SEQ ID NO: 10 is provided in SEQ ID NO: 11. Ncrglu1 belongs to the glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO: 11) has a 22-amino acid signal peptide, as predicted by SignalP version 4.0 (Nordahl Petersen et al. 2011, *Nature Methods*, 8: 785-786). The presence of the signal peptide indicates that Ncrglu1 is a secretory enzyme. The predicted amino acid sequence of the mature form of Ncrglu1 is shown in SEQ ID NO: 12.

[0457] Expression of α-glucosidase Ncrglu1 in Neurospora crassa

[0458] The synthesized Ncrglu1 gene was cloned into the pTrex3gM expression vector (described in U.S. Patent Application Publication 2011 / 0136197), and the resulting plasmid was named pJG296. The sequence of the Ncrglu1 gene was confirmed by DNA sequencing.

[0459] The plasmid pJG296 was transformed into a tetrad-deleted *Trichoderma reesei* strain (described in WO05 / 001036) using the gene gun method (Te′o VS et al., J Microbiol Methods, 51:393-399, 2002). The protein containing SEQ ID NO: 12 was predicted to be secreted into extracellular culture medium, and the filtered medium was used for SDS-PAGE and α-glucosidase activity assays to confirm enzyme expression.

[0460] The discovery of Rasamsonia composticola α-glucosidase TauSec098

[0461] Strains of *Rasamsonia composticola* were selected as a potential source of other enzymes for various industrial applications. A gene identified in *Rasamsonia composticola* encodes an α-glucosidase (designated “TauSec098”), and the sequence of this gene 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 family 31 of glycosyl hydrolases based on a PFAM search (pfam.sanger.ac.uk web link) and contains an N-terminal CBM 20 domain. At the N-terminus, the protein (SEQ ID NO: 14) has a 22-amino acid signal peptide, as predicted by SignalP version 4.0 (Nordahl Petersen et al. 2011, *Nature Methods*, 8: 785-786). The presence of the signal peptide indicates that TauSec098 is a secretory enzyme. The amino acid sequence of the predicted mature form of TauSec098 is shown in SEQ ID NO: 15.

[0462] Expression of Rasamsonia composticola α-glucosidase TauSec098

[0463] The TauSec098 gene was synthesized and cloned into the Trichoderma reesei expression vector pGXT (pTTT-derived plasmid) 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.

[0464] The plasmid pGX256-TauSec098 was transformed into a tetrad-deleted *Trichoderma reesei* strain (described in WO05 / 001036) using protoplast transformation (Te'o et al., J. Microbiol. Methods 51:393-9, 2002). Transformants 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; ferrous(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 (approximately 50-100 colonies) appeared within about one week. After growth on acetamide plates, spores of the transformants were collected and transferred to fresh acetamide agar plates. After 5 days of growth on acetamide plates, 1×10⁻⁶ spores were transferred to fresh acetamide agar plates. 8Spores were inoculated into 30 ml of glucose / sophorose assay medium in 250 mL shake flasks. The shake flasks were incubated at 28 °C with shaking for 5 days. The supernatant obtained from these cultures was used to confirm the expression (SDS PAGE) and activity of the mature TauSec098 enzyme (SEQ ID NO: 15).

[0465] The discovery of Rasamsonia composticola α-glucosidase TauSec099

[0466] Strains of *Rasamsonia composticola* were selected as a potential source of other enzymes for various industrial applications. A gene identified in *Rasamsonia composticola* encodes an α-glucosidase (designated “TauSec099”), and the sequence of this gene is provided in SEQ ID NO: 16. The corresponding protein encoded by SEQ ID NO: 16 is provided in SEQ ID NO: 17. TauSec099 belongs to the glycosyl hydrolase family 31 based on a PFAM search (pfam.sanger.ac.uk web link). At the N-terminus, the protein (SEQ ID NO: 17) has a 17-amino acid signal peptide, as predicted by SignalP version 4.0 (Nordahl Petersen et al. 2011, *Nature Methods*, 8: 785-786). The presence of the signal peptide indicates that TauSec099 is a secretory enzyme. The amino acid sequence of the predicted mature form of TauSec099 is shown in SEQ ID NO: 18.

[0467] Expression of Rasamsonia composticola α-glucosidase TauSec099

[0468] The TauSec099 gene was synthesized and cloned into the Trichoderma reesei expression vector pGXT (pTTT-derived plasmid) 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.

[0469] The plasmid pGX256-TauSec099 was transformed into a tetrad-deleted *Trichoderma reesei* strain (described in WO05 / 001036) using protoplast transformation (Te'o et al., J. Microbiol. Methods 51:393-9, 2002). Transformants 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; ferrous(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 (approximately 50-100) appeared within about one week. After growth on acetamide plates, spores of the transformants were collected and transferred to fresh acetamide agar plates. After 5 days of growth on acetamide plates, 1×10⁻⁶ spores were transferred to fresh acetamide agar plates. 8 Spores were inoculated into 30 ml of glucose / sophorose determinant medium in 250 mL shake flasks. The shake flasks were incubated at 28 °C with shaking for 5 days. The supernatant obtained from these cultures was used to confirm the expression (SDS PAGE) and activity of the mature TauSec099 enzyme (SEQ ID NO: 18).

[0470] Sequence of Bifidobacterium longum α-glucosidase BloGlu1

[0471] The α-glucosidase gene “BloGlu1” was identified from *Bifidobacterium longum* subspecies JDM301. The nucleic acid sequence of the BloGlu1 gene (SEQ ID NO: 19, GENBANK accession number NC014169.1, complementary sequence from positions 140600 to 142414) and the amino acid sequence of the putative protein encoded by SEQ ID NO: 19 (SEQ ID NO: 20) are present in GENBANK accession number YP_003660432.1.

[0472] Expression of α-glucosidase BloGlu1 in Bifidobacterium longum

[0473] The DNA sequence encoding the entire BloGlu1 protein (SEQ ID NO: 20) was optimized for expression in Bacillus subtilis, then synthesized by Generay Biotech Co. (Shanghai, China) (resulting in SEQ ID NO: 21) and inserted into the p3JM plasmid, resulting in p3JM-BloGlu1. The p3JM-BloGlu1 plasmid contains the aprE promoter to drive the expression of the optimized BloGlu1 sequence (SEQ ID NO: 21).

[0474] The plasmid p3JM-BloGlu1 was used to transform Bacillus subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), and the transformed cells were plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies with the correct insert fragment, as confirmed by PCR and sequencing, were selected and fermented in 250-mL shake flasks containing MBD medium (MOPS-based defined component medium supplemented with an additional 5 mM CaCl2) to express the BloGlu1 protein (SEQ ID NO: 20).

[0475] Sequence of Bifidobacterium longum α-glucosidase BloGlu2

[0476] The α-glucosidase gene BloGlu2 was identified from Bifidobacterium longum. The amino acid sequence of BloGlu2 (SEQ ID NO: 22) is available in the NCBI database (GENBANK accession number WP_007054665.10).

[0477] Expression of α-glucosidase BloGlu2 in Bifidobacterium longum

[0478] The DNA sequence encoding the BloGlu2 protein was optimized for expression in Bacillus subtilis, then synthesized by Generay Biotech Co. (resulting in SEQ ID NO: 23) and inserted into the p3JM plasmid, resulting in p3JM-BloGlu2. SEQ ID NO: 23 encodes the amino acid sequence of SEQ ID NO: 24. The p3JM-BloGlu2 plasmid contains the aprE promoter to drive the expression of the optimized BloGlu2 sequence (SEQ ID NO: 23).

[0479] The plasmid p3JM-BloGlu2 was used to transform Bacillus subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), and the transformed cells were plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies with the correct insert fragment, as confirmed by PCR and sequencing, were selected and fermented in 250-mL shake flasks containing MBD medium (MOPS-based defined component medium supplemented with an additional 5 mM CaCl2) to express the BloGlu2 protein (SEQ ID NO: 24).

[0480] The sequence of Bifidobacterium longum α-glucosidase BloGlu3

[0481] The α-glucosidase gene “BloGlu3” was identified from Bifidobacterium longum subspecies F8. The nucleic acid sequence of the BloGlu3 gene (SEQ ID NO: 25, GENBANK accession number NC_021008.1, positions 2130627 to 2132441) and the amino acid sequence of the putative protein encoded by SEQ ID NO: 25 (SEQ ID NO: 26) are present in GENBANK accession number YP_007768249.1.

[0482] Expression of α-glucosidase BloGlu3 in Bifidobacterium longum

[0483] The DNA sequence encoding the entire BloGlu3 protein (SEQ ID NO: 26) was optimized for expression in Bacillus subtilis, then synthesized by Generay Biotech Co. (resulting in SEQ ID NO: 27) and inserted into the p3JM plasmid, resulting in p3JM-BloGlu3. The p3JM-BloGlu3 plasmid contains the aprE promoter to drive the expression of the optimized BloGlu3 sequence (SEQ ID NO: 27).

[0484] The plasmid p3JM-BloGlu3 was used to transform Bacillus subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), and the transformed cells were plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies with the correct insert fragment, as confirmed by PCR and sequencing, were selected and fermented in 250-mL shake flasks containing MBD medium (MOPS-based defined component medium supplemented with an additional 5 mM CaCl2) to express the BloGlu3 protein (SEQ ID NO: 26).

[0485] The sequence of Bifidobacterium pseudolongum α-glucosidase BpsGlul

[0486] The α-glucosidase gene BpsGlu1 was identified from Bifidobacterium pseudolongum. The amino acid sequence of BpsGlu1 (SEQ ID NO: 28) is available in the NCBI database (GENBANK accession number WP_022858408.1).

[0487] Expression of α-glucosidase BpsGlu1 in Bifidobacterium pseudolongum

[0488] The DNA sequence encoding the BloGlu1 protein was optimized for expression in Bacillus subtilis, then synthesized by Generay Biotech Co. (resulting in SEQ ID NO: 29) and inserted into the p3JM plasmid, resulting in p3JM-BpsGlul. SEQ ID NO: 29 encodes the amino acid sequence of SEQ ID NO: 30. The p3JM-BpsGlu1 plasmid contains the aprE promoter to drive the expression of the optimized BpsGlu1 sequence (SEQ ID NO: 29).

[0489] Plasmid p3JM-BpsGlu1 was used to transform Bacillus subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), and the transformed cells were plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies with the correct insert fragment, as confirmed by PCR and sequencing, were selected and fermented in 250-mL shake flasks containing MBD medium (MOPS-based defined component medium supplemented with an additional 5 mM CaCl2) to express the BpsGlu1 protein (SEQ ID NO: 30).

[0490] The sequence of Bifidobacterium thermophilum α-glucosidase BthGlu1

[0491] The α-glucosidase gene “BthGlu1” was identified from Bifidobacterium thermophilum RBL67. The nucleic acid sequence of the BthGlu1 gene (SEQ ID NO: 31, GENBANK accession number NC_020546.1, positions 150690 to 152495) and the amino acid sequence of the putative protein encoded by SEQ ID NO: 31 (SEQ ID NO: 32) are present in GENBANK accession number YP_007592840.1.

[0492] Expression of α-glucosidase BthGlu1 in Bifidobacterium thermophilum

[0493] The DNA sequence encoding the entire BthGlu1 protein (SEQ ID NO: 32) was optimized for expression in Bacillus subtilis, then synthesized by Generay Biotech Co. (resulting in SEQ ID NO: 33) and inserted into the p3JM plasmid, resulting in p3JM-BthGlu1. The p3JM-BthGlu1 plasmid contains the aprE promoter to drive the expression of the optimized BthGlu1 sequence (SEQ ID NO: 33).

[0494] The plasmid p3JM-BthGlu1 was used to transform Bacillus subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), and the transformed cells were plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies with the correct insert fragment, as confirmed by PCR and sequencing, were selected and fermented in 250-mL shake flasks containing MBD medium (MOPS-based defined component medium supplemented with an additional 5 mM CaCl2) to express the BthGlu1 protein (SEQ ID NO: 32).

[0495] Sequence of Bifidobacterium breve α-glucosidase BbrGlu2

[0496] The α-glucosidase gene BbrGlu2 was identified from Bifidobacterium breve. The amino acid sequence of BbrGlu2 (SEQ ID NO: 34) is available in the NCBI database (GENBANK accession number WP_003827971.1).

[0497] Expression of α-glucosidase BbrGlu2 in Bifidobacterium breve

[0498] The DNA sequence encoding the BbrGlu2 protein was optimized for expression in Bacillus subtilis, then synthesized by Generay Biotech Co. (resulting in SEQ ID NO: 35) and inserted into the p3JM plasmid, resulting in p3JM-BbrGlu2. SEQ ID NO: 35 encodes the amino acid sequence of SEQ ID NO: 36. The p3JM-BbrGlu2 plasmid contains the aprE promoter to drive the expression of the optimized BbrGlu2 sequence (SEQ ID NO: 35).

[0499] Plasmid p3JM-BbrGlu2 was used to transform Bacillus subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), and the transformed cells were plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies with the correct insert, as confirmed by PCR and sequencing, were selected and fermented in 250-mL shake flasks containing MBD medium (MOPS-based defined component medium supplemented with an additional 5 mM CaCl2) to express SEQ ID NO: 36.

[0500] The sequence of Bifidobacterium breve α-glucosidase BbrGlu5

[0501] The α-glucosidase gene “BbrGlu5” was identified from *Bifidobacterium breve* ACS-071-V-Sch8b. The nucleic acid sequence of the BbrGlu5 gene (SEQ ID NO: 37, GENBANK accession number NC_017218.1, complementary sequence at positions 2241075 to 2242895) and the amino acid sequence of the putative protein encoded by SEQ ID NO: 37 (SEQ ID NO: 38) are present in GENBANK accession number YP_005583701.1.

[0502] Expression of Bifidobacterium α-glucosidase BbrGlu5

[0503] The DNA sequence encoding the entire BbrGlu5 protein (SEQ ID NO: 38) was optimized for expression in Bacillus subtilis, then synthesized by Generay Biotech Co. (resulting in SEQ ID NO: 39) and inserted into the p3JM plasmid, resulting in p3JM-BbrGlu5. The p3JM-BbrGlu5 plasmid contains the aprE promoter to drive the expression of the optimized BbrGlu5 sequence (SEQ ID NO: 39).

[0504] The plasmid p3JM-BbrGlu5 was used to transform Bacillus subtilis cells (degUHy32, ΔnprB, Δvpr, Δepr, ΔscoC, ΔwprA, Δmpr, ΔispA, Δbpr), and the transformed cells were plated on Luria agar plates supplemented with 5 ppm chloramphenicol. Colonies with the correct insert fragment, as confirmed by PCR and sequencing, were selected and fermented in 250-mL shake flasks containing MBD medium (MOPS-based defined component medium supplemented with an additional 5 mM CaCl2) to express the BbrGlu5 protein (SEQ ID NO: 38).

[0505] Purification of α-glucosidase from expression culture

[0506] AclGlu1 and NcrGlu1

[0507] Two chromatographic steps were used to purify both AclGlu1 (SEQ ID NO: 6) and NcrGlu1 (SEQ ID NO: 12) α-glucosidases. For each purification, the crude fermentation broth in the shake flask was concentrated, followed by the addition of ammonium sulfate to a final concentration of 2M. The solution was loaded onto a 50-mL phenyl HP column pre-equilibrated with 20 mM Tris (pH 8.0) and 2M ammonium sulfate. The target protein (SEQ ID NO: 6 or SEQ ID NO: 12) was eluted from the column with 1M ammonium sulfate and 20 mM Tris (pH 8.0). The fractions were combined, concentrated using a VIVAFLOW 200 ultrafiltration system (Sartorius Stedim), and buffer-exchanged to 20 mM Tris (pH 8.0) (buffer A). The resulting solution was applied to a 40-mL Q HP column pre-equilibrated with buffer A. The target protein was eluted from the column with buffer A containing 0.3M NaCl. The fractions containing the target protein were then combined and concentrated using a 10K AMICONULTRA-15 device, and stored at -20°C in 40% glycerol until use.

[0508] NfiGlu1

[0509] The NfiGlu1α-glucosidase (SEQ ID NO: 9) was purified using two hydrophobic interaction chromatographic steps. The crude fermentation broth in shake flasks was concentrated, followed by the addition of ammonium sulfate to a final concentration of 1 M. The solution was loaded onto a 50-mL phenylHP column pre-equilibrated with 20 mM Tris (pH 8.0) and 1 M ammonium sulfate. The target protein (SEQ ID NO: 9) was allowed to flow through the column. The eluent fractions were combined, followed by the addition of ammonium sulfate to a final concentration of 2 M. The solution was loaded onto the same phenylHP column pre-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 and 20 mM Tris (pH 8.0). The fractions containing the target protein were then combined and concentrated using a 10K AMICON ULTRA-15 instrument and stored at -20°C in 40% glycerol until use.

[0510] TauSec098 and TauSec099

[0511] Both TauSec098 (SEQ ID NO: 15) and TauSec099 (SEQ ID NO: 18) α-glucosidases were purified by hydrophobic interaction chromatography. For each purification, ammonium sulfate was added to approximately 180 mL of concentrated crude fermentation broth in a 7-L fermenter to a final concentration of 1 M. This solution was then loaded onto a 50-mL HIPREP phenyl-FF agarose column (GE Healthcare) pre-equilibrated with 20 mM sodium acetate (pH 5.0) and 1 M ammonium sulfate (buffer A). After washing with the same buffer for three column volumes (CVs), the column was eluted stepwise with three CVs each of 75%, 50%, and 0% buffer A, followed by two CVs of elution with Milliq H2O. All fractions were analyzed by SDS-PAGE. The target protein (SEQ ID NO: 15 or SEQ ID NO: 18) was predominantly present in the elution fraction, which was concentrated and buffer-replaced using a 10 kDa AMICON ULTRA-15 instrument to remove excess ammonium sulfate. At -80°C, the final product with a purity greater than 90% is stored in 40% glycerol until use.

[0512] BloGlu1, BloGlu2 and BloGlu3

[0513] BloGlu1 (SEQ ID NO: 20), BloGlu2 (SEQ ID NO: 24), and BloGlu3 (SEQ ID NO: 26) α-glucosidases were purified in three steps. For each purification, 1 L of crude fermentation broth from a DASGIP fermenter was concentrated, followed by the addition of ammonium sulfate to 60% saturation. The solution was stirred at 4°C for 1 hour and then centrifuged at 8000 × g for 30 minutes. The resulting precipitate was resuspended in 20 mM Tris (pH 8.0, buffer A). Ammonium sulfate was added to the final solution to a final concentration of 1 M; this formulation was then loaded into 40 mL HiPrep pre-equilibrated with 20 mM Tris (pH 8.0) and 1 M ammonium sulfate (buffer B). TM Phenyl-FF column. After washing, elution was performed stepwise with three column volumes each of 75%, 50%, and 0% buffer B and H2O. All fractions were analyzed by SDS-PAGE and activity assays. Fractions containing the target protein (SEQ ID NO: 20, SEQ ID NO: 24, or SEQ ID NO: 26) were combined, concentrated, and then loaded onto a HiLoad column pre-equilibrated with 20 mM sodium phosphate (pH 7.0) and 0.15 M NaCl. TM 26 / 60 Superdex TM75 column. The eluent fractions containing the target protein were then combined and concentrated using a 10K AMICON ULTRA-15 apparatus and stored at -20°C in 40% glycerol until use.

[0514] BpsGlu1 and BthGlu1

[0515] Both BpsGlu1 (SEQ ID NO: 30) and BthGlu1 (SEQ ID NO: 32) α-glucosidase were purified in two steps. For each purification, 1-L of crude fermentation broth from a DASGIP fermenter was concentrated, followed by the addition of ammonium sulfate to 60% saturation. The solution was stirred at 4°C for 1 hour and then centrifuged at 8000×g for 30 minutes. The resulting precipitate was resuspended in 20 mM Tris (pH 8.0, buffer A). Ammonium sulfate was added to the final solution to a final concentration of 1 M; this formulation was then loaded into 40-mL HiPrep pre-equilibrated with 20 mM Tris (pH 8.0) and 1 M ammonium sulfate (buffer B). TM Phenyl-FF column. After washing, elution was performed stepwise with three column volumes each of 75%, 50%, and 0% buffer B and H2O. All fractions were analyzed by SDS-PAGE and activity assay. The target protein (SEQ ID NO: 30 or SEQ ID NO: 32) was present in the eluent of the 0% buffer B elution step; this eluent was combined and concentrated using a 10K AMICON ULTRA-15 instrument. The final product with a purity greater than 95% was stored in 40% glycerol at -20°C until use.

[0516] BbrGlu2 and BbrGlu5

[0517] Both BbrGlu2 (SEQ ID NO: 36) and BbrGlu5 (SEQ ID NO: 38) α-glucosidases were purified using a four-step process. For each purification, 1-L of crude fermentation broth from a DASGIP fermenter was concentrated, followed by the addition of ammonium sulfate to 60% saturation. The solution was stirred at 4°C for 1 hour and then centrifuged at 8000×g for 30 minutes. The resulting precipitate was resuspended in 20 mM HEPES (pH 7.0, buffer A). Ammonium sulfate was added to the final solution to a final concentration of 1 M; this formulation was then loaded into HiPrep pre-equilibrated with 20 mM HEPES (pH 7.0) and 1 M ammonium sulfate. TMOn a phenyl-FF column, the target protein (SEQ ID NO: 36 or SEQ ID NO: 38) was eluted from the column with 0.5M ammonium sulfate. The fractions were combined, concentrated using a VIVAFLOW 200 ultrafiltration system (Sartorius Stedim), and exchanged with buffer A. The resulting solution was then applied to a HiPrep solution pre-equilibrated with buffer A. TM Q FF16 / 10 column. Elute the target protein from the column using buffer A containing a linear gradient of 0–0.5 M NaCl. Combine the fractions containing the target protein, concentrate, and then load them into HiLoad buffer pre-equilibrated with 20 mM HEPES (pH 7.0) and 0.15 M NaCl. TM 26 / 60 Superdex TM The fractions containing the target protein were then combined and concentrated using a 10K AMICON ULTRA-15 device and stored in 40% glycerol at -20°C until use.

[0518] Therefore, various other α-glucosidases were expressed and purified. The hydrolytic activity of these α-glucosidases against α-1,5-glucosyl-fructose bonds and α-1,3 and / or α-1,6-glucosyl-glucose bonds was tested (in Examples 11, 12, 15 and 16 provided below).

[0519] Example 11

[0520] Testing the hydrolytic activity of α-glucosidase on various glycosidic bonds

[0521] This embodiment discloses a method for testing whether α-glucosidases possess hydrolytic activity exceeding that of previous enzymes of this class (EC 3.2.1.20). The α-glucosidase of Example 10 exhibits hydrolytic activity against α-1,5-glucosyl-fructose bonds and α-1,3 and α-1,6-glucosyl-glucose bonds.

[0522] Substrate specificity of α-glucosidase

[0523] The substrate specificity of each α-glucosidase disclosed in Example 10 was analyzed based on the release of glucose from specific substrates (isomaltose, maltose, panose, Leuconostoc disaccharide, or Aspergillus niger) upon incubation with the substrate and α-glucosidase. The glucose release rate was determined using the coupled glucose oxidase / peroxidase (GOX / HRP) method (1980, Anal. Biochem. 105: 389-397). The glucose release was quantified as the rate at which the peroxide produced by the reaction of the coupled GOX / HRP enzyme with glucose oxidized 2,2′-azino-di-3-ethylbenzothiazoline-6-sulfonic acid (ABTS).

[0524] Each substrate solution was prepared by mixing 9 mL of substrate solution (1% water, w / v) with 1 mL of 0.5 M (pH 5.0) sodium acetate buffer and 40 μL of 0.5 M calcium chloride in a 15-mL conical tube. A GOX / HRP coupled enzyme solution containing ABTS was prepared in 50 mM sodium acetate buffer (pH 5.0) to a final concentration of 2.74 mg / mL ABTS, 0.1 U / mL HRP, and 1 U / mL GOX. A series of dilutions of each α-glucosidase sample and glucose standard were prepared in Milliq water. For Aspergillus niger, only one dose of 10 ppm was used to test the α-glucosidase sample due to the limited stock solution of substrate. Each α-glucosidase sample (10 μL) was transferred to a new microtiter plate (Corning 3641) containing 90 μL of substrate solution pre-incubated at 50 °C and 600 rpm for 5 min. The reaction was carried out at 50°C in a THERMOMIXER (Eppendorf) for 10 minutes (for isomaltose, maltose, panose, and Aspergillus niger substrates) or 60 minutes (for Leuconostoc disaccharide substrate) with shaking (600 rpm). Then, 10 μL of each reaction mixture and 10 μL of serially diluted standard glucose were rapidly transferred to new microtiter plates (Corning 3641), followed by the addition of 90 μL of ABTS / GOX / HRP solution. The microtiter plates containing the reaction mixtures were rapidly measured at 405 nm at 11-second intervals for 5 minutes using a SOFTMAX PRO plate reader (Molecular Devices). For each enzyme concentration, the output is the reaction rate V. o Linear regression was used to determine the curve V. o The slope relative to enzyme dosage. The specific activity of each α-glucosidase was calculated using Equation 1 based on the glucose standard curve:

[0525] Specific activity (units / mg) = slope (enzyme) / slope (standard) × 1000 (1),

[0526] One unit = 1 μmol glucose / minute.

[0527] For Aspergillus niger, the reaction rate value at an enzyme dosage of 10 ppm is used directly to indicate enzyme activity.

[0528] Using the methods described above, the specificity of each α-glucosidase for each substrate was determined. The activities of oligo-1,6-glucosidase (purchased from Megazyme, see Table 4) and transglucosidase (TG L-2000, see Table 4) for each substrate were also determined. The results of this analysis are presented in Table 17.

[0529] Table 17

[0530] The activities of various α-glucosidases on different substrates

[0531]

[0532]

[0533] For Aspergillus niger, each enzyme is used at a dose (10 ppm).

[0534] Interestingly, it has been found that α-glucosidase, in addition to exhibiting hydrolytic activity on α-1,4-glucosyl-glucose bonds (maltose), also exhibits hydrolytic activity on α-1,6-glucosyl-glucose bonds (isomaltose), α-1,3-glucosyl-glucose bonds (Aspergillus niger), and α-1,5-glucosyl-fructose bonds (Leuconostoc mesenteroides) (Table 17).

[0535] Therefore, α-glucosidases possess hydrolytic activities exceeding those previously described in EC 3.2.1.20. Specifically, α-glucosidases exhibit hydrolytic activity against α-1,5-glucosyl-fructose bonds and α-1,3 and α-1,6-glucosyl-glucose bonds.

[0536] Example 12

[0537] Leuconosticol disaccharide and oligosaccharides in the filtrate of the dextran hydrolysis reaction with α-glucosidase

[0538] This example describes the use of α-glucosidase to hydrolyze Leuconostoc disaccharide and other oligosaccharides present in the filtrate obtained from the dextran synthesis reaction. Specifically, the effect of the α-glucosidase disclosed in Example 10 on the hydrolysis of Leuconostoc disaccharide and oligosaccharides DP2, DP3 and HS (high sugar, DP4+) in the filtrate of the insoluble dextran (poly-α-1,3-glucan) synthesis reaction was investigated.

[0539] Isolation and analysis of oligosaccharides used for testing α-glucosidase activity

[0540] First, a concentrated filtrate of the dextran synthesis reaction was prepared according to Example 1.

[0541] In summary, oligosaccharides were separated from the concentrated filtrate by chromatography, and the glycosidic bond characteristics were analyzed. Chromatographic separation using a strongly acidic cation exchange resin was employed to separate the oligosaccharide fraction from the concentrated filtrate. The physical parameters of the column used for this separation were as follows: FINEX CS11GC, #227 resin; Na... + Ionic form; 5% divinylbenzene (crosslinked); 0.34 mm particle size; 1.64 m bed length; 0.093 m column diameter.

[0542] More specifically, the concentrated sugar solution (i.e., concentrated filtrate) described in Table 3 was filtered and diluted with tap water to a concentration of 25 g dry solids / 100 g solution. Before adding this sugar solution to the column resin, the resin was washed with six bed volumes (BV) of sodium chloride solution (three BV of 10 wt% sodium chloride, followed by three BV of 5 wt% sodium chloride) to convert the resin to its sodium form. The sugar solution (0.6 L) was then fed into the column, followed by elution with water at a flow rate of 50 L / h. The operating conditions for the chromatographic separation were summarized as follows: 0.6 L feed volume, 25 g dry solids / 100 g solution, column temperature 65 °C, flow rate 50 mL / min. The oligosaccharide solution eluted between 11 and 21 minutes. A small amount of salt—indicating increased conductivity—eluted simultaneously. The product distribution of the oligosaccharide fraction thus prepared was determined by HPLC analysis. In summary, the fraction contained >89% oligosaccharides containing three or more hexose units and less than 1.5% identifiable monosaccharides and disaccharides. The fraction was concentrated to a total dry weight of 317 g / L using a thin-film evaporator (LCICorporation, Charlotte, NC), followed by rotary evaporation using a ROTAVAPOR (R-151; Buchi, New Castle, DE). The product distribution of the concentrated fraction as determined by HPLC is shown in Table 18.

[0543] Table 18

[0544] Product distribution of concentrated oligosaccharide fractions

[0545]

[0546] Preliminary screening of α-glucosidases that hydrolyze dextran oligomers

[0547] The activities of eleven different α-glucosidases (Example 10) and two reference enzymes, namely oligo-1,6-glucosidase (purchased from Megazyme) and transglucosidase (TG L-2000), were evaluated for the purified oligosaccharide fractions prepared above (Table 18). Each α-glucosidase (dose 1 mg / mL) was incubated at pH 5.0 and 60°C in a solution containing oligosaccharide substrate (2.9% dry solids) and 2 mM calcium chloride. After 24 hours of incubation, each reaction was quenched by adding 50 μL of 0.5 M NaOH.

[0548] The oligosaccharide / monosaccharide contents after quenching reaction were determined as follows. Samples from each reaction were diluted 5-fold with water for HPLC analysis. HPLC separation was performed at 85°C using an Agilent 1200 series HPLC system with an AMINEX HPX-42A column (300 mm × 7.8 mm). Samples (10 mL) were applied to the HPLC column and separated using an isogradient Milli-Q water as the mobile phase at a flow rate of 0.6 mL / min. A refractive index detector was used to detect the oligosaccharide products. The figures in Table 19 below reflect the individual DP values. n The average peak area percentage (obtained from each sample, in duplicate) is part of the total DPI to DP7.

[0549] Table 19

[0550] Analysis of oligosaccharides in dextran filtrate after α-glucosidase treatment

[0551]

[0552] As indicated by shading in Table 19, the oligosaccharide contents of the reaction generally favor smaller-particle sugars compared to the control reaction without the enzyme (“blank”). These results indicate that α-glucosidases can be used to hydrolyze oligosaccharides and their fractions contained in dextran synthesis reactions. Furthermore, based on the bond characteristics of oligosaccharides (Examples 3 and 4) and the activity of α-glucosidases against various glycosidic bonds other than α-1,4 bonds (Example 11), it is evident that α-glucosidases can be used to break down oligosaccharides having α-1,5-glucosyl-fructose bonds and / or α-1,3 and α-1,6-glucosyl-glucose bonds. The results provided in Table 19 also indicate that fungal α-glucosidases exhibit better hydrolytic activity towards soluble oligosaccharides compared to bacterial α-glucosidases.

[0553] Confirmation of the hydrolytic activity of α-glucosidase on oligosaccharide products from dextran synthesis.

[0554] A reaction was carried out comprising one or two α-glucosidases and a concentrated filtrate obtained from the synthesis reaction of poly-α-1,3-glucan (Table 3). The dosage of the α-glucosidase was 4 ppm of enzyme, or for blends, the individual enzymes were 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 also contained 2 mM calcium chloride (pH 5.0) and was carried out at 60°C or 65°C. After incubation for 23 hours, the reaction was quenched by adding 50 μL of 0.5 M NaOH.

[0555] The oligosaccharide / monosaccharide contents after quenching reactions were determined as follows. Samples from each reaction were diluted 25-fold with water for HPLC analysis. HPLC separation was performed at 85°C using an Agilent 1200 series HPLC system with an AMINEX HPX-42A column (300 mm × 7.8 mm). Samples (10 mL) were applied to the HPLC column and separated using an isogradient Milli-Q water as the mobile phase at a flow rate of 0.6 mL / min. A refractive index detector was used to detect the oligosaccharide products. The figures in Table 20 below reflect the individual DP values. n The average percentage of peak area (from each of the two copies) is included as part of the total. The results provided in Table 20 generally confirm the activity of the specific α-glucosidase as discussed above (regarding the results provided in Table 19).

[0556]

[0557] Therefore, α-glucosidase can be used to hydrolyze Leuconostoc disaccharide and other oligosaccharides present in fractions (e.g., filtrates) obtained from dextran synthesis reactions such as poly-α-1,3-glucan synthesis reactions.

[0558] Example 13

[0559] Extreme separation of oligosaccharides / Leuconostoc disaccharides from GTF-S / MUT3325 reverse derivatives

[0560] Sucrose (4.50 kg) was dissolved in distilled deionized water to a final total volume of 9.5 L. The resulting solution was heated at 80 °C for 5 minutes with stirring and then cooled to 47 °C. With stirring, 500 g of crude extract containing 0.6 g / L of gtf-S enzyme (GTF0459, SEQ ID NO: 42) and 15.0 mL of crude extract containing 10 g / L of mutant enzyme (MUT3325, SEQ ID NO: 47) were added (see General Methods for Enzyme Preparation). With stirring, 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. The reaction temperature and pH were maintained at 47°C and pH 5.5-6.0, respectively, until the sucrose conversion was >95% according to HPLC analysis. The reaction mixture was then immediately adjusted to pH 7.0-7.5 and heated to 90°C for 20 minutes, followed by cooling to 25°C and immediate filtration to remove particles and precipitate. The resulting filtrate was maintained at 5°C prior to IEX / SEC column chromatography using the following resin and conditions: FINEX CS 11GC SAC(Ca 2+The column was prepared using the following method: column id = 9.3 cm, column bed height = 1.58 m, T = 70 °C, flow rate = 51 mL / min, linear flow rate = 0.44 m / h, feed volume = 0.6 L = 171 g, feed RI-DS = 25.1 g / 100 g, sampling interval = 3 min. The column fractions collected between 30 min and 67 min were combined, concentrated to 66% dissolved solids by evaporation, and analyzed by HPLC as described in the general method. Table 21 shows the oligosaccharide and monosaccharide components of the separated fractions thus prepared.

[0561] Table 21

[0562] Analysis of oligomer / Leuconostoc disaccharide fractions obtained from the GTF-S / MUT3325 reaction

[0563]

[0564] In this embodiment, a dextran synthesis reaction is used to produce at least one soluble α-glucan product. This soluble product is produced by the synergistic action of both glucosyltransferase (GTF0459, SEQ ID NO: 42) and α-glucan hydrolase (MUT3325, SEQ ID NO: 47), both present in glucosyltransferase reactions. This embodiment also illustrates a chromatographic fraction prepared from the dextran synthesis reaction. This fraction was therefore used in Examples 15 and 16 below to test α-glucosidase activity.

[0565] Example 14

[0566] Separation of oligomers / Leuconostoc disaccharides from Gtf-C reactants

[0567] Sucrose (4.50 kg) was dissolved in distilled deionized water to a final total volume of 9.5 L. The resulting solution was heated at 80 °C for 5 minutes with stirring and then cooled to 47 °C. With stirring, 500 g of crude extract containing 0.41 g / L of GTF-C enzyme (GTF0088BsT1, SEQ ID NO: 45) was added (see General Methods for Enzyme Preparation). With stirring, 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. The reaction temperature and pH were maintained at 47 °C and pH 5.5–6.0, respectively, until the sucrose conversion was >95% according to HPLC analysis. The reaction mixture was then immediately adjusted to pH 7.0 to 7.5 and heated to 90 °C for 20 minutes, then cooled to 25 °C and immediately filtered to remove particles and precipitate. Using the following resin and conditions, maintain the resulting filtrate at 5°C prior to IEX / SEC column chromatography: FINEX CS 11GC SAC(Ca 2+The column was prepared using the following method: column id = 9.3 cm, column bed height = 1.58 m, T = 70 °C, flow rate = 50 mL / min, linear flow rate = 0.44 m / h, feed volume = 0.6 L = 171 g, feed RI-DS = 25.8 g / 100 g, sampling interval = 3 min. The column fractions collected between 34 min and 72 min were combined, concentrated to 67% dissolved solids by evaporation, and analyzed by HPLC as described in the general method. Table 22 shows the oligosaccharide and monosaccharide components of the separated fractions thus prepared.

[0568] Table 22

[0569] Analysis of oligomers / Leuconostoc disaccharide fractions obtained from the Gtf-C reaction

[0570]

[0571] In this embodiment, a dextran synthesis reaction is used to produce at least one soluble α-glucan product. This embodiment also illustrates a chromatographic fraction prepared from the dextran synthesis reaction that produces the soluble α-glucan product. This fraction was therefore used in Examples 15 and 16 below to test α-glucosidase activity.

[0572] Example 15

[0573] Preliminary screening of α-glucose was performed using oligosaccharide fractions derived from Gtf-S / MUT3325 and Gtf-C reactions from Leuconostoc mesenteroides. glycosidase

[0574] This example describes the use of α-glucosidase to hydrolyze Leuconostoc disaccharide and other oligosaccharides present in chromatographic fractions obtained from dextran synthesis reactions that produce soluble α-glucan products. Specifically, the effect of the α-glucosidase disclosed in Example 10 on the hydrolysis of Leuconostoc disaccharide and oligosaccharides in the fractions prepared in Examples 13 and 14 was investigated.

[0575] A total of twelve α-glucosidases and two reference enzymes (oligo-1,6-glucosidase and TGL-2000 transglucosidase) were screened using oligomer / Leuconostoc melanogaster fractions obtained from the GTF-S / MUT3325 (Example 13) and GTF-C (Example 14) reactions as substrates. All enzymes (α-glucosidases and reference enzymes) were used at the same protein concentration. Each α-glucosidase was incubated at pH 5.5 and 47°C in a solution containing oligomer / Leuconostoc melanogaster disaccharide substrate (10% dry solids) and 2 mM calcium chloride (100 ppm). After 21 hours of incubation, each reaction was quenched by adding 50 μL of 0.5 M NaOH.

[0576] The oligosaccharide / monosaccharide contents after quenching reactions were determined as follows. Samples from each reaction were centrifuged, and the supernatant was diluted 25-fold with water for HPLC analysis (general method). The percentages recorded in Table 23 reflect the individual DP values. n The percentage of average peak area (obtained from the analysis of each sample, in duplicate) was included as part of the total. The results showed that fungal α-glucosidases exhibited better hydrolytic activity against dextran oligomers compared to bacterial α-glucosidases.

[0577]

[0578]

[0579] As indicated by shading in Table 23, the oligosaccharide contents of the reaction generally favor smaller-particle sugars compared to the control reaction without the enzyme (“blank”). These results indicate that α-glucosidases can be used to hydrolyze oligosaccharides and their fractions contained in dextran synthesis reactions, particularly the chromatographic fractions of dextran synthesis reactions that produce soluble α-glucan products. Furthermore, based on the bond characteristics of oligosaccharides (Examples 13 and 14) and the activity of α-glucosidases against various glycosidic bonds other than α-1,4 bonds (Example 11), it is evident that α-glucosidases can be used to break down oligosaccharides having α-1,5-glucosyl-fructose bonds, and possibly α-1,3 and α-1,6-glucosyl-glucose bonds. The results provided in Table 23 also indicate that fungal α-glucosidases exhibit better hydrolytic activity towards soluble oligosaccharides compared to bacterial α-glucosidases.

[0580] Therefore, α-glucosidase can be used to hydrolyze Leuconostoc disaccharide and other oligosaccharides present in fractions (e.g., chromatographic fractions) of reactions derived from glucan synthesis reactions, such as the synthesis of soluble α-glucan products.

[0581] Example 16

[0582] Oligosaccharide-based α-glucosidase from Gtf-S / MUT3325 and Gtf-C reaction was used for sorting and sieving of Leuconostoc melanin.

[0583] This embodiment, based on Example 15, describes the use of α-glucosidase to hydrolyze Leuconostoc disaccharide and other oligosaccharides present in the chromatographic fraction of a glucan synthesis reaction yielding soluble α-glucan products.

[0584] The efficacy of sugar compositions derived from enzymes containing the same protein concentration was evaluated by analyzing the reactions they produced. Oligomers / Leuconostoc disaccharide fractions resulting from the reaction of GTF-S / MUT3325 and GTF-C (Example 15) The most hydrolyzable α- Portugal glycosidaseα-glucosidase (4 ppm; for blends, the ratio of the two enzymes was 1:1 and the total amount was 4 ppm) and oligomer / Leuconostoc mesylate substrate (10% ds) were incubated at 60 °C and 65 °C, respectively, in the presence of 2 mM calcium chloride, at pH 5.5. After 23 hours of incubation, the reaction was quenched by adding 50 μL of 0.5 mM NaOH.

[0585] The oligosaccharide / monosaccharide contents after quenching reactions were determined as follows. Samples from each reaction were centrifuged and the supernatant was diluted 25-fold with water for HPLC analysis (general method). The percentages recorded in Table 24 (below) reflect the individual DP contents. n The percentage of average peak area (obtained from the analysis of each sample, in duplicate) was included as part of the total. The results showed that TauSec098 was effective for hydrolyzing DP2 to DP7 oligomers when incubated at 65°C, and TauSec099 was superior to TG L-2000 in the hydrolysis of Leuconostoc mesenteroides. Blends of TauSec098 and TauSec099 (or TG L-2000) effectively hydrolyzed oligomers and Leuconostoc mesenteroides to produce DP1.

[0586] Therefore, α-glucosidase can be used to hydrolyze Leuconostoc disaccharide and other oligosaccharides present in fractions (e.g., chromatographic fractions) of reactions derived from glucan synthesis reactions, such as the synthesis of soluble α-glucan products.

[0587]

[0588]

Claims

1. A method for reducing (i) the amount of Leuconostoc disaccharide present in a dextran synthesis reaction or (ii) its fractions, wherein the method comprises: The dextran synthesis reaction or its fraction is contacted with the transglucosidase shown in SEQ ID NO:1, wherein the amount of Leuconostoc disaccharide in the dextran synthesis reaction or its fraction is reduced compared to the amount of Leuconostoc disaccharide present before the contact, and wherein the Leuconostoc disaccharide is a byproduct of the dextran synthesis reaction.

2. The method according to claim 1, wherein the transglucosidase is immobilized.

3. The method according to claim 1, wherein the concentration of Leuconostoc disaccharide after the contact is less than 50% of the concentration of Leuconostoc disaccharide present before the contact.

4. The method according to any one of claims 1-3, wherein the dextran synthesis reaction produces at least one insoluble α-glucan product.

5. The method according to claim 4, wherein at least 50% of the glycosidic bonds of the insoluble α-glucan product are α-1,3-glycosidic bonds.

6. The method according to claim 4, wherein at least 80% of the glycosidic bonds of the insoluble α-glucan product are α-1,3-glycosidic bonds.

7. The method according to claim 4, wherein at least 95% of the glycosidic bonds of the insoluble α-glucan product are α-1,3-glycosidic bonds.

8. The method according to claim 4, wherein the insoluble α-glucan product has a number-average degree of polymerization of at least 100.

9. The method of claim 4, wherein the method comprises contacting the dextran synthesis reaction with the transglucosidase.

10. The method of claim 4, wherein the method comprises contacting the fraction with the transglucosidase.

11. The method according to claim 10, wherein the fraction is the filtrate of the dextran synthesis reaction.

12. The method according to claim 10, wherein the fraction is the supernatant of the dextran synthesis reaction.

13. The method according to claim 1, wherein the dextran synthesis reaction produces at least one soluble α-glucan product, which is: (i) a product of a glucosyltransferase, or (ii) a product of the synergistic action of a glucosyltransferase and an α-glucan hydrolase, wherein the α-glucan hydrolase is capable of hydrolyzing dextran polymers having one or more α-1,3-glycosidic bonds or one or more α-1,6-glycosidic bonds.

14. The method according to claim 1, wherein the fraction is a chromatographic fraction of the dextran synthesis reaction.

15. The method of claim 1, wherein the method comprises contacting the dextran synthesis reaction with the transglucosidase.

16. The method of claim 1, wherein the method comprises contacting the fraction with the transglucosidase.

17. A method for enriching fructose present in fractions of a dextran synthesis reaction, the method comprising: (a) The method according to claim 1 produces the fraction of the dextran synthesis reaction, thereby providing a fructose-containing hydrolysis fraction; as well as (b) Separate fructose from the hydrolyzed fraction of step (a) to obtain a composition with a higher fructose concentration than that of the fraction of step (a).

18. A fermentation method, the method comprising: (a) The method according to claim 1 for producing the fraction of the dextran synthesis reaction; (b) Obtaining a product by microbial fermentation of the fraction described in step (a), wherein the fermentation is performed after or concurrently with step (a); and (c) Optionally, the product of step (b) is separated; wherein the product yield of (b) is increased compared to the product yield of fermenting fractions of the glucan synthesis reaction that have not been contacted with the transglucosidase.

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