Compositions and methods for producing 1, 2-beta-oligoglucan

Abstract

Disclosed herein are compositions and methods for the production of a beta-1, 2-oligoglucan. The composition comprises glucose-1-phosphate, a primer molecule, and a [beta]-glucan phosphorylase having [beta]-1, 2-glucan phosphorylase activity. For example, the betaGP having beta-1, 2-glucan phosphorylase activity may have a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to at least one of SEQ ID NO: 10, 11, 13, 15, or 16.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of European application number 22192796.5 filed on August 30, 2022 and EP 22213127.8 filed on December 13, 2022, which are incorporated herein by reference in their entirety.

[0003] References to electronically submitted sequence listings

[0004] The contents of the sequence listing XML file named "PT-1130-WO-PCT.xml", which is 35,438 bytes in size and was created on August 29, 2023 and electronically filed with this application, are incorporated herein by reference in their entirety. Background Art

[0005] In nature, β-glucans are produced by bacteria and play an important role in the invasion and immunomodulation of infected mammalian or plant cells. Commercially, most β-glucans are produced by cell wall extracts of yeast, fungi and plants, although there are some limited fermentation-based synthesis methods. Purified β-glucans are usually obtained by an acid hydrolysis step followed by selective precipitation using an organic solvent. The extraction process and biological sources of β-glucans lead to significant changes in their physicochemical properties and functional properties, including their branching patterns, molecular weight distribution, viscosity and concentration in biological matrices. Although some glycosyltransferases are specifically used for the synthesis of β-glucans, the high cost of their nucleotide-activated donor sugars is a serious limitation to their commercial development. Therefore, there is a need in the art for more consistent, reproducible and commercially relevant compositions and methods for producing β-glucans and particularly β-1,2-oligoglucans. Summary of the invention

[0006] The present disclosure provides a method for producing 1,2-β-oligoglucans, the method comprising contacting α-D-glucose-1-phosphate (G1P) with beta (β)-glucan-phosphorylase (βGP) to produce 1,2-β-oligoglucans. The method may further include a step of contacting a substrate with alpha (α)-glucan-phosphorylase (αGP) in the presence of an inorganic phosphate (e.g., sodium phosphate or potassium phosphate) to produce glucose-1-phosphate. The substrate may have a degree of polymerization (DP) equal to or greater than 4. The substrate may be selected from the group consisting of: maltodextrin, starch liquefact, trehalose, sucrose, cellulose, cellodextrin, cellobiose, and combinations thereof. The βGP G1P contacting step may be performed at a pH between 6.0 and 7.5, and / or the substrate αGP contacting step may be performed at a pH between 6.5 and 8.0.

[0007] The present disclosure also provides a composition comprising i) beta (β)-glucan-phosphorylase (βGP); ii) α-D-glucose-1-phosphate (G1P); and iii) a primer molecule. The composition may further comprise 1,2-β-oligoglucan, a phosphatase inhibitor (e.g., sodium molybdate), a buffer and / or a reducing agent. The primer molecule may be selected from the group consisting of: D-glucose, sophorose, laminaribiose, cellobiose, gentiobiose, and combinations thereof.

[0008] The present disclosure also provides a composition comprising alpha(α)-glucan-phosphorylase (αGP), inorganic phosphate, and a substrate (eg, maltodextrin, starch, starch liquefact, trehalose, sucrose, cellulose, cellodextrin, cellobiose, and combinations thereof).

[0009] In the compositions and methods described herein, the αGP can be a glycoside hydrolase 94 enzyme. The αGP can have an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: 1, 2, 3, 4, 5, or 18, preferably at least 80%, at least 85%, at least 95%, or at least 95% identical to at least one of SEQ ID NOs: 1, 2, or 5, or most preferably at least 90% identical to SEQ ID NO: 5.

[0010] In the compositions and methods described herein, βGP can be a glycosyltransferase 35 enzyme. βGP can have an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to at least one of SEQ ID NOs: 10, 11, 13, 15, and 16, preferably at least 90% identical to SEQ ID NO: 11.

[0011] The present disclosure also provides use of the αGP and / or βGP compositions described herein for producing the 1,2-β-oligoglucan compositions described herein.

[0012] Also provided herein is a 1,2-β-oligoglucan composition produced by the method according to any one of claims 1 to 5 and 10 to 11, wherein the composition has a polydispersity between 2 and 40, a degree of polymerization (DP) of about 6-150, and a viscosity between 800 mPas and 1200 mPas at 50° C. Generally, the 1,2-β-oligoglucan composition is indigestible.

[0013] The present disclosure further provides a vector comprising a nucleic acid encoding a polypeptide that is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 5. The present disclosure also provides a cell comprising the vector.

[0014] The present disclosure further provides a vector comprising a nucleic acid encoding a polypeptide that is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 11. The present disclosure also provides a cell comprising the vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0016] The drawings illustrate various aspects discussed in this document generally by way of example and not by way of limitation.

[0017] Figure 1 Shown is an SDS-PAGE gel of A1, A3, A5, A6 and A8 expression products from 100 mL overnight culture of E. coli BL21(DE3) cells grown at 20°C or 30°C and chemically induced with 1 mM IPTG.

[0018] Figure 2 Shown is an SDS-PAGE gel of αGPase after heat treatment for 1 hour at 60° C. For each enzyme, the soluble and insoluble fractions are depicted in the left and right columns, respectively.

[0019] Figure 3 Crude cell extracts on 12% SDS-PAGE gels are shown before (left) and after (right) HIS-tag purification. Arrows point to the positions of the three thermostable αGP candidates.

[0020] Figure 4 Shown is the variation of specific activity of αGP candidate enzymes as a function of temperature.

[0021] Figure 5 Shown are the specific activities of the αGP candidate enzymes retained after incubation for the indicated time periods at 55° C. Standard deviations were ≤7% (A1), ≤14% (A3), and ≤20% (A8).

[0022] Figure 6 Shown are the specific activities of candidate αGPases on two different maltodextrin substrates.

[0023] Figure 7 The Michaelis-Menten graph of A1αGP is shown.

[0024] Figure 8 The Michaelis-Menten plot of A3αGP is shown.

[0025] Fig. 9 The Michaelis-Menten plot of A8αGP is shown.

[0026] Fig.10 Michaelis-Menten plots of A8 αGPase at maximum maltodextrin concentrations of 24% (left) and 60% (right) are shown.

[0027] Fig.11 Shown is an SDS-PAGE gel of βGP expression products from 100 mL overnight cultures (B1, B2, B3, B4, B5, B6 and B8) or 250 mL overnight cultures (B7, B11, B12, B13 and B14) of E. coli BL21 (DE3) cells grown at 20°C and chemically induced with 1 mM IPTG. "a" indicates the soluble crude extract fraction, and "b" indicates the insoluble crude extract fraction. The solid arrows show the soluble protein bands, and the dotted arrows show the insoluble protein bands.

[0028] Fig.12 Shown is an SDS-PAGE gel of βGPase after heat treatment for 1 hour at 60° C. For each enzyme, the soluble (“hS”) and insoluble (“hl”) fractions are depicted in the left and right columns, respectively.

[0029] Fig.13 The release of phosphate (Pi) and glucose (Glc) over time as a result of the assay described in Example 4 is shown.

[0030] Fig.14 Thin layer chromatograms of the reaction products of the B6 assay summarized in Example 5 and Table 10 are shown.

[0031] Fig.15 Image showing the results of anion exchange chromatography of the activity of B6 on sophorose. The individual components in the reaction mixture are included (bottom three rows). The formation of glucans of different DP is evidenced by a "row of peaks" that clearly does not belong to any individual component in the assay mixture.

[0032] Fig.16 A graph showing the relationship between temperature or pH and B6 enzyme activity is shown.

[0033] Fig.17 Shown is a graph representing the thermal stability of B6 after incubation at 50° C. Standard deviation ≤ 17%.

[0034] Fig.18 Michaelis-Menten plot showing the activity of B6 on the sophorose substrate.

[0035] Fig.19 HIS-tag purification of βGPases B7, B11, B12, B13 and B14 is shown.

[0036] Fig. 20TLC plates loaded with reaction mixtures are shown for the reactions of B7, B11, B12, B13 and B14 enzymes on each of glucose, sophorose, laminaribiose, gentiobiose and cellobiose. Arrows point to the products formed. Inactive reactions have two spots in the columns corresponding to G1P and substrate.

[0037] Fig.21 An anion exchange chromatogram showing the activity of the B7 enzyme on the sophorose substrate is shown.The text refers to known reaction mixture components (eg sophorose and G1P) and reaction products (eg oligosaccharides and polysaccharides formed).

[0038] Fig. 22 An anion exchange chromatogram showing the activity of the B12 enzyme on a glucose substrate is shown. The text refers to known peaks (e.g. glucose, gentiobiose / cellobiose, sophorose / laminarinose and G1P) and reaction products (e.g. disaccharides formed: β-1,2 linked sophorose and / or β-1,3 linked laminarinose).

[0039] Fig.23 An anion exchange chromatogram showing the activity of the B12 enzyme to form laminaribiose on a glucose substrate is shown.

[0040] Fig.24 An anion exchange chromatogram showing the activity of the B13 enzyme on the laminaribiose substrate is shown. The text refers to known peaks (eg glucose, laminaribiose and G1P) and reaction products (eg sugars formed).

[0041] Fig.25 An anion exchange chromatogram showing the activity of the B13 enzyme on the sophorose substrate is shown. The text refers to known peaks (eg glucose, sophorose) and reaction products (oligosaccharides formed).

[0042] Fig.26 An anion exchange chromatogram showing the activity of the B13 enzyme on glucose is shown. The text refers to known peaks (eg glucose and G1P) and reaction products (eg sugars formed).

[0043] Fig. 27 An anion exchange chromatogram showing the activity of the B6 enzyme on the combined substrate syrup as outlined in Example 8 is shown.

[0044] Fig.28 Shown is the chromatogram obtained by HPLC fingerprinting of debranched Zulkowsky starch maltodextrins.

[0045] Fig.29 Shown is the chromatogram obtained by HLPC fingerprinting of debranched soluble starch maltodextrin.

[0046] Fig.30Shown is the chromatogram obtained by HPLC fingerprinting of debranched DE 4-7 maltodextrin.

[0047] Fig.31 Shown is the chromatogram obtained by HPLC fingerprinting of debranched DE 13-17 maltodextrin.

[0048] Fig.32 The chromatogram obtained by HPLC fingerprinting of debranched maltodextrin 01910 is shown.

[0049] Fig.33 The chromatogram obtained by HPLC fingerprinting of debranched maltodextrin 01912 is shown.

[0050] Fig.34 Diagram showing the conversion of debranched soluble starch to G1P using A8 αGPase.

[0051] Fig.35 Diagram showing the conversion of debranched Zulkowsky starch to G1P using A8 αGPase.

[0052] Fig.36 Diagram showing the conversion of debranched MDX DE 4-7 to G1P using A8 αGPase.

[0053] Fig.37 Diagram showing the conversion of debranched MDX DE 13-17 to G1P using A8 αGPase.

[0054] Fig.38 Shown are pictures of β-glucans synthesized starting from 0.5M, 1M or 1.5M G1P and using crude extracts of B6 βGP released by sonication (left) or homogenization (right).

[0055] Fig.39 Graph showing the effect of temperature and Sumizyme GOP on β-glucan synthesis.

[0056] Fig.40 Graph showing the effect of Sumizyme GOP and / or sodium molybdate on β-glucan synthesis.

[0057] Fig.41 The 1H-NMR spectrum of β-1,2-glucan produced using B6 βGPase is shown.

[0058] Fig.42 Shown is the 13C-NMR spectrum of β-1,2-glucan produced using B6 βGPase.

[0059] Fig.43Shown are a 120 g sample of β-1,2-glucan (top) and a chromatogram of oligosaccharides (middle) and a high molecular weight GPC analysis of β-1,2-glucan.

[0060] Fig.44 Shown is a graph of shear stress versus shear rate for β-1,2-glucan samples over the temperature range of 20°C-80°C.

[0061] Fig.45 Shown is a graph of viscosity versus shear rate for β-1,2-glucan samples over the temperature range of 20°C-80°C.

[0062] Fig.46 The Newtonian viscosity of β-1,2-glucan at 20°C-30°C is shown compared to glucose (top), sucrose (middle) and standard maltodextrin (bottom).

[0063] Fig.47 Shown is the glucose release from isomaltulose, sucroseman, promitor 70 and β-1,2-glucan in an in vitro digestibility assay.

[0064] Fig.48 Shown is the synthesis of β-glucan starting from 0.5M G1P and pure B6 using glucose as primer.

[0065] Fig.49 Shown is the synthesis of β-glucan starting from 0.2 M G1P and pure B6 using glucose as primer.

[0066] Fig.50 Graph showing the molecular weight distribution of β-glucan produced by the reaction of 0.2 M (top) or (0.5 M) G1P with pure B6 after 72 hours reaction time.

[0067] Fig.51 The reaction scheme used in Example 12 is shown.

[0068] Fig.52 β-glucan synthesis under the conditions described as outlined in Example 12 is shown.

[0069] Fig.53 The results of viscosity measurements of the starch and / or β-glucan compositions as outlined in Example 13 are shown.

[0070] Fig.54 The results of viscosity measurements of the starch and / or β-glucan compositions as outlined in Example 13 are shown.

[0071] Fig.55 A phylogenetic tree of αGP is shown.

[0072] Figure 56A- Fig.56D SDS-PAGE gel images showing the retained protein bands of (A) AtGP, TtGP and TaGP and (C) weak protein bands of TsGP and strong protein bands of TmGP after incubation at 60°C for 1 h. In FIG. 56A and FIG. 56C , "c" indicates the soluble fraction after heat treatment, and "d" indicates the insoluble fraction after heat treatment. FIG. 56B and FIG. Fig.56D AtGP, TtGP, TaGP, TsGP and TmGP purified by affinity (His6-tag) chromatography are shown. Arrows indicate protein bands.

[0073] Fig.57 The effect of temperature on the activity of TmGP and TsGP compared to TaGP is shown. The temperature profile was determined using 50 mM phosphate buffer and 2% maltodextrin mixture as substrate at pH 7. The relative activity was calculated by the percentage of the value to the maximum value.

[0074] Fig.58 The 1H-NMR spectrum of β-1,2-glucan produced using B7 βGPase is shown. The boxed portion of the NMR spectrum is Fig.59 Medium zoom.

[0075] Fig.59 Show Fig.58 Magnified view of the boxed portion of the NMR spectrum.

[0076] Fig.60 The 13C-NMR spectrum of β-1,2-glucan produced using B7βGPase is shown. The boxed portion of the NMR spectrum is Fig.61 Medium zoom.

[0077] Fig.61 Show Fig.60 Magnified view of the boxed portion of the NMR spectrum.

[0078] Fig.62 The 1H-NMR spectrum of β-1,2-glucan produced using B13βGPase is shown. The boxed portion of the NMR spectrum is Fig.63 Medium zoom.

[0079] Fig.63 Show Fig.62 Magnified view of the boxed portion of the NMR spectrum.

[0080] Fig.64 The C13-NMR spectrum of β-1,2-glucan produced using B13 βGPase is shown.

[0081] Fig.65 Show Fig.64Magnified view of the boxed portion of the NMR spectrum. DETAILED DESCRIPTION

[0082] Reference will now be made in detail to certain aspects of the presently disclosed subject matter, examples of which are partially illustrated in the accompanying drawings.While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0083] In this document, the terms "a", "an" or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of an inconsistency between the usages of this document and those documents so incorporated by reference, the usage in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0084] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly listed as the limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y or about Z".

[0085] Unless expressly stated, ppm (parts per million), percentages and ratios are based on weight. Percentages based on weight are also referred to below as wt% or (wt)%.

[0086] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. As used herein, each of the following terms has the meaning associated therewith as defined below.

[0087] The present disclosure relates to β-1,2-oligoglucan compositions and compositions and methods for preparing the β-1,2-oligoglucans. Generally speaking, the composition for preparing β-1,2-oligoglucans comprises α-D-glucose-1-phosphate (G1P, also known as "alpha-D-glucose-1-phosphate"), a primer molecule, and a β-glucan-phosphorylase (βGP) enzyme. The method for preparing β-1,2-oligoglucans described herein comprises incubating a composition comprising G1P, a primer molecule, and βGP at a temperature and time sufficient to produce β-1,2-oligoglucans. The present disclosure also provides compositions for synthesizing G1P from maltodextrin, which compositions comprise maltodextrin, phosphate, and α-glucan-phosphorylase (αGP). The method for preparing G1P described herein comprises incubating a composition comprising maltodextrin, phosphate, and αGP at a temperature and time sufficient to produce G1P. The present disclosure further provides a composition comprising the produced β-1,2-oligoglucans.

[0088] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the overall primary, secondary, tertiary and quaternary amino acid sequences and structures necessary to give the macromolecule its function and properties. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (either independently or as part of a biosynthetic pathway) is understood to include the cofactors, coenzymes and metals necessary for the enzyme to function properly. An overview of amino acids and their three letter and one letter symbols as understood in the art is provided in Table 1. The amino acid names, three letter symbols and one letter symbol are used interchangeably herein.

[0089] Table 1: Amino Acid Three-Letter and One-Letter Symbols

[0090]

[0091]

[0092] Variant or sequence with basic identity or homology with polypeptide as described herein can be used for implementing disclosed pigment, composition and method.Such sequence can be referred to as variant or modified sequence.That is, peptide sequence can be modified but still retains the ability of showing desired activity.Usually, variant or modified sequence can comprise with wild-type, naturally occurring peptide sequence or with variant polypeptide as described herein for or greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity.

[0093] As used herein, the phrases "% sequence identity", "% identity" and "percent identity" are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well known. The generation of sequence alignments and sequence identities includes global alignments and local alignments performed using computational methods. Alignments can be performed using BLAST (National Center for Biological Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using standard protein BLAST with the following default parameters: maximum target sequence: 100; short query: automatically adjust parameters for short input sequences; expected threshold: 10; word length: 6; maximum number of matches within the query range: 0; matrix: BLOSUM62; gap penalty: (existence: 11, extension: 1); component adjustment: conditional component score matrix adjustment; filter: not selected; mask: not selected. The % nucleic acid sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within query range: 0; Match / mismatch score: 1, -2; Gap penalty: linear; Filter: Low complexity region; Mask: Mask for lookup table only. Sequences with an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently, to have XX% sequence identity with the reference sequence.

[0094] Polypeptide or polynucleotide sequence identity can be measured over the length of an entire defined polypeptide sequence (e.g., as defined by a particular SEQ ID number), or can be measured over a shorter length, for example, over the length of a fragment (e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues) taken from a larger defined polypeptide sequence. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown in this article, table, figure, or sequence listing can be used to describe the length over which a percentage identity can be measured.

[0095] The polypeptides disclosed herein may include "variant" polypeptides, "mutants" and "derivatives thereof". As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of the polypeptide in nature, as distinguished from a variant or mutant form. As used herein, a "variant", "mutant" or "derivative" refers to a polypeptide molecule having an amino acid sequence that is different from a reference protein or polypeptide molecule. A variant or mutant may have an insertion, deletion or substitution of one or more amino acid residues relative to a reference molecule.

[0096] The amino acid sequence of the polypeptide variant, mutant, derivative or fragment considered herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. "Conservative amino acid substitutions" are those substitutions in which the amino acid is substituted with a different amino acid, wherein the substitution is predicted to interfere with the properties of the reference polypeptide to the minimum. In other words, conservative amino acid substitutions substantially retain the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of the substitution, such as a beta sheet or alpha helical conformation, (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the volume of the side chain.

[0097] The composition for synthesizing β-1,2-oligoglucans comprises G1P, primer molecules and β-glucan phosphorylase as described herein. The composition may further comprise a buffer and / or a reducing agent. Suitable buffers may include, but are not limited to, phosphate buffers (e.g., potassium phosphate buffers, sodium phosphate buffers or preferably citric acid Na2HPO4 buffer systems pH 5.5-7.6). Suitable reducing agents may include, but are not limited to, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), ascorbic acid, cysteine, sodium bisulfite, SO2 and combinations thereof. The composition may have a pH between 6.5 and 7.5.

[0098] As used herein, "G1P" and "glucose-1-phosphate" are used interchangeably and refer to a glucose molecule with a phosphate group on the 1' carbon. G1P can be present in the compositions and methods described herein at a concentration between 0.05M and 2.0M, between 0.1M and 1.75M, or between 0.2M and 1.5M.

[0099] As used herein, "βGP", "β-GP", "beta-glucan-phosphorylase" and "β-glucan-phosphorylase" are used interchangeably and refer to an enzyme that reversibly catalyzes the phosphorylation of glycosidic linkages in β-glucans to form glucose-1-phosphate. The reverse reaction catalyzes the synthesis of β-glucans by phosphorylation transfer of glucose from glucose-1-phosphate to an acceptor primer molecule (e.g., glucose, sophorose, laminaribiose, cellobiose, etc.). βGP enzymes may be regioselective, for example, selectively catalyzing linkages at the β-1,2 positions of glucose in the primer molecule. As used herein, enzymes that catalyze the regioselective addition of glucose at the β-1,2 positions of the primer molecule from G1P are considered to have "β-1,2-glucan phosphorylase activity". βGP can be annotated in the Carbohydrate-Active Enzyme Database (CAZY) as belonging to glycoside hydrolase family 94 (GH94). βGP having β-1,2-glucan phosphorylase activity may also be referred to in the art as laminaribiose phosphorylase. βGP having β-1,2-glucan phosphorylase activity can have a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to at least one of SEQ ID NO: 10, 11, 13, 15 or 16.

[0100] The βGP polypeptide having β-1,2-glucan phosphorylase activity may be the Paenibacillus sp. laminaribiose phosphorylase (PsLBP) of SEQ ID NO: 10, or may be derived from the PsLBP of SEQ ID NO: 10. The βGP polypeptide having β-1,2-glucan phosphorylase activity may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 10.

[0101] The βGP polypeptide having β-1,2-glucan phosphorylase activity may be the Rhizobium tropici β-1,2-glucan phosphorylase (RtSOGP) of SEQ ID NO: 11, or may be derived from the RtSOGP of SEQ ID NO: 11. The βGP polypeptide having β-1,2-glucan phosphorylase activity may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 11.

[0102] The βGP polypeptide having β-1,2-glucan phosphorylase activity may be Clostridium saccharoperbutylaceonicum β-1,2-glucan phosphorylase (CsSOGP) of SEQ ID NO: 13, or may be derived from CsSOGP of SEQ ID NO: 13. The βGP polypeptide having β-1,2-glucan phosphorylase activity may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 13.

[0103] The βGP polypeptide having β-1,2-glucan phosphorylase activity may be the Paenibacillus stellifer β-glucan phosphorylase (PsGP) of SEQ ID NO: 15, or may be derived from the PsGP of SEQ ID NO: 15. The βGP polypeptide having β-1,2-glucan phosphorylase activity may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 15.

[0104] The βGP polypeptide having β-1,2-glucan phosphorylase activity may be Beutenbergia cavernae β-1,2-glucan phosphorylase (BcSOGP) of SEQ ID NO: 16, or may be derived from BcSOGP of SEQ ID NO: 16. The βGP polypeptide having β-1,2-glucan phosphorylase activity may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0105] As used herein, "primer molecules" refer to monosaccharides, disaccharides or polysaccharides containing D-glucose. Primer molecules may be, but are not limited to, D-glucose, sophorose, laminaribiose, cellobiose, gentiobiose and combinations thereof. Suitable primers are known and described in the art. See, for example, Ubiparip et al. ("β-glucan phosphorylases in carbohydrate synthesis," Applied Microbiology and Biotechnology, 2021, 105: 4073-4087). Without wishing to be bound by any particular theory or mode of action, it is believed that the primer molecules act as an initial scaffold, and additional sugar molecules are subsequently added to produce β-1,2-oligoglucans. If the βGP enzyme is provided to the composition as a crude cell extract or lysate, the primer molecules may be residual glucose in the extract / lysate, and no additional primer molecules are required. Primer molecules may also be referred to as receptor molecules in the art. In the compositions and methods described herein, the concentration of the primer molecules may be at least 1 mM, 2 mM, 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM or at least 300 mM. In the compositions and methods described herein, the primer molecules may be present at a concentration of up to 5 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM or up to 200 mM. Without being bound by any particular theory or mode of action, the concentration of the primer molecules and G1P may be modulated to produce β-1,2-oligoglucans of a specific degree of polymerization. For example, as shown in Example 11 below, a combination of low concentration primer molecules can be used to produce β-1,2-oligoglucans with a high degree of polymerization.

[0106] The composition for synthesizing β-1,2-oligoglucans may optionally include a phosphatase inhibitor. Suitable phosphatase inhibitors are known and described in the art. For example, the phosphatase inhibitor may be sodium molybdate (Na2MoO4), (NH4)6Mo7O 24 , ATP, Cu 2+ (e.g., CuSO4), GDP, GTP, HgCl2, iodoacetic acid, Na3AsO4, Na3VO4, NaF, sodium citrate, tartaric acid, or a combination thereof. Phosphatase inhibitors such as sodium molybdate can be added to the composition for synthesizing β-1,2-oligoglucans at a concentration between 1 mM-500 mM, between 50 mM-400 mM, or between 100 mM-300 mM.

[0107] The composition for synthesizing β-1,2-oligoglucans can be used in a method for preparing β-1,2-oligoglucans, as described herein. The method comprises incubating a composition comprising glucose-1-phosphate (G1P), a primer molecule, and βGP having β-1,2-glucan phosphorylase activity for a time and under conditions sufficient to produce β-1,2-oligoglucans. Based on the disclosure herein, a skilled person will understand the time and conditions suitable for producing β-1,2-oligoglucans. The composition may be incubated at a temperature between 30°C and 70°C, between 35°C and 65°C, between 37°C and 60°C, between 40°C and 57°C, or between 40°C and 50°C. The composition may be incubated at a temperature of about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C, about 57°C, about 60°C, about 65°C. The composition may have a pH between 5.5 and 7.5. The pH of the composition can be about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5. The composition can be incubated for at least 30 minutes, at least 1 hour, at least 5 hours, at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, or at least 44 hours. The composition can be incubated with shaking or stirring.

[0108] The present disclosure also provides a composition for synthesizing G1P from maltodextrin. The composition for synthesizing G1P comprises a glucan substrate as described herein, an inorganic phosphate and an α-glucan phosphorylase. The composition may additionally comprise a buffer and / or a reducing agent. Suitable buffers may include, but are not limited to, phosphate buffers (e.g., K2HPO4 / KH2PO4 or Na2HPO4 / NaH2PO4). Suitable reducing agents may include, but are not limited to, dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), ascorbic acid, cysteine, sodium bisulfite, SO2 and combinations thereof. The composition may have a pH between 6.5 and 8.

[0109] The glucan substrate may be maltodextrin, starch (e.g., starch liquefact), glycogen, amylose, amylopectin, trehalose, sucrose, laminaribiose, cellulose, cellodextrin, cellobiose, or a combination thereof. Preferably, the glucan substrate is maltodextrin. The glucan substrate may be present in a composition for synthesizing G1P at a concentration of 100mM-2000mM, 150mM-1800mM, or 200mM-1000mM. The glucan substrate concentration may also be based on the dry matter percentage of the composition. For example, maltodextrin may be added so that the dry matter percentage of the composition is between 1%-30%, between 2% and 25%, or between 5% and 20%. The glucan substrate may be added so that the dry matter percentage of the composition is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or about 20%. Generally, the glucan substrate used in the composition for synthesizing G1P will have a degree of polymerization (DP) of at least 5. Without wishing to be bound by any particular theory or mode of action, glucan substrates having a DP of 4 or less cannot serve as substrates for the αGPase that synthesizes G1P.

[0110] The glucan substrate (e.g., maltodextrin) can be debranched before being added to the composition for synthesizing G1P. Suitable methods for debranching maltodextrin and other glucan substrates are known and described in the art. See, for example, Ling Hii et al. ("Pullulanase: Role in starch hydrolysis and potential industrial applications," Enzyme Research, 2012, 921362) and Moller et al. ("Structure and function of α-glucan debranching enzymes," Cell. Mol. Life Sci., 2016, 73: 2619-2641). For example, maltodextrin can be debranched with a pullulanase (such as OPTIMAX® under the trade name). TM L-1000) and / or isoamylase to debranch the maltodextrin.

[0111] Inorganic phosphate can be provided in the composition in any suitable form. Suitable forms of inorganic phosphate include, but are not limited to, sodium phosphate (Na2HPO4), potassium phosphate (KH2PO4), other salts and combinations thereof. Inorganic phosphate can be present in the composition at a concentration of 100mM-2000mM, 150mM-1800mM or 200mM-1000mM. Inorganic phosphate can be present in the composition at an initial concentration of about 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM, 1100mM, 1200mM, 1300mM, 1400mM, 1500mM, 1600mM, 1700mM, 1800mM, 1900mM or about 2000mM.

[0112] The glucan substrate (e.g., maltodextrin) and the inorganic phosphate may be present in the composition for synthesizing G1P in any suitable ratio. For example, maltodextrin and the inorganic phosphate may be present in the composition in a starting molar ratio of 1:0.25, 1:0.5, or 1:1. Maltodextrin and the inorganic phosphate may be present in the composition in a starting molar ratio of between 1:0.1 and 1:2, between 1:0.2 and 1:1.5, between 1:0.25 and 1:1.25, or between 1:0.5 and 1:1.

[0113] As used herein, "αGP", "α-GP", "alpha-glucan-phosphorylase" and "α-glucan-phosphorylase" are used interchangeably and refer to an enzyme that reversibly catalyzes the phosphorylative cleavage of α-1,4 glycosidic linkages to form glucose-1-phosphate. See Scheme 1. α-1,4 glycosidic linkages can be cleaved in substrates such as glycogen, starch, and maltodextrin. αGP can be annotated in the Carbohydrate-Active Enzyme Database (CAZY) as belonging to glycosyltransferase family 35 (GT35). αGP can have a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to at least one of SEQ ID NO: 1, 2, 3, 4, 5 or 18. The αGP may have a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 1, 2, 5, or 18. The αGP may have a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 1, 2, or 5.

[0114]

[0115] The αGP polypeptide may be the Anaerolinea thermophila αGP (AtGP) of SEQ ID NO: 1, or may be derived from the AtGP of SEQ ID NO: 1. The αGP polypeptide may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0116] The αGP polypeptide may be the Thermobaculum terrenum αGP (TtGP) of SEQ ID NO: 2, or may be derived from the TtGP of SEQ ID NO: 2. The αGP polypeptide may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0117] The αGP polypeptide may be the Thermincola potens αGP (TpGP) of SEQ ID NO: 3, or may be derived from the TpGP of SEQ ID NO: 3. The αGP polypeptide may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0118] The αGP polypeptide may be the Thermodesulfobacterium geofontis αGP (TgGP) of SEQ ID NO: 4, or may be derived from the TgGP of SEQ ID NO: 4. The αGP polypeptide may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0119] The αGP polypeptide may be the Thermosipho africanus αGP (TaGp) of SEQ ID NO: 5, or may be derived from the TaGp of SEQ ID NO: 5. The αGP polypeptide may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0120] The αGP polypeptide may be the Thermosiphomelanesiensis αGP (TmGP) of SEQ ID NO: 18, or may be derived from the TmGP of SEQ ID NO: 18. The αGP polypeptide may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0121] The composition for synthesizing G1P can be used in a method for preparing G1P, as described herein. The method comprises incubating a composition comprising a glucan substrate, an inorganic phosphate, and an αGP polypeptide for a time and under conditions suitable for producing G1P. Based on the disclosure herein, a skilled person will understand the time and conditions suitable for producing G1P from a glucan substrate. The composition may be incubated at a temperature between 30°C and 70°C, between 35°C and 65°C, between 37°C and 60°C, between 40°C and 57°C, or between 40°C and 50°C. The composition may be incubated at a temperature of about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C, about 57°C, about 60°C, about 65°C. The composition may have a pH between 5.5 and 8.0, between 6.0 and 7.5, or between 7.0 and 7.5. The pH of the composition may be about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. The composition may be incubated for at least 30 minutes, at least 1 hour, at least 5 hours, at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 20 hours, at least 24 hours, at least 30 hours, at least 35 hours, at least 40 hours, or at least 44 hours. The composition may be incubated under shaking or stirring. The method may further include the step of debranching the glucan substrate (e.g., maltodextrin) prior to G1P synthesis or simultaneously with G1P synthesis in the same reaction vessel.

[0122] Generally, the β-1,2-oligoglucans produced by the compositions and methods described herein are characterized by a polydispersity (Mw / Mn) between 2 and 40 (e.g., 4-30, 6-20, 8-15, or any value or subrange therein), a degree of polymerization (DP) of at least 150 (e.g., at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, between 150-400, or between 200 and 350), a molecular weight between 20 kDa and 200 kDa (e.g., between 25 kDa and 150 kDa, between 40 kDa and 125 kDa, or between 50 kDa and 100 kDa), and a viscosity greater than 200 cP (e.g., between 200 cP and 350 cP, or between 250 cP and 350 cP), when measured at 30°C, 50 rpm stirring, and 30% dry matter concentration. Alternatively, the β-1,2-oligoglucans produced by the compositions and methods described herein are characterized by a polydispersity (Mw / Mn) between 2 and 40 (e.g., 4-30, 6-20, 8-15, or any value or subrange therein), a degree of polymerization (DP) between 3 and 150 (e.g., 3-150, 6-100, or 10-50), a molecular weight between 20 kDa and 200 kDa (e.g., 25 kDa and 150 kDa, 40 kDa and 125 kDa, or 50 kDa and 100 kDa), and a viscosity greater than 200 cP (e.g., 200 cP and 350 cP, or 250 cP and 350 cP), when measured at 30°C, 50 rpm stirring, and 30% dry matter concentration.

[0123] The β-1,2-oligoglucans produced by the compositions and methods described herein are non-digestible. As used herein, "non-digestible" refers to compositions having less than 1%, less than 5%, less than 7.5%, or less than 10% glucose release when evaluated using an in vitro digestion assay as described in Garcia-Campayo et al., "Digestion of food ingredients and food using an in vitro model integrating intestinal mucosal enzymes," Food and Nutrition Sciences, 2018, 9: 711-734.

[0124] The produced β-1,2-oligoglucans can be used in β-1,2-oligoglucan compositions. β-1,2-oligoglucan compositions can be used to prepare food products, beverage products and / or animal feed products. For example, the β-1,2-oligoglucan composition can be used to replace part or all of a bulking agent, fiber or another low-calorie ingredient used in the preparation of a food, beverage or animal feed product. The β-1,2-oligoglucan composition can be used as a prebiotic or immunostimulant, or can be used to prepare a prebiotic or immunostimulant composition. Without wishing to be bound by any particular theory or mode of action, it is believed that due to the non-digestible nature of the β-1,2-oligoglucans produced by the compositions and methods described herein, the β-1,2-oligoglucans can be used to replace other calorie components of food, beverages and / or animal feed products, but retain the beneficial bulking agent or fiber properties of the food, beverage and / or animal feed products.

[0125] As used herein, the terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence" and "nucleic acid" are used interchangeably and refer to a nucleotide sequence or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense strand or the antisense strand. The DNA polynucleotide may be a cDNA or a genomic DNA sequence.

[0126] If in its natural state or when operated by methods known to those skilled in the art, a polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof, then the polynucleotide is said to encode a polypeptide. The antisense strand of such a polynucleotide is also considered to encode the sequence.

[0127] Those skilled in the art understand that the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some aspects, the polynucleotide (i.e., the polynucleotide encoding the αGP polypeptide or the βGP polypeptide) can be codon-optimized to be expressed in a specific cell, and the specific cell includes but is not limited to a plant cell, a bacterial cell, a fungal cell, or an animal cell. Although polypeptides encoded by polynucleotide sequences found in various species are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptide described herein can be used. Therefore, non-naturally occurring sequences can be used. These sequences may be desirable, for example, to enhance expression in a heterologous expression system of a polypeptide or protein. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, genetic codes, and human hands can also be used to generate degenerate coding sequences.

[0128] Also provided herein are polynucleotides encoding αGP polypeptides. The polynucleotides may encode any of the αGP polypeptides described herein, for example, the polynucleotides may encode a polypeptide that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to at least one of SEQ ID NOs: 1, 2, 3, 4, 5, and 18. The polynucleotides encoding αGP polypeptides may be cDNA sequences encoding a polypeptide that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to at least one of SEQ ID NOs: 1, 2, 3, 4, 5, and 18.

[0129] Also provided herein are polynucleotides encoding βGP polypeptides. The polynucleotides may encode any of the βGP polypeptides described herein, for example, the polynucleotides may encode a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to at least one of SEQ ID NOs: 10, 11, 13, 15, and 16. The polynucleotide encoding the βGP polypeptide may be a cDNA sequence encoding a polypeptide that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to at least one of SEQ ID NOs: 1, 2, 3, 4, 5, and 18.

[0130] The polypeptide described herein can be provided as a part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which can be single-stranded or double-stranded and can represent a sense strand or an antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods, which include polynucleotide sequences derived from at least two different natural sources, or they can be synthetic. Therefore, the construct can include new modifications to endogenous genes introduced by, for example, genome editing techniques. The construct can also include recombinant polynucleotides produced using, for example, a recombinant DNA method. The construct can be a vector including a promoter operably connected to a polynucleotide encoding a heat-unstable EforRed polypeptide. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide connected thereto. A vector can be a plasmid, which refers to a circular double-stranded DNA loop, into which an additional DNA fragment can be integrated.

[0131] Also provided are cells comprising any polynucleotide, construct or vector described herein. The cell can be a prokaryotic cell or a eukaryotic cell. Suitable prokaryotic cells include bacterial cells, for example, Escherichia coli and Bacillus subtilis cells. Suitable eukaryotic cells include, but are not limited to, fungal cells, plant cells and animal cells. Suitable fungal cells include, but are not limited to, Fusarium venenatum, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Trichodermareesei, Issatchenkia orientalis and Aspergillus niger cells. For example, cells comprising a polynucleotide encoding at least one of SEQ ID NO: 1, 2, 3, 4, 5, 10, 11, 13, 15, 16, or 18 can be used to produce αGP and / or βGP polypeptides for use in the compositions and methods described herein. Suitable methods for cell-based protein expression are known and described in the art, and one of skill in the art will understand how to appropriately express and purify any of the polypeptides described herein from a cell-based or cell-free system.

[0132] Example

[0133] The present invention is further described in detail by reference to the following experimental examples. Unless otherwise stated, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all changes that become apparent due to the teachings provided herein.

[0134] Example 1 - Selection of alpha(α)-glucan-phosphorylase (αGP) and beta(β)-glucan-phosphorylase (βGP) select

[0135] About 1600 sequences were extracted from the carbohydrate-active enzyme database (CAZY) for the selection of possible αGPs (CAZY annotated glycosyltransferase family 35 members (GT35)) and about 1000 sequences were used to select possible βGPs (CAZY annotated glycoside hydrolase family 94 members (GH94)). Sequence extraction and creation of lists with unique sequences were performed computationally. The lists were used for sequence alignment and for the construction of protein phylogenetic trees in the ClustalOmega EMBL-EBI online tool (multiple sequence alignment). The tree was visualized in an online tool for the display, annotation and management of phylogenetic trees - iTOL (Interactive Tree of Life).

[0136] The selection of candidate sequences partially focuses on genes and enzymes from thermophilic sources, in view of which these enzymes should show stability and activity at higher temperatures. Candidate βGP is selected based on the possible probability that the selected enzyme will perform the desired activity. It is also selected by using the enzyme sequence previously characterized. 7 kinds of selected αGP and 12 kinds of selected βGP are summarized in Table 2. Use ProtParam (ExPASy, SIB Bioinformatics Resource Portal (SIB Bioinformatics Resource Portal)) online tool to calculate protein size.

[0137] Table 2 .

[0138]

[0139]

[0140] Example 2-Expression, stability and activity of αGP

[0141] The synthetic genes encoding the αGPases described in Table 2 were codon optimized for expression in E. coli and subcloned into a pET30a(+) plasmid vector with a 6-HIS-tag and a linker (SEQ ID NO: 20) at the N-terminus. E. coli BL21(DE3) cells were transformed with the pET30a(+) vector and grown under conditions of chemical induction of enzyme expression.

[0142] Figure 1 The SDS-PAGE in 5 showed that 3 of the 5 enzyme candidates (A1, A3 and A8) were very well expressed in the soluble fraction, with A3 being the most abundant. Similar results were observed for expression at both 20°C and 30°C. Although A5 and A6 did not show clearly visible bands on the gel, the low-expressing variants may still be active, and these enzymes were tested further.

[0143] Initial stability testing was performed by incubating the crude protein extract at 60°C for 1 hour. After incubation, the extract was centrifuged to precipitate the denatured protein. The soluble fraction and the insoluble fraction (the insoluble fraction contained the denatured protein) were examined by SDS-PAGE ( Figure 2 ). The results demonstrated that the A1, A3 and A8 enzymes were still present in the soluble fraction at the correct molecular weight and were therefore stable at 60°C.

[0144] The activity of αGP enzymes was analyzed using glucose-1-phosphatase assay. Two types of maltodextrins with different molecular weights (Mw) and polydispersities (Mw / Mn) were used as substrates (Table 3). MD1 is a maltodextrin substrate with a polydispersity of 23.2, and MD2 is a maltodextrin substrate with a polydispersity of 16.3. Initial results demonstrated that all selected enzymes showed activity at 37°C and 57°C for both maltodextrin substrates. In the assays performed at 57°C, the activity of all proteins was higher.

[0145] (Table 4)

[0146] Table 3 .

[0147] Substrate Maltodextrin 1(MDX1) Maltodextrin 2(MDX2) Weight average molecular weight (Mw) 32768 18404 Number average molecular weight (Mn) 1413 1129 Polydispersity 23.2 16.3

[0148] Table 4 .

[0149]

[0150] Example 3 - Characterization of αGPase

[0151] The thermostable enzyme candidates A1, A3 and A8 were further characterized to determine specific activity, residual activity after prolonged incubation at elevated temperature, the effect of pH on activity, and the effect of temperature on activity. Figure 3 Results of His-tag purification of crude soluble fraction enzyme extracts and A1, A3 and A8 enzyme candidates are shown. Approximate enzyme concentrations from His-tag purifications are shown in Table 5.

[0152] Table 5 .

[0153]

[0154]

[0155] The purified enzymes were further examined to determine specific activity (U / mg), temperature range, pH range, and residual activity after extended incubation at 55°C. The results showed that all three enzymes had the highest activity between 55°C and 65°C ( Figure 4). The temperature and pH of maximum activity and the activity of each enzyme at 55°C and 65°C are reported in Table 6. Unless otherwise indicated, the assays reported in Table 6 were performed with 2% maltodextrin substrate in 50 mM phosphate buffer at pH 7.

[0156] Table 6 .

[0157] Enzyme characteristics A1 A3 A8 The temperature of maximum activity ( Figure 4 ) 55℃ 65℃ 65℃ pH of highest activity 7 7.5 7 Activity at 55℃ (U / mg) 19.6±0.5 5.6±0.7 99.7±9.1 Activity at 65℃ (U / mg) 5.8±0.3 12.2±0.3 132.5±8.4

[0158] The residual specific activity of all αGP candidates after prolonged incubation at 55°C was determined. Figure 5 The specific activity of the enzymes after 0, 24 or 44 hours of incubation at 55°C is shown. The time is the enzyme incubation time rather than the assay run time. After 44 hours of incubation, A3, A1 and A8 maintained about 80%, 40% and 20% of the specific activity, respectively, however, the A8 candidate enzyme still had a higher specific activity at all time points. The specific activity of the A8 enzyme decreased by about 70% after 24 hours of incubation at 55°C, and decreased by about 80% after 44 hours of incubation at 55°C.

[0159] The substrate specificity of the A1, A3, and A8 enzyme candidates was also tested using two different maltodextrin substrates. Figure 6 ) The properties of the two different substrates are summarized in Table 3. The A1 and A3 candidate enzymes had similar activities on the two maltodextrin substrates, while A8 had an initial activity on the MDX2 substrate that was approximately 60% lower. ( Figure 6 ).

[0160] Example 4-αGPase Kinetic Parameters

[0161] In addition, the kinetic parameters of αGPase candidates A1, A3 and A8 were characterized. See Tables 7 and Figure 7-Figure 9 Enzyme A8 was not inhibited by the substrate (maltodextrin), while the inhibitor constant was the highest for A1. Similarly, A8 had the best kinetic properties compared to A1 and A3, with the lowest Michaelis constant (K m ), the highest metabolic conversion number (K cat ) and the highest K cat / K m Compare.

[0162] Table 7 .

[0163] Enzymes A1 A3 A8 <![CDATA[K m (mM)]]> 0.2 0.09 0.07 <![CDATA[V max (U mg -1 )]]> 25 17 105 <![CDATA[K cat (s -1 )]]> 38 24 178 <![CDATA[K cat / K m (s -1 mM -1 )]]> 171 259 2519 <![CDATA[K i (mM)]]> 35 5 -

[0164] Enzyme A8 was further tested at maltodextrin substrate concentrations up to 60%. Fig.10These results demonstrate that the A8 enzyme is inhibited by substrate at higher concentrations, probably starting at about 25%-30% maltodextrin. Assays with a maximum maltodextrin concentration of 24% did not show significant substrate inhibition. However, when the maximum maltodextrin concentration was increased to 60%, substrate inhibition was evident starting at a concentration of about 30%. Fig.10 The 60% graph in , which takes into account all substrate concentrations, appears to drop more rapidly than the 25% graph. However, it is more likely that the specific activity remains fairly constant up to a substrate concentration of about 30%, above which it drops off sharply. Fig.10 The kinetic properties of the assay shown in are reported in Table 8.

[0165] Table 8 .

[0166] Maximum concentration of maltodextrin 24％ 60％ <![CDATA[K m ]]> 0.17% Maltodextrin 0.33% Maltodextrin <![CDATA[V max (U / mg)]]> 125 158 <![CDATA[K i ]]> - 40% Maltodextrin

[0167] Example 5-Expression, stability and activity of βGP

[0168] The synthetic genes encoding the βGP enzymes described in Table 2 were codon optimized for expression in E. coli and subcloned into a pET30a(+) plasmid vector with a 6-HIS-tag at the N-terminus. E. coli BL21(DE3) cells were transformed with the pET30a(+) vector and grown under conditions of chemical induction of enzyme expression. Fig.11 SDS-PAGE in showed that, except B2, all enzyme candidates were expressed in the soluble fraction.

[0169] Initial stability testing was performed by incubating the crude protein extract at 60°C for 1 hour. After incubation, the extract was centrifuged to precipitate the denatured protein. The soluble fraction and the insoluble fraction (the insoluble fraction contained the denatured protein) were examined by SDS-PAGE ( Fig.12 ). The results demonstrated that the B4 enzyme was still present in the soluble fraction at the correct molecular weight and was therefore stable at 60°C.

[0170] The activity of the βGP enzyme was measured using a combination of the Gawronski phosphate release assay (which measures phosphate concentration, see Gawronski et al., "Microtiter assay for glutamine synthetase biosynthetic activity using organic phosphate detection," Analytical Biochem. April 1, 2004, 327(1): 114-8) and the GOD-POD assay (which measures glucose concentration). The absence of βGP activity will result in the release of equal concentrations of phosphate and glucose over time, while a higher concentration of phosphate relative to glucose will indicate that the candidate enzyme has the desired βGP activity. Endpoint measurements for both assays were performed using 1 mM laminaribiose as a substrate at 37°C for 2 minutes, 6 minutes, 15 minutes, and 30 minutes of incubation. Results ( Fig.13 ) showed significantly higher concentrations of phosphate release compared to glucose in 4 of the 7 candidate enzymes (B1, B4, B5 and B6), indicating that these enzymes may have the desired activity. The B6 candidate showed the highest difference in glucose and phosphate release over time.

[0171] The activity of B4βGP enzyme was tested on three different substrates (laminarinose, sophorose and cellobiose) using a combination of the Gawronski phosphate release assay (which measures phosphate concentration) and the GOD-POD assay (which measures glucose concentration), as described above. The assay was performed at 55°C using heat-treated crude cell extracts (60°C for 1 hour). The specific activity (U / mg) of each of the three substrates is reported in Table 9. The different substrates can also be referred to as primers and are acceptor molecules that transfer glucose from G1P. In other words, they are primers for the resulting oligosaccharides or polysaccharides prepared using glucose molecules from G1P.

[0172] Table 9 .

[0173]

[0174] In addition to the phosphate and glucose release assays, the activity of the B6 enzyme was evaluated by a product detection method using thin layer chromatography (TLC). The experiments were performed by incubating the enzyme / primer-substrate / G1P mixture at 35°C and 45°C for 20 min or 40 min, after which a small amount was loaded onto the TLC and a chromatogram was generated to detect the formed products. Cellobiose, sophorose, gentiobiose, laminaribiose, trehalose, glucose, maltose and isomaltulose were used as substrates together with 1 mM glucose-1-phosphate (G1P) in 50 mM MOPS buffer (pH 7) at a concentration of 1 mM. As Fig.14As shown, the B6 enzyme is active on sophorose. Fig.14 The TLC samples of B6 are summarized in Table 10. When sophorose was used as substrate, the black spots indicated by the arrows show the possible formation of >DP10 glucans. The smear above the spots is probably smaller DP glucans that are "too heavy" to be pulled up onto the slide. This activity of B6 on sophorose was confirmed by anion exchange chromatography, as shown in Table 10. Fig.15 shown.

[0175] Table 10 .

[0176] TLC column sample 1 Glucose-1-phosphate (G1P) 2 Sophorose substrate alone 3 Results of the B6 assay using sophorose substrate 4 Cellobiose substrate alone 5 B6 Assay Results Using Cellobiose Substrate 6 Trehalose substrate alone 7 Results of B6 assay using trehalose substrate 8 Glucose substrate alone 9 B6 Assay Results Using Glucose Substrate 10 Gentianobiose alone 11 B6 assay results using gentiobiose substrate 12 Maltose substrate alone 13 Results of B6 assay using maltose substrate 14 Laminariabiose substrate alone 15 B6 Assay Results Using Laminaribiose Substrate 16 Isomaltulose substrate alone 17 B6 assay results using isomaltulose substrate 18 MDX-Maltodextrin Blend 19 Blank-B6 enzyme and G1P

[0177] Example 6 - Characterization of βGPase

[0178] The B6 candidate βGPase was further characterized to identify the optimal activity temperature, pH, and kinetic parameters. Fig.16 As shown, B6 has peak activity at approximately 50°C and between pH 6.5 and 7. Fig.17 The thermostability of the B6 enzyme was demonstrated. After incubation at 50°C for 15 minutes, the B6 enzyme showed a loss of activity and after 3 hours at 50°C more than 80% of its initial activity was lost.

[0179] Using the Gawronski phosphate release assay (which measures phosphate concentration), the B6 enzyme showed high activity and substrate inhibition with an inhibitor constant of 14.3 mM sophorose. The Michaelis-Menten kinetics are reported in Tables 11 and Fig.18 middle.

[0180] Table 11 .

[0181] Enzymes B6 <![CDATA[K m (mM)]]> 0.07 <![CDATA[V max (U mg -1 )]]> 24 <![CDATA[K cat (s -1 )]]> 50 <![CDATA[K cat / K m (s -1 mM -1 )]]> 717 <![CDATA[K i (mM)]]> 14

[0182] Example 7 - Characterization of βGPase

[0183] βGPase candidates B7, B11, B12, B13 and B14 were purified using HIS-tag purification ( Fig.19 ). The B7, B12, B13 and B14 enzymes were present in reasonable amounts in the elution sample, however purification (and / or expression) of B11 was unsuccessful.

[0184] B7, B11, B12, B13 and B14 were screened for activity on β-disaccharides (cellobiose, gentiobiose, laminaribiose and sophorose), α-disaccharides (isomaltose, maltose, maltulose, sucrose, trehalose) and glucose, focusing on product detection by TLC. The reaction was carried out at 30°C for 20 minutes, after which 1 μl was loaded and developed by TLC. The final concentration of substrate in the reaction mixture was 5 mM, and the G1P concentration was 50 mM. TLC demonstrated that B12 was active on glucose by forming disaccharides. B7 and B13 were active on sophorose, B7 mainly by forming oligosaccharides and B13 mainly by forming polysaccharides. B13 was also active on laminaribiose by producing mainly polysaccharides, while no enzyme (according to TLC analysis) was active on cellobiose and gentiobiose ( Fig. 20 ). Subsequently, the samples were analyzed by anion exchange chromatography. The results confirmed the hypothesized activity ( Figure 21-26 ), and in addition showed that B13 was also active on glucose, forming oligosaccharides and polysaccharides. No enzyme was active on α-disaccharide substrates. Anion exchange chromatography also demonstrated that the disaccharide formed by B12 was laminaribiose.

[0185] Example 8-Characterization of B6 on mixed substrates

[0186] A mixed substrate syrup composition was prepared using a β-glucosidase from Aspergillus niger sold under the trade name "Novozyme 188". Anion exchange chromatography indicated that the substrate syrup consisted primarily of trehalose, with sophorose, laminaribiose, and gentiobiose present in lower concentrations, as reported in Table 12. The B6 enzyme was active on the substrate syrup, resulting in the production of polysaccharides such as Fig. 27 shown.

[0187] Table 12 .

[0188] Substrate syrup components approximate% Gentianobiose 2 Laminariabiose 3.7 Cellobiose 2 Trehalose 54 Sophora flavescens 3.1 Unidentified 38

[0189] Example 9 - Synthesis of G1P from maltodextrin and phosphate catalyzed by αGP A8

[0190] Various maltodextrins (Cargill, Incorporated and Sigma Aldrich) were used as substrates for the synthesis of G1P. Maltodextrins have different average chain lengths and degrees of branching. The A8αGPase cannot bypass branches in branched substrates, and therefore, it is believed that branched substrates hinder the synthesis of G1P. In order to increase the synthesis of G1P using A8αGP, maltodextrins were debranched at pH 4.8 and 50°C before the assay was performed (debranching method described below).

[0191] like et al. ("α-1,4-D-glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties, and molecular shape of the enzyme form solution X-ray scattering," Biochem J, 1997, 773-783) reported that α-glucan phosphorylase cannot use maltodextrin chains with a degree of polymerization (DP) of 4 or less (DP≤4) as substrates. Therefore, only a portion of the total glucose units present in maltodextrin can be used by αGPase.

[0192] Using maltodextrin C DRY MD from Cargill TM 01910 ("maltodextrin 01910") was used for the initial determination in this example. Based on calculations using Formula 1 as shown below, it is estimated that a maximum of 63% of the maltodextrin 01910 supplied to the A8 catalytic reaction can be converted to G1P. Formula 1 accounts for the fact that chains with DP=4 (or less) cannot serve as substrates for the A8 enzyme. This calculation is based on the molecular weight distribution obtained using low molecular weight gel permeation chromatography (Table 13).

[0193] Table 13 .

[0194]

[0195]

[0196] Formula 1:

[0197]

[0198] Formula 2:

[0199] Usable glucose (%) = slice area (5) × fraction of usable glc units

[0200] The fraction of usable glucose units in each DP range calculated using Formula 1 is then used in Formula 2 to determine the total percentage of usable glucose in the maltodextrin. The DP ranges and fractions of usable glucose units are reported in Table 14, and the usable glucose (glc) units are reported in Table 15.

[0201] Table 14 .

[0202] DP range Low DP High DP Fraction of glucose units that can be used 1-5 1 5 0.07 6-9 6 9 0.47 10-19 10 19 0.72 20-45 20 45 0.88 46-125 46 125 0.95 126-280 126 280 0.98 281-600 281 600 0.99 601-1500 601 1500 1.00 >1500 1500 3000 1.00

[0203] Table 15 .

[0204] DP range Slice area (%) Usable Glc (%) 1-5 21.5 1.4 6-9 23.0 10.7 10-19 10.5 7.6 20-45 8.0 7.0 46-125 12.1 11.5 126-280 10.7 10.5 281-600 8.0 7.9 601-1500 4.2 4.2 >1500 2.0 2.0 total 62.9

[0205] In the case where the maltodextrin substrate is branched, the above calculation may be an overestimate, as the calculation does not take into account the branch bonds. Six different debranched maltodextrins were investigated as substrates in the enzymatic conversion to produce G1P. After debranching, all maltodextrins were analyzed by HPLC fingerprinting (Ag+ column) and high molecular weight gel permeation chromatography (GPC). The results are summarized in Tables 16 and 17. The corresponding chromatograms are shown in Figure 28-Figure 33 middle.

[0206]

[0207]

[0208] GPC molecular weight distribution data showed that the number average molecular weight (Mn) of the 01910 and 01912 debranched maltodextrins was approximately 1000 Daltons, while the remaining maltodextrins had larger Mn, varying between 5000 Daltons and 200,000 Daltons.

[0209] Screening of dry substrate, reaction temperature, enzyme dosage and Na2HPO4 / MDX molar ratio on G1P production

[0210] The production of G1P was performed in a two-step process: first a debranching step in which the substrate maltodextrin was debranched using two types of debranching enzymes, followed by a G1P production step. All tested maltodextrins were incubated during 5 hours at pH 4.8-5 and 50°C using a combination of 0.2% pullulanase and 0.1% isoamylase (based on maltodextrin concentration).

[0211] The G1P synthesis reaction step aims to achieve almost complete consumption of phosphate in the reaction by adding excess carbohydrate (ie, maltodextrin). The reaction was performed at pH 7 with different ratios of maltodextrin 01910 / Na2HPO4 (1:0.25, 1:0.50 and 1:1).

[0212] The influence of dry matter type in reaction temperature, enzyme dosage and reaction mixture is evaluated. Sampling is taken after 24 hours reaction time. G1P productive rate is measured based on the amount of phosphate consumed (i.e., residual Na2HPO4 measured is deducted from the initial phosphate concentration supplied to the reaction mixture). In addition, the G1P assay described in the people such as Silverstein ("Purification and mechanismof action of sucrose phosphorylase," Journal of Biological Chemistry, 1967, 242 (6): 1338-1346, herein referred to as "Silverstein G1P assay") is used to measure the G1P productive rate, to confirm the data of Na2HPO4 amount based on consumption. Substrate conversion percentage is measured according to G1P productive rate based on initial maltodextrin substrate concentration.

[0213] Five experiments (Trials 1-5) were performed with 10 wt% or 20 wt% dry matter (ds) 01910 maltodextrin (MDX) solutions.MDX 01910 (6.46 g; 92.81% ds) was dissolved in a total of 20 g demi-water and the pH was adjusted to pH 4.8 with 0.1 M HCl.

[0214] 0.2% OPTIMAX TM L-1000 pullulanase (total 0.012 g based on 6 g dry MDX) and 0.1% isoamylase (total 0.006 g based on 6 g dry MDX) were added to the MDX solution and incubated at 50°C for 5 hours. No deactivation step was performed between two consecutive steps. Next, the debranched MDX solution was subjected to G1P conversion by adding 250 mM / L, 500 mM / L or 1000 mM / L Na2HPO4. After homogenization (vortexing) of the suspension, the pH was measured and, if necessary, adjusted to pH 7 using 0.1 M NaOH solution. Finally, the reaction was started by adding 64 U to 128 U enzyme / g dry maltodextrin or 12.8 U / ml to 24 U enzyme / ml reaction mixture of α-glucan phosphorylase (A8). Incubations were performed at 50°C or 60°C for 24 hours in a 30 ml (32 g) scale thermomixer (950 rpm shaking speed). After 24 hours of incubation, the enzyme was inactivated by increasing the temperature of the reaction mixture to 90°C for 5 minutes. The data (reaction conditions; analysis) are summarized in Table 18.

[0215]

[0216] The results showed higher activity at 60°C (Test 9.1 and Test 9.2). This temperature corresponds to the peak activity temperature of the thermostable α-glucan phosphorylase (A8). When the initial concentration of Na2HPO4 was doubled (from 250 mM to 500 mM) in the reactions performed at 60°C and 20% ds, an increase in the G1P yield / substrate conversion (11.8% for reaction 9.4) was observed (Test 9.1 and Test 9.4).

[0217] Experiment 9.3 was conducted at 10% dry matter and 60°C with approximately equimolar amounts of Na2HPO4 and MDX, and demonstrated a significant increase in maltodextrin conversion (18.5%). To further elucidate the contribution of low dry substrate % to equimolar amounts of Na2HPO4 and MDX, Experiment 9.5 was conducted with 20% dry matter. To accommodate the increased phosphate concentration and increased dry matter %, the reaction used 24U / mL A8 enzyme. To achieve the 24U / mL concentration, the A8 enzyme was freeze-dried and added to the reaction in freeze-dried form. The results of Experiment 9.5 showed no further increase in maltodextrin conversion, indicating that the reaction may be limited by the equilibrium conversion of phosphate (approximately 20%).

[0218] In Trials 9.1 to 9.4, good correlation was observed between the analysis of maltodextrin conversion to G1P and the analysis of phosphate consumption. However, Trial 9.5 showed a large difference between the two analytical methods. The phosphate test kit appears to be sensitive to foreign substances in the enzymatic reaction solution. Due to this sensitivity, the remaining experiments and examples use the Silverstein G1P assay as a tool for determining the amount of G1P produced after the reaction.

[0219] In order to shift the equilibrium of the A8 reaction in favor of G1P synthesis, the addition of excess Na2HPO4 and higher reaction temperatures (70°C) were investigated. Samples were taken periodically during the reaction (17 hours - 24 hours - 48 hours) and the G1P concentration was determined. These determinations were made with 10 wt% or 15 wt% dry matter 01912 maltodextrin solutions. MDX 01912 (1.073 g total; 93.16% ds) was dissolved in 5 g demineralized water total and the pH was adjusted to pH 4.8 with 0.1 M HCl.

[0220] 0.2% OPTIMAX TM L-1000 pullulanase (total 0.002 g based on 1 g dry MDX) and 0.1% isoamylase (total 0.001 g based on 1 g dry MDX) were added to the MDX solution and incubated for 5 hours at 50° C. No deactivation step was performed before the G1P conversion reaction.

[0221] Next, the debranched 01912MDX solution was subjected to G1P conversion by adding 1000 mmol / L to 2000 mmol / L Pi. After homogenization (vortexing) of the suspension, the pH was measured and, if necessary, adjusted to pH 7 using 0.1 M NaOH solution. The reaction was started by adding A8 enzyme (64 U enzyme / g dry maltodextrin or 12.8 U / ml reaction mixture). The reaction was incubated at a temperature of 50°C or 70°C for 17 hours, 24 hours or 48 hours in a 5 ml (6.6 g) scale thermomixer (950 rpm shaking speed). The enzyme was inactivated by increasing the temperature of the reaction mixture to 90°C for 5 minutes. The Silverstein G1P assay was used to measure the G1P concentration. The data (reaction conditions; analysis) are summarized in Table 19.

[0222] The results indicate that increasing the phosphate concentration to 2000mmol / L did not enhance the rate of G1P production. This may be due to the high concentration of Na2HPO4 causing the reaction mixture to be viscous and insoluble. Similarly, too high an insoluble salt concentration can lead to enzyme deactivation, as in the case of Experiment 9.7. The most appropriate phosphate concentration for this reaction system is 1000mmol / L. i , while keeping the maltodextrin concentration constant (Test 9.6). Evaluation of the G1P production over time showed no further increase in G1P synthesis. Due to the equilibrium constant of the reaction, it seems that only ≤ 20% of the phosphate is converted to G1P. The results of Test 9.8 are similar to those of Test 9.6, where the dry matter % is reduced and the reaction temperature is increased to 70°C.

[0223]

[0224] Screening for substrate type, Pi source and pH for G1P production

[0225] As reported by Bae et al. ("Facile synthesis of G1P from starch by Thermus Caldophilus GK24 α-glucan phosphorylase," Process Biochemistry 40 (2005) 3707-3713), soluble starch may be a better substrate, giving higher G1P yields than maltodextrins 01910 and 01912. Likewise, a more soluble potassium phosphate substrate rather than sodium phosphate may be beneficial.

[0226] To investigate G1P synthesis by the A8 enzyme using a soluble starch substrate (5% w / v dry matter) and potassium phosphate, four additional experiments were performed at different soluble starch and KH2PO4 molar ratios (1:2.5, 1:3.5) and at two different pH values ​​(pH 7 and 8).

[0227] Soluble starch (1.108 g total; 90.25% ds) was dissolved in 10 g total demineralized water and the pH was adjusted to pH 4.8 with 0.1 M HCl. 0.2% OPTIMAX TM L-1000 pullulanase (total 0.002 g based on 1 g dry soluble starch) and 0.1% isoamylase (total 0.001 g based on 1 g dry soluble starch) were added to the soluble starch solution and incubated for 5 hours at 50° C. No deactivation step was performed before the G1P synthesis reaction.

[0228] Next, debranched soluble starch solution (1.725g debranched soluble starch solution containing 0.1572g dry weight starch) and 2500mmol / L to 3500mmol / L KH2PO4 were used in the G1P conversion reaction. After the suspension was homogenized (vortexed), pH was measured and adjusted to pH 7 using 0.1M NaOH solution. The reaction was started by adding the A8 enzyme (64U enzyme / g dry soluble starch) from the crude cell lysate. The reaction was incubated at a temperature of 60°C in a thermal mixer (950rpm oscillation speed) of 10ml (+ / -10.4g) scale within the reaction time of 6 hours, 17 hours and 48 hours. The enzyme was inactivated by increasing the temperature of the reaction mixture to 90°C for 5 minutes. The G1P concentration in the reaction product was measured using the Silverstein G1P assay. The results of the reaction conditions are summarized in Table 20.

[0229] The results indicate that the G1P yield is higher in the reactions performed at pH 7. Experiment 9.10 shows a high soluble starch conversion (24% after 17 hours) where the molar ratio of soluble starch:KH2PO4 is 1:3.5. This is a 5% increase in G1P yield compared to the previous reaction experiments using Na2HPO4 and maltodextrin 01912. The reason for the decrease in G1P yield after 24 hours, as observed in Experiments 9.9 and 9.10, is not entirely clear. The A8 enzyme cell lysis fragments may contain contaminants (e.g., phosphatases) that are responsible for the decomposition of G1P, or it may be attributed to the presence of precipitation in the non-homogeneous crude enzyme sample.

[0230]

[0231]

[0232] Based on the results of Experiment 9.10, an additional reaction (Experiment 9.13) was performed identical to Experiment 9.10 but with shorter reaction times (5 hours and 8 hours) and triple the amount of A8 αGPase (192 U / g dry soluble starch). Experiment 9.13 showed very high conversion, up to 44.8% after 8 hours. In Experiment 9.14, the A8 enzyme concentration was tripled. The reaction conditions of Experiment 9.14 were identical to Experiment 9.13, but the incubation time was varied based on the A8 concentration.

[0233] Increasing the reaction time demonstrated equilibrium in the formation of G1P over a period of 3 to 24 hours. An equilibrium of approximately ±30% conversion of soluble starch to G1P was reached over a period of 24 hours (see Tables 21 and Fig.34 ).

[0234] In addition to soluble starch, other substrates with longer average chain lengths were also tested. Commercially available Zulkowsky starch (i.e., potato starch treated with glycerol at 190°C, see K.Zulkowsky, "Verhalten der Starke gegen Glycerin," Ber.Deutsch.Chem.Ges., 13, 1395, 1880)) and two other maltodextrin types with different dextrose equivalents (13.0-17.0 and 4.0-7.0 dextrose equivalents from Sigma-Aldrich) were evaluated using reaction conditions similar to those of test 9.14. All substrates were debranched before the A8 catalytic reaction. Zulkowsky starch reaction (test 9.15) was performed using A8 αGP (192U enzyme / g dry Zulkowsky starch) cell fragment lysate. Experiments using 13-17 and 4-7 dextrose equivalent maltodextrins (Tests 9.16 and 9.17, respectively) were performed using cell debris suspensions with A8 inclusion bodies. The G1P concentration in the reaction products was evaluated using the Silverstein G1P assay, and the presence of glucose was measured using the GOPOD-FORMAT program from Megazyme. The formation of glucose in these assays may be due to the presence of contaminants in the enzyme preparation. The results for the reaction conditions are summarized in Tables 22, 23, 24, 25, and 26. Figure 35-Figure 37 G1P and glucose production during the course of the reactions are shown for Experiment 9.15, Experiment 9.16 and Experiment 9.17, respectively.

[0235] Under the reaction conditions of test 9.15, a G1P yield of 44% was achieved and maintained for an incubation period of 7 to 10 hours, also confirming the thermostability of the active A8 enzyme. The use of Zulkowsky starch increased the G1P yield by 1.5 times compared to tests 9.16 and 9.17. Given that the amount of glucose formed was kept to a minimum, it is likely that the crude A8 cell lysate used in the assay of test 9.15 was free of phosphatase contamination.

[0236] However, it is likely that the cell debris suspension with A8 inclusion bodies used in Experiments 9.16 and 9.17 was contaminated with phosphatases (which convert G1P to glucose) because glucose levels increased throughout the assay time points (see Tables 24, 25 and 26 and Fig.36 and Fig.37 ). This may result in a lower overall yield of G1P.

[0237]

[0238]

[0239] Table 25 .

[0240] Reaction time (minutes) Maltodextrin DE 4-7 to G1P (%) % Glucose Formation 0 0 0 90 29.8 3.78 360 26.7 9.06 720 22.7 15.48 3060 16 37.39 3510 15.5 39.48 4320 15.3 44.32

[0241] Table 26 .

[0242] Reaction time (minutes) Maltodextrin DE 13-17 to G1P (%) % Glucose Formation 0 0 0 90 32.9 1.94 330 31.2 6.23 738 28.5 10.84 3060 21.4 24.4 3600 20.9 26.36 4320 20.7 27.3

[0243] Example 10 - Synthesis of β-glucan from primer molecules catalyzed by G1P and β-glucan phosphorylase B6

[0244] This example demonstrates the production of 1,2-β-oligoglucans using the B6βGP enzyme. For this example, the B6 enzyme was supplied to the reaction as a crude cell extract to stabilize the enzyme at elevated reaction temperatures. This crude cell extract also contains some residual glucose, which can act as a primer molecule during β-glucan synthesis. Oligoglucan assembly requires a starting or "primer" molecule on which the oligoglucans will be constructed by transferring glucose molecules. The crude cell extract will contain residual glucose as this primer molecule, but if the B6 enzyme is purified from the cell extract (e.g., His-tag purification, etc.), a separate primer molecule (sophorose or glucose) is added to the reaction.

[0245] Effect of substrate concentration on β-glucan synthesis

[0246] In a first example, the effect of substrate (G1P) concentration in this second reaction of the method was evaluated. Three 30 ml reactions were performed starting with 0.5 M, 1 M or 1.5 M G1P (Sigma-Aldrich) to evaluate the effect of substrate concentration on β-glucan synthesis. These G1P concentrations corresponded to 14%, 26% and 40% dry matter, respectively.

[0247] α-D-Glucose-1-phosphate (G1P, 98% purity) was dissolved in about 30 ml of 100 mM pH 7 phosphate buffer, pH was adjusted with 1 M HCl. 0.1 mL of dithiothreitol (DTT) 1 M was added to protect the enzyme from oxidation. The reaction mixture was prepared in a 50 ml FALCON tube and mixed by Vortex to obtain a homogeneous solution.

[0248] The activity and protein content of crude B6 enzyme cell lysates prepared by ultrasonic treatment were measured. The corresponding activity of 30 U / ml enzyme solution was determined by Gawronski phosphate release assay. The protein content of 10 mg protein / ml was quantified using the Pierce BCA kit from Thermofisher.

[0249] 3 ml (90 U) of β-glucan phosphorylase B6 were added to 30 ml of the reaction mixture so that the enzyme concentration in the reaction solution was 3 U enzyme / ml substrate. No primer syrup or sophorose (primer molecules) were added due to the presence of residual glucose in the crude cell lysate. The tubes were placed in a preheated thermomixer (900 rpm shaking speed) at 40°C. Samples were taken after a period of 24 hours of incubation and heated at 90°C for 10 minutes to inactivate the B6 enzyme. Precipitation (0.6 wt.%, based on the starting weight of G1P) was noticed during the reaction and after enzyme deactivation. This haze, which was insoluble in H2O / DMSO (10% / 90%) solution, probably originated from the enzyme and was not related to the formation of β-glucan. (See Fig.38 ).

[0250] To evaluate the difference between sonication and homogenization applied during cell lysis, a second set of reactions (3) was performed under the same reaction conditions, but homogenized cleaved β-glucan phosphorylase was supplied to the reaction mixture. The enzyme activity was measured to be 22.32 U / ml and 8.2 mg protein / ml enzyme.

[0251] The resulting β-glucan concentration for each reaction was determined based on the concentration of residual G1P (phosphoglucomutase / glucose-6-dehydrogenase assay) and the concentration of glucose (glucose oxidase / peroxidase assay) in the reaction products. The molecular weight distribution of the resulting β-glucan was determined using high molecular weight GPC.

[0252] Because phosphate, G1P and β-glucan peaks overlap on high molecular weight GPC (Table 27), G1P and phosphate need to be removed from the reaction product before analysis. The reaction product was treated with 0.5% acid phosphatase from malt extract (Aldrich) at 37°C and pH 4.8 for 24 hours. After acid phosphatase treatment, phosphate and glucose were removed by dialysis. G1P and phosphate can also be removed by using a guard column before the GPC analytical column.

[0253] Table 27 .

[0254]

[0255]

[0256] The β-glucan yield, glucose concentration and oligosaccharide molecular weight (MW) distribution of the above reactions are summarized in Tables 28 and 29.

[0257]

[0258] A higher β-glucan yield (57.2%) was obtained in the reaction with a lower initial substrate concentration (0.5 M G1P, test 10.1). However, the glucose concentration in the reaction product doubled (9.28%) compared to test 10.2. The reaction of test 10.3 with an initial G1P concentration of 1.5 M showed a low β-glucan conversion (5.98%). This may be due to the high dry matter concentration in the reaction mixture, which was too viscous for efficient enzyme activity.

[0259] When comparing reactions performed with enzymes prepared under different cleavage methods, sonication resulted in a higher percentage of β-glucan conversion (52.6% in test 10.2 compared to 33.4% in test 10.5). However, reactions of crude extracts prepared by homogenization resulted in the production of larger β-glucans with a degree of polymerization (DP) as high as 630 (test 10.5). In summary, the data demonstrate that crude enzyme extracts prepared by sonication or homogenization produce β-glucan products.

[0260] Effects of Temperature and Phosphatase Inhibitors on β-Glucan Synthesis

[0261] To evaluate the effect of phosphatase inhibitors and the absence of glucose as a primer, the B6 enzyme was mixed with Pre-incubate GOP glucose oxidase with catalase mixture and / or sodium molybdate. 200 mM sodium molybdate and / or 10 mg (per ml B6 enzyme) The GOP glucose oxidase and catalase mixture was incubated with the B6 enzyme at room temperature (approximately 22° C.) at a pH between 6.5 and 7 for 5 hours.

[0262] After pre-incubation of B6 enzyme with phosphatase inhibitors, reactions were prepared containing 3 U / ml B6 enzyme, 1 MG1P, 100 mM phosphate buffer pH 7, and 0.1 mL DTT per 30 mL reaction mixture. Reactions also contained sodium molybdate and / or GOP glucose oxidase and catalase mixture, which was included in the B6 enzyme pre-incubation process (i.e., inhibitors were not removed before starting the reaction). The reaction was carried out at 40°C or 50°C for 24 hours. The β-glucan yield, glucose concentration and MW distribution of the resulting β-glucan are summarized in Tables 30 and 31.

[0263]

[0264] Results The use of a mixture of GOP glucose oxidase and catalase reduced glucose formation and increased β-glucan yield (up to about 60%). The β-glucan synthesized in the reaction with the GOP glucose oxidase and catalase mixture inhibitor had a higher molecular weight (Test 10.7) than the equivalent reaction in the absence of the glucose oxidase and catalase mixture (Test 10.1). Similar results were observed using the sodium molybdate inhibitor (see Fig.40 and Tables 30 and 31). In experiment 10.8, 3 U / ml of B6 enzyme was added at the beginning of the reaction, and the reaction was supplemented with an additional 3 U / ml of B6 enzyme after 17 hours of incubation. This experiment resulted in an increase in β-glucan yield of approximately 2%-4%. The reaction carried out at 50°C (experiment 10.9) resulted in a lower β-glucan yield and a higher amount of glucose, but the synthesized β-glucan had a higher molecular weight. (See Fig.39 and Table 31). Although GOP glucose oxidase and catalase mixture and sodium molybdate inhibitor were both added to Experiment 10.10, but this combination did not further improve the β-glucan yield or increase the molecular weight of the resulting β-glucan.

[0265] In order to confirm that the synthesized β-glucan is a linear β-1,2-oligoglucan, the isopropanol precipitated purified product from Experiment 10.8 was freeze-dried for analysis using NMR. The structural characteristics of β-1,2-glucan were confirmed by 1H-NMR and 13C-NMR. ( Fig.41 and Fig.42). 1D 1H and 13C spectra were recorded on an Avance II Bruker spectrometer operating at 400 MHz 1H frequency and equipped with a 5 mm 1H / BB BBO probe with Topspin 2.1 in an ICON environment. The sample temperature was set at 25°C and controlled within ±0.1°C with a Eurotherm 2000VT controller. The sample was prepared by NMR as follows: 18.3 mg of β-glucan was weighed and dissolved in 598.86 μl of D2O and 1.14 μl of tBuOH as internal standard for a final concentration of 20 mM in a total volume of 600 μl. After addition of the solvent, the sample was vortexed and centrifuged several times. The resulting solution was transferred to a high precision 5 mm NMR tube (Norell). The 1D 1H-NMR and 13C-NMR measurements were consistent with the spectra obtained by Kakajima et al. (“1,2-β-Oligoglucan phosphorylase from Listeria innocua,” PLoS One, 9(3), e92353, 2014).

[0266] B6βGP concentration

[0267] To evaluate the effect of reduced enzyme concentration and enzyme activity, experiments were performed with homogenized crude enzyme lysates at concentrations of 3 U / mL, 2 U / mL, 1 U / mL, or 0.5 U / mL of the reaction mixture. In addition, the B6 enzyme used in these experiments was from a separate growth and preparation of E. coli cells to test the batch-to-batch reproducibility of B6 enzyme activity. These reactions contained GOP glucose oxidase and catalase mixture. The results are summarized in Table 32 and Table 33.

[0268]

[0269] Comparison of Experiment 10.17 with Experiment 10.13 demonstrated the reproducibility of the B6 enzyme between preparations. Although the molecular weight of the synthesized β-glucan was higher at lower enzyme concentrations, the overall β-glucan yield was highest when the 3 U / ml concentration was used. In Experiment 10.17, the G1P substrate and B6 enzyme were combined separately with GOP glucose oxidase and catalase mixtures were pre-incubated together, however this additional step did not further improve β-glucan yield.

[0270] Large-scale β-glucan synthesis using B6βGP

[0271] Use a starting concentration of 100 g G1P, 3 U / ml pre-incubated B6 enzyme, 15 mg (per ml B6 enzyme) Five large-scale experiments were performed with a mixture of GOP glucose oxidase and catalase and 1 M DTT in 100 mM phosphate buffer pH 7. The B6 enzyme was mixed with GOP glucose oxidase and catalase mixtures were pre-incubated together at room temperature at a pH between 6.5-7 for 5 hours. Each reaction was performed at 40°C for 24 hours. After the reaction, the reaction mixture was heated at 90°C for 10 minutes to deactivate the B6 enzyme. The results are summarized in Tables 34 and 35.

[0272] The β-glucan products of all five tests were isolated from the reaction mixture by ethanol precipitation, dried and combined. Approximately 120 g of the dried sample was used for testing in Example 11.

[0273]

[0274] The reaction solution was cooled to 37°C and the pH was adjusted from 7.23 to 4.8 by adding 19.2% HCl. The conversion of unreacted G1P to glucose occurred by additional incubation with 0.5 g of acid phosphatase at 37°C for 24 hours. At regular time intervals, the phosphate content was measured until a constant value was obtained. When no additional phosphate was released, a constant phosphate measurement was used as an indicator of the completion of the reaction. After heating the reaction mixture at 70°C for 10 minutes, the solution was filtered on a buchner funnel with filter paper. After washing with 50 ml of demineralized water, there was a precipitate or flocculent material which represented only 0.1% of the total G1P (dried product).

[0275] A total of 400 ml of filtered reaction solution was recovered with a conductivity of 54.6 mS / cm. Dialysis membrane standard RC tubes MWCO 6-8kD were filled with 100 ml of reaction mixture each. Dialysis was performed for 24 hours against tap water (flowing), conductivity, phosphate and glucose (rod method) were measured. The final dialysis took place in milli Q water, followed by concentration in a rotary evaporator to about 50 ml or 50% ds, measured on an IR balance.

[0276] Example 11 - Characterization of β-1,2-oligoglucans synthesized from B6 βGP

[0277] The combined β-glucan samples obtained from Experiments 10.18 to Experiment 10.22 of Example 10 were further analyzed. Fig.43 ). Table 36 summarizes the overall molecular weight distribution of the combined β-glucan products.

[0278] Table 36 .

[0279]

[0280] In trials 10.18 to 10.22, an average of 56% of G1P was converted to β-glucan, while the conversion to glucose was only 1.7%. The purity of the β-glucan was 98.5% and the DP was 157. 76% of the MW was in the MW range of 2500 kDa to 200000 kDa.

[0281] The viscosity and shear thinning characteristics of the synthesized β-glucan were also analyzed. β-glucan syrup was prepared with 50% dry matter (prepared from samples prepared in Tests 10.18-10.22) with 20 g of powder and 20 g of low conductivity water. The solution was stirred and equilibrated at 60°C until the solution obtained optical clarity. Prior to analysis, the samples were stored at 20°C. Isothermal viscosity was measured at 20°C (reference temperature) and at a set of temperatures in the range of 30°C-80°C (in duplicate; Fig.44 and Fig.45 ). The cooling water bath was set to 15°C. Fig.46 As shown, the viscosity and shear thinning of the produced β-glucan are higher than sucrose or commercially available maltodextrin.

[0282] The digestibility of the β-glucan product was evaluated using an in vitro digestion assay and compared to the digestibility of sucrose, isomaltulose, and Promitor 70 soluble corn fiber. Samples were subjected to 72-hour in vitro digestion in triplicate and glucose release was analyzed using a glucose oxidase colorimetric assay. Fig.47 As shown, β-glucan was non-digestible with minimal glucose release within 72 hours of incubation. Glucose released from β-1,2-glucan (as % total glucose) after 72 hours was 5%, glucose released from isomaltulose was 97%, glucose released from sucroseman was 66%, and glucose released from Promitor 70 was 27%. These results provide encouraging evidence that the digestibility of β-1,2-glucan may be very limited. The 5% glucose release from the β-glucan sample may be due to residual glucose in the Fig.47 The indicated time points were autohydrolyzed from rat intestinal powder (RIP).

[0283] Example 12 - β-glucan synthesis from G1P and pure B6 using glucose as a primer

[0284] While the previous reactions were performed with crude cell lysates containing B6 enzyme, the experiments in this example used His-tagged purified B6 enzyme. Reactions used a starting concentration of 0.2M or 0.5M G1P and 5mM, 10mM, 20mM, 100mM, 200mM, or 300mM glucose as primers. No DTT or GOP glucose oxidase and catalase mixture. The reaction was carried out in 100 mM phosphate buffer pH 7. 0.32 ml (5.2 mg / ml and 140 U / ml) of purified B6 enzyme was added to the reaction mixture. This resulted in the addition of the same 3 U / ml reaction mixture of B6 enzyme as performed in the previous experiment. The reaction was carried out at 40°C for 24 hours, 48 ​​hours and 72 hours, followed by enzyme deactivation at 90°C for 10 minutes. The results are summarized in Fig.48 and Fig.49 And in Table 37.

[0285]

[0286] For reactions started with 0.5 M G1P, lower concentrations of glucose (e.g., 20 mM) were associated with significantly lower β-glucan synthesis and glucose consumption, whereas higher initial glucose concentrations (e.g., 100 mM-300 mM) were associated with high glucose consumption (up to about 91%, Fig.48 However, for the reactions started with 0.2 M G1P, glucose consumption at all initial glucose concentrations was at least about 50% at 48 hours ( Fig.49 ).

[0287] The data collected for the 0.5M G1P reaction indicate that the higher the initial glucose concentration, the faster the reaction reaches its equilibrium. For 300mM, 200mM, and 100mM initial glucose concentrations at 0.5M G1P, the total G1P concentration decreases until approximately 52% of the initial G1P is converted to β-glucan between 48 hours and 72 hours. Given that the glucose concentration did not increase significantly over time (i.e., above the initial glucose primer concentration), there are no side reactions in the presence of pure B6 that cleave G1P to glucose. Fig.49 As shown, an initial 0.2M G1P concentration converted to an equilibrium of approximately 35% β-glucan, while consuming only approximately 70% of the initial glucose supply. This suggests that once a glucose molecule is used as a primer by B6 and converted to DP 2, this disaccharide becomes the preferred acceptor for the enzyme to continue β-glucan synthesis.

[0288] GPC analysis showed that the weight average molecular weight (MW) of the synthesized β-glucan depended on the concentration of glucose added as a primer (see Tables 37 and Fig.50 The lowest concentration of glucose as primer (10 mM) produced the largest polymer (DP = 2700). These reactions using pure enzyme produced longer β-glucans than reactions performed using crude enzyme, and β-glucan size correlated with primer concentration.

[0289] Example 12 - Synthesis of β-glucan from maltodextrin

[0290] This example demonstrates the overall method for producing β-glucan from maltodextrin via a G1P intermediate.In this example, two enzymatic processes were performed in the same reaction vessel to test whether the equilibrium of the first reaction can be shifted to the right (ie, toward the production of G1P) when G1P is consumed in the second reaction.

[0291] This one-pot approach requires that both enzymes be active at the same pH and temperature. The reactions were performed at 40°C and pH 7. Given that the B6 βGPase has a higher activity than the A8 αGPase at 40°C, the concentration of the A8 enzyme was increased in the reaction to account for the difference in activity. Enzyme ratio, phosphate concentration, maltodextrin type and concentration, glucose concentration, and incubation time were variables in the synthesis of β-glucans by both enzymes.

[0292] Reaction of crude A8 and B6 with MDX 01912

[0293] Prior to the reaction, maltodextrin 01912 was debranched by incubation with 0.1% isoamylase and 0.2% pullulanase at pH 4.8 and 50°C for 5 hours. 128 U of A8 αGPase (10 ml, approximately 12 U / ml-13 U / ml) was added to the pH 7 reaction mixture containing debranched maltodextrin 01912 (6.5% dry matter) and Na2HPO4, and incubated at 60°C for 4 hours to produce G1P before adding B6 βGPase. Approximately 21.1% G1P (percentage of maltodextrin conversion) was produced during this incubation. After cooling to 40°C, the used 18U (0.43mL, 42U / ml) of B6 enzyme pretreated with a mixture of GOP glucose oxidase and catalase, and the reaction was maintained at 40°C. Samples were taken for analysis at 8 hours and 24 hours. After 24 hours, an additional 18U of B6 was added to the reaction, and a final sample was taken out at 48 hours for analysis. Prior to analysis, the sample was treated with a combination of glucoamylase and pullulanase to hydrolyze any remaining maltodextrin, and the sample was passed through a mixed bed resin to remove G1P and phosphate from the reaction. The sample was then characterized by high molecular weight GPC and HPLC fingerprints (Ag+ column). The reaction scheme is shown in Fig.51 and the results are summarized in Tables 38 and 39.

[0294]

[0295]

[0296] Reaction of A8 and B6 purified with HIS-tag with Zulkowsky starch or low-viscosity branched dextrin

[0297] Prior to the reaction, Zulkowsky and maltodextrin DE1 were individually debranched by incubation with 0.1% isoamylase and 0.2% pullulanase at pH 4.8 and 50°C for 5 hours. Four separate reactions were performed containing: (i) 5.2% MDXDE1 and 1.3% glucose; (ii) 5.2% Zulkowsky starch and 1.3% glucose; (iii) 5.85% MDX DE1 and 0.65% glucose; or (iv) 5.85% Zulkowsky starch and 0.65% glucose; each reaction contained 6.5% dry matter and an equimolar amount of KH2PO4. 128U of His-tagged purified A8αGPase (21ml, approximately 6U / ml) was added to each of the pH 7 reaction mixtures and incubated at 60°C for 4 hours before adding B6βGPase to produce G1P. After cooling to 40°C, 18U (0.12mL, approximately 160U / ml) of His-tagged purified B6 enzyme was added and the reaction was maintained at 40°C. Samples were taken for analysis at 7.5 hours, 19.5 hours, 24 hours and 48 hours. After 24 hours, an additional 18U of B6 was added to the reaction. Prior to analysis, the sample was treated with a combination of glucoamylase and pullulanase to hydrolyze any remaining maltodextrin, and the sample was passed through a mixed bed resin to remove G1P and phosphate from the reaction. The sample was then characterized by high molecular weight GPC and HPLC fingerprints (Ag+ column). The results are summarized in Tables 40 and 41 and Fig.52 middle.

[0298] Reaction of purified A8 and B6 with Zulkowsky starch using immobilized HIS-tag

[0299] The purified A8 and B6 enzymes of the His-tag immobilized by Duolite A-568 carrier were loaded onto separate glass jacketed columns (2 cm×14 cm) in series to separate the G1P synthesis catalyzed by A8 and the β-glucan synthesis catalyzed by B6. The reaction temperature in both columns was maintained at 50° C. using circulating water reflux. A pH 7 substrate solution with 7% dry matter and containing 3.2% debranched Zulkowsky starch, 0.8% glucose (as a primer) and 3.2% KH2PO4 was circulated through the column at 3 BV / hour using a peristaltic pump. Samples were taken after 7 hours, 28 hours and 52 hours. Prior to oligosaccharide analysis, the samples were incubated overnight at 50° C., pH 4.5 with 0.2% glucoamylase and 0.1% pullulanase. The results are summarized in Tables 42 and 43.

[0300] Table 43.

[0301]

[0302]

[0303] Table 43 .

[0304]

[0305] Example 13 - β-Glucan Viscosity

[0306] The viscosity of a pure 30% ds DP 150 β-glucan sample (produced as described in Example 10) was measured as a function of temperature using a rapid viscometer under stirring at 50 rpm. For comparison, the viscosity of various maltodextrin samples was also measured. Fig.53 As reported in , DP 150 β-1,2-oligoglucan had a significantly higher viscosity at all temperatures than any of the maltodextrin samples tested.

[0307] Similarly, the viscosity of the β-glucan samples was compared to a mixture of 50% 30DE syrup and 50% DP 150 β-glucan to which various maltodextrin samples were added. (See Fig.54 ) Although the mixture had a slightly higher viscosity than the maltodextrin sample, the pure β-glucan sample had a higher viscosity than all samples tested. The 50 / 50 mixture sample simulated the β-glucan composition immediately after B6 enzyme synthesis and before purification. This sample represents the presence of residual starch and lower molecular weight oligosaccharides present in the reaction product before purification of the synthesized β-1,2-oligoglucan.

[0308] Example 14 - Characterization of αGP

[0309] The enzymes from Thermosyphon melanesiensis (TmGP) and Thermosynechococcus sp. (TsGP) were recombinantly expressed in E. coli and analyzed for their biochemical and kinetic characteristics. When analyzed, TmGP and TsGP were most identical to TaGP, 84% and 56%, respectively, compared to all putative α-GPase sequences in the NCBI database (available on the World Wide Web at ncbi.nlm.nih.gov) (Tables 44 and 45). Fig.55 ).

[0310] Table 44: Percent Identity

[0311]

[0312] For detailed biochemical and kinetic characterization, TsGP and TmGP were purified by His-tag affinity chromatography ( Fig.56DAs expected, the enzymes showed a single protein band on the SDS-PAGE gel with an estimated molecular weight (MW) of 99 kDa and 96 kDa, respectively (Table 45). The optimum temperature for TmGP (60°C) was found to be similar to that for TaGP. TmGP had a very low tolerance to temperature changes as it retained more than 50% of its specific activity at temperatures between 55°C and 60°C (Table 45). TsGP had the lowest T value of all the α-GPs analyzed in this work. opt =50°C (Table 45). In contrast to TmGP, thermal stability assessment of TsGP by SDS-PAGE did not produce a strong protein band after incubation at 60°C for 1 hour (Figure 56C). The optimal pH for TmGP was found to be pH 6.5, which is lower than the optimal pH for TaGP (pH opt =8). However, TmGP maintained >50% activity at pH 8 (Table 45). In Table 45, pH and temperature profiles were determined using 50 mM phosphate buffer and 2% maltodextrin mixture as substrates, theoretical molecular weights were calculated using the ExPSAY server ProtParam tool, and the concentration of the enzyme purified by affinity chromatography was determined by the Protein A280 method.

[0313] Table 45 .

[0314]

[0315] The specific activity of TmGP was 1 / 2.5 of that of TaGP (Table 45). In addition, TmGP was obtained at a yield of almost 1 / 2 of that of TaGP (Table 45). The affinity of TmGP for maltodextrin was equal to that of TaGP, while the catalytic efficiency was 1 / 1.7 (Table 46). In contrast to TaGP, TmGP was catalyzed by a maltodextrin having a K of about 53%. i The values ​​of 1.0 and 1.0 were slightly inhibited by high maltodextrin concentrations (Table 46). TsGP, although it had the lowest Michaelis constant at 0.03 mM maltodextrin compared to all α-GPs analyzed in this work, showed a significant decrease in the expression of 1.0 at 2 mg·L -1 The protein yield of soluble protein is expressed as 5 U·mg -1 The low specific activity is 1 / 26 lower than that of TmGP (Table 45).

[0316] Table 46 .

[0317]

[0318] Example 15 - Structural Analysis of β-Glucan

[0319] 1,2-β-glucan compositions were prepared by adding 0.7 mg of enzyme B7 or enzyme B13 to a solution of 650 mM G1P and 5 mM sophorose in 50 mM MOPS buffer pH 7. The solution was kept at 35 ° C and shaken for 24 hours. After 24 hours, the reaction was stopped by heating at 100 ° C for 5 minutes. After the thermal deactivation of the enzyme, the enzyme and residual salts were removed by centrifugation and mixed bed resin treatment. The β-glucan composition was further purified by isopropanol precipitation and vacuum oven drying. After being dissolved in water, glucose and fructose were removed from the dried composition by dialysis. The β-glucan composition purified by dialysis was then freeze-dried, and the oligosaccharide composition of the freeze-dried product was analyzed using high performance liquid chromatography (HPLC) and low molecular weight gas chromatography (LMW GPC). The LMW GPC results of the β-glucan composition produced by enzyme B7 are reported in Table 47, and the results of the β-glucan composition produced by enzyme B13 are reported in Table 48. The HPLC data are reported in Table 49.

[0320] Table 47: B7 β-glucan product LMWGPC

[0321]

[0322]

[0323] Table 48: B13 β-glucan product LMWGPC

[0324]

[0325] Table 49: HPLC Oligosaccharide Analysis

[0326] sample B7 B13 DP 100％ 100％ DP 10 0％ 0％ DP 9 0％ 0％ DP 8 0％ 0％ DP 7 0％ 0％ DP 6 0％ 0％ DP 5 0％ 0％ DP 4 0％ 0％ DP 3 0％ 0％ DP 2 0％ 0％ Dextrose 0％ 0％

[0327] Additionally, the B7 and B13 β-glucan products were analyzed by NMR using the method outlined in Example 10. The structural identity of the β-1,2-glucans produced by the B7 enzyme or the B13 enzyme was confirmed by 1H-NMR and 13C-NMR. Figure 58-Figure 65 ).

Claims

1. A method for producing 1,2-β-oligoglucan, the method comprising: α-D-glucose-1-phosphate (G1P) is contacted with beta(β)-glucan-phosphorylase (βGP) to produce 1,2-β-oligoglucans.

2. The method according to claim 1, further comprising the step of contacting the substrate with alpha(α)-glucan-phosphorylase (αGP) in the presence of an inorganic phosphate (e.g., sodium phosphate or potassium phosphate) to produce the glucose-1-phosphate.

3. The method of claim 2, wherein the substrate has a degree of polymerization (DP) equal to or greater than 4.

4. The method of claim 2 or 3, wherein the substrate is selected from the group consisting of maltodextrin, starch liquefact, trehalose, sucrose, cellulose, cellodextrin, cellobiose, and combinations thereof.

5. The method according to any one of claims 1 to 4, wherein the βGP G1P contacting step is performed at a pH between 6.0 and 7.5 and / or the substrate αGP contacting step is performed at a pH between 6.5 and 8.

0.

6. A composition comprising i) beta(β)-glucan-phosphorylase (βGP); ii) α-D-glucose-1-phosphate (G1P); and iii) Primer molecules.

7. The composition according to claim 3, further comprising 1,2-β-oligoglucan, a phosphatase inhibitor (eg sodium molybdate), a buffer and / or a reducing agent.

8. The composition of claim 6 or claim 7, wherein the primer molecule is selected from the group consisting of D-glucose, sophorose, laminaribiose, cellobiose, gentiobiose, and combinations thereof.

9. A composition comprising alpha(α)-glucan-phosphorylase (αGP), inorganic phosphate, and a substrate (e.g., maltodextrin, starch, starch liquefact, trehalose, sucrose, cellulose, cellodextrin, cellobiose, and combinations thereof).

10. The composition and method according to any one of claims 2 to 5 and 9, wherein the αGP is a glycoside hydrolase 94 enzyme; and / or wherein the αGP has an amino acid sequence that is at least 80%, at least 85%, at least 90% or at least 95% identical to at least one of SEQ ID NO: 1, 2, 3, 4, 5 or 18, preferably a sequence that is at least 80%, at least 85%, at least 95% or at least 95% identical to at least one of SEQ ID NO: 1, 2 or 5, or most preferably a sequence that is at least 90% identical to SEQ ID NO:

5.

11. The method according to any one of claims 1 to 10, wherein the βGP is a glycosyltransferase 35 enzyme; and / or The βGP has an amino acid sequence that is at least 80%, at least 85%, at least 90% or at least 95% identical to at least one of SEQ ID NOs: 10, 11, 13, 15 and 16, preferably at least 90% identical to SEQ ID NOs: 11, 13 or 16.

12. A 1,2-β-oligoglucan composition produced by the method according to any one of claims 1 to 5 and 10 to 11, wherein the composition has a polydispersity between 2 and 40, a degree of polymerization (DP) of about 6-150 and a viscosity between 800 mPas and 1200 mPas at 50°C.

13. The 1,2-β-oligoglucan composition according to claim 12, wherein the composition is non-digestible.

14. Use of the composition according to any one of claims 6 to 8 or 10 to 11 for producing the 1,2-β-oligoglucan composition according to claim 12 or 13.

15. A vector comprising a nucleic acid encoding a polypeptide that is at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO:

5.

16. A vector comprising a nucleic acid encoding a polypeptide that is at least 80%, at least 85%, at least 90% or at least 95% identical to at least one of SEQ ID NOs: 11, 13 and 16.

17. A cell comprising the vector according to claim 15 or 16.

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