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

Abstract

Disclosed herein are compositions and methods for producing β-1,2-oligoglucans. The compositions include glucose-1-phosphate, a primer molecule, and a β-glucan phosphorylase having β-1,2-glucan phosphorylase activity. For example, the 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 NOs: 10, 11, 13, 15, or 16.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of European Patent Application Nos. 22192796.5, filed August 30, 2022, and 22213127.8, filed December 13, 2022, which are incorporated herein by reference in their entireties.

[0002] (Reference to electronically submitted sequence listing) The contents of the Sequence Listing XML file entitled "PT-1130-WO-PCT.xml", created on August 29, 2023, and submitted electronically with this application, having a size of 35,438 bytes, are hereby incorporated by reference in their entirety. [Background technology]

[0003] 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 from yeast, fungal, and plant cell wall extracts, although a few limited fermentation-based synthetic methods exist. Purified β-glucans are typically obtained by an acidic hydrolysis step followed by selective precipitation using organic solvents. The extraction process and biological origin of β-glucans result in significant variation in their physicochemical and functional properties, including their branching pattern, molecular weight distribution, viscosity, and concentration in biological matrices. Several glycosyltransferases have been specialized for the synthesis of β-glucans, but the high cost of their nucleotide-activated donor sugars is a significant limitation to their commercial use. Therefore, there is a need in the art for more consistent, reproducible, and commercially viable compositions and methods for producing β-glucans, particularly β-1,2-oligoglucans. Summary of the Invention

[0004] The present disclosure provides a method for producing 1,2-beta-oligoglucan, the method comprising contacting α-D-glucose-1-phosphate (G1P) with beta (β)-glucan phosphorylase (βGP) to produce 1,2-beta-oligoglucan. The method may further comprise contacting the substrate with α-glucan phosphorylase (αGP) in the presence of inorganic phosphate (e.g., sodium phosphate or potassium phosphate) to produce glucose-1-phosphate. The substrate may have a degree of polymerization (DP) of 4 or greater. The substrate may be selected from the group consisting of maltodextrin, starch liquefaction product, trehalose, sucrose, cellulose, cellodextrin, cellobiose, and combinations thereof. The βGP G1P contacting step may be carried out at a pH of 6.0 to 7.5, and / or the substrate αGP contacting step is carried out at a pH of 6.5 to 8.0.

[0005] 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 a 1,2-beta-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.

[0006] The present disclosure also provides compositions comprising alpha (α)-glucan phosphorylase (αGP), inorganic phosphate, and a substrate (e.g., maltodextrin, starch, starch liquefaction, trehalose, sucrose, cellulose, cellodextrin, cellobiose, and combinations thereof).

[0007] In the compositions and methods described herein, αGP can be a glycoside hydrolase 94 enzyme. αGP can have an amino acid sequence 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.

[0008] In the compositions and methods described herein, βGP can be a glycosyltransferase 35 enzyme. βGP can have an amino acid sequence 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.

[0009] The present disclosure also provides the use of an αGP composition and / or a βGP composition described herein to produce a 1,2-beta-oligoglucan composition described herein.

[0010] Also provided herein is a 1,2-beta-oligoglucan composition produced by the method of any one of claims 1-5 and 10-11, the composition having a polydispersity of 2-40, a degree of polymerization (DP) of about 6-150, and a viscosity of 800-1200 mPas at 50° C. Generally, the 1,2-beta-oligoglucan composition is not digestible.

[0011] 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.

[0012] 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 explanation of the drawings]

[0013] 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.

[0014] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed in the present document. [Figure 1] FIG. 1 shows an SDS-PAGE gel of A1, A3, A5, A6, and A8 expression products from a 100 mL overnight culture of E. coli BL21(DE3) cells grown at either 20° C. or 30° C. and chemically induced with 1 mM IPTG. [Figure 2] 1 shows an SDS-PAGE gel of αGP enzyme after heat treatment at 60° C. for 1 hour. For each enzyme, the soluble and insoluble fractions are shown in the left and right columns, respectively. [Figure 3] Crude cell extracts on a 12% SDS-PAGE gel before (left) and after (right) HIS-tag purification. Arrows indicate the positions of the three thermostable αGP candidates. [Figure 4] FIG. 1 shows the specific activity of αGP candidate enzymes as a function of temperature. [Figure 5] Figure 1 shows the specific activity of candidate αGP enzymes retained after incubation at 55°C for specified periods, with standard deviations of 7% (A1), 14% (A3), and 20% (A8). [Figure 6] FIG. 1 shows the specific activity of candidate αGP enzymes on two different maltodextrin substrates. [Figure 7] This is a Michaelis-Menten graph for A1αGP. [Figure 8]This is a Michaelis-Menten graph for A3αGP. [Figure 9] This is a Michaelis-Menten graph for A8αGP. [Figure 10] Michaelis-Menten graphs for the A8αGP enzyme at maximum maltodextrin concentrations of 24% (left) and 60% (right). [Figure 11] Figure 1 shows 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. Solid arrows indicate soluble protein bands, and dashed arrows indicate insoluble protein bands. [Figure 12] Figure 1 shows an SDS-PAGE gel of βGP enzyme after heat treatment for 1 hour at 60° C. For each enzyme, the soluble ("hS") and insoluble ("hI") fractions are shown in the left and right lanes, respectively. [Figure 13] FIG. 1 shows the release of phosphate (Pi) and glucose (Glc) over time as a result of the assay described in Example 4. [Figure 14] 1 is a thin layer chromatography chromatogram of the reaction products of the B6 assay outlined in Example 5 and Table 10. [Figure 15] Image of anion exchange chromatography results for B6 activity against sophorose. Individual components in the reaction mixture are included (three bottom lines). The formation of glucans of different DP was demonstrated by "trains of peaks" that did not clearly belong to any of the individual components in the assay mixture. [Figure 16] 1 is a graph showing the relationship between temperature or pH and B6 enzyme activity. [Figure 17] 1 is a graph showing the thermal stability of B6 after incubation at 50° C. The standard deviation is ≦17%. [Figure 18]Michaelis-Menten graph of B6 activity on sophorose substrate. [Figure 19] FIG. 1 shows HIS-tag purification of βGP enzymes B7, B11, B12, B13, and B14. [Figure 20] Figure 1 shows TLC plates with reaction mixtures loaded for the reactions of B7, B11, B12, B13, and B14 enzymes on glucose, sophorose, laminaribiose, gentiobiose, and cellobiose, respectively. Arrows point to the products formed. Reactions with no activity have two spots in the columns corresponding to G1P and the substrate. [Figure 21] 1 is an anion exchange chromatogram showing the activity of the B7 enzyme on sophorose substrate. The text refers to known reaction mixture components (e.g., sophorose and G1P) and reaction products (e.g., oligosaccharides and polysaccharides formed). [Figure 22] Anion-exchange chromatogram showing the activity of the B12 enzyme on a glucose substrate. The text refers to known peaks (e.g., glucose, gentiobiose / cellobiose, sophorose / laminaribiose, and G1P) and reaction products (e.g., disaccharides formed: β-1,2-linked sophorose and / or β-1,3-linked laminaribiose). [Figure 23] 1 is an anion exchange chromatogram showing the activity of B12 enzyme on glucose substrate to form laminaribiose. [Figure 24] 1 shows an anion exchange chromatogram demonstrating the activity of the B13 enzyme on the laminaribiose substrate. The text refers to known peaks (e.g., glucose, laminaribiose, and G1P) and reaction products (e.g., sugars formed). [Figure 25] 1 is an anion exchange chromatogram showing the activity of the B13 enzyme on sophorose substrate. The text refers to known peaks (e.g., glucose, sophorose) and reaction products (oligosaccharides formed). [Figure 26]1 is an anion exchange chromatogram showing the activity of the B13 enzyme on glucose. The text refers to known peaks (e.g., glucose and G1P) and reaction products (e.g., sugars formed). [Figure 27] 1 is an anion exchange chromatogram showing the activity of B6 enzyme on the combined substrate syrup outlined in Example 8. [Figure 28] 1 is a chromatogram obtained by HPLC fingerprinting of debranched Zulkowsky starch maltodextrin. [Figure 29] Chromatogram obtained by HLPC fingerprinting of debranched soluble starch maltodextrin. [Figure 30] 1 is a chromatogram obtained by HPLC fingerprinting of debranched DE 4-7 maltodextrin. [Figure 31] 1 is a chromatogram obtained by HPLC fingerprinting of debranched DE 13-17 maltodextrin. [Figure 32] 1 is a chromatogram obtained by HPLC fingerprinting of debranched maltodextrin 01910. [Figure 33] 1 is a chromatogram obtained by HPLC fingerprinting of debranched maltodextrin 01912. [Figure 34] 1 is a graph of the conversion of debranched soluble starch to G1P using A8αGP enzyme. [Figure 35] 1 is a graph of the conversion of debranched Zulkowsky starch to G1P using A8αGP enzyme. [Figure 36] 1 is a graph of the conversion of debranched MDX DE 4-7 to G1P using A8αGP enzyme. [Figure 37] 1 is a graph of the conversion of debranched MDX DE 13-17 to G1P using A8αGP enzyme. [Figure 38]Photographs of β-glucans synthesized using crude B6βGP extracts starting with 0.5, 1, or 1.5 M G1P and released by sonication (left) or homogenization (right). [Figure 39] 1 is a graph of the effect of temperature and Sumizyme GOP on β-glucan synthesis. [Figure 40] 1 is a graph of the effect of Sumizyme GOP and / or sodium molybdate on β-glucan synthesis. [Figure 41] 1H-NMR spectrum of β-1,2-glucan produced using B6βGP enzyme. [Figure 42] 13C-NMR spectrum of β-1,2-glucan produced using B6βGP enzyme. [Figure 43] FIG. 1 shows a 120 g sample of β-1,2-glucan (top) and a chromatogram of oligosaccharides (middle) and high molecular weight GPC analysis of β-1,2-glucan. [Figure 44] 1 is a graph of shear stress versus shear rate for β-1,2-glucan samples over a temperature range of 20-80° C. [Figure 45] 1 is a graph of viscosity versus shear rate for β-1,2-glucan samples over a temperature range of 20 to 80° C. [Figure 46] FIG. 1 shows the Newtonian viscosity at 20-30° C. of β-1,2-glucan compared to glucose (top), sucrose (middle), and standard maltodextrin (bottom). [Figure 47] FIG. 1 shows glucose release from isomaltulose, sucromalt, promitor 70, and β-1,2-glucan in an in vitro digestibility assay. [Figure 48] FIG. 1 shows β-glucan synthesis starting from 0.5 M G1P and pure B6 using glucose as a primer. [Figure 49] FIG. 1 shows β-glucan synthesis starting from 0.2 M G1P and pure B6 using glucose as a primer. [Figure 50]Graph of molecular weight distribution of β-glucan produced by reaction of 0.2M (top) or (0.5M) G1P with pure B6 after 72 hours of reaction time. [Figure 51] 1 is the reaction scheme used in Example 12. [Figure 52] FIG. 1 shows β-glucan synthesis under the conditions listed as outlined in Example 12. [Figure 53] FIG. 1 shows viscosity measurements for the listed starch and / or β-glucan compositions as outlined in Example 13. [Figure 54] FIG. 1 shows viscosity measurements for the listed starch and / or β-glucan compositions as outlined in Example 13. [Figure 55] FIG. 1 is a diagram of the phylogenetic tree of αGP. [Figure 56A] Figure 56 shows SDS-PAGE gel images of stable (A) conserved protein bands of AtGP, TtGP, and TaGP, and (C) weak protein bands of TsGP and strong protein bands of TmGP after 1 hour incubation at 60° C. In Figures 56A and 56C, "c" indicates the soluble fraction, and "d" indicates the insoluble fraction after heat treatment. [Figure 56B] Figure 56 shows SDS-PAGE gel images of stable (A) conserved protein bands of AtGP, TtGP, and TaGP, and (C) a weak protein band of TsGP and a strong protein band of TmGP after incubation at 60°C for 1 hour. Figures 56B and 56D show AtGP, TtGP, TaGP, TsGP, and TmGP purified by affinity (His6-tag) chromatography. Arrows indicate the protein bands. [Figure 56C] Figure 56 shows SDS-PAGE gel images of stable (A) conserved protein bands of AtGP, TtGP, and TaGP, and (C) weak protein bands of TsGP and strong protein bands of TmGP after 1 hour incubation at 60° C. In Figures 56A and 56C, "c" indicates the soluble fraction, and "d" indicates the insoluble fraction after heat treatment. [Figure 56D] Figure 56 shows SDS-PAGE gel images of stable (A) conserved protein bands of AtGP, TtGP, and TaGP, and (C) a weak protein band of TsGP and a strong protein band of TmGP after incubation at 60°C for 1 hour. Figures 56B and 56D show AtGP, TtGP, TaGP, TsGP, and TmGP purified by affinity (His6-tag) chromatography. Arrows indicate the protein bands. [Figure 57] Figure 1 shows the effect of temperature on TmGP and TsGP activity compared to TaGP. Temperature profiles were determined using 50 mM phosphate buffer and 2% maltodextrin mixture at pH 7 as substrate. Relative activity is calculated as a percentage of the maximum value. [Figure 58] 59 is a 1H-NMR spectrum of β-1,2-glucan produced using the B7βGP enzyme. The boxed portion of the NMR spectrum is shown enlarged in FIG. 59. [Figure 59] FIG. 59 is an enlarged view of the boxed portion of the NMR spectrum in FIG. 58. [Figure 60] 61 is a 13C-NMR spectrum of β-1,2-glucan produced using the B7βGP enzyme. The boxed portion of the NMR spectrum is shown enlarged in FIG. 61. [Figure 61] This is an enlarged view of the boxed area of ​​the NMR spectrum in Figure 60. [Figure 62] 63 is a 1H-NMR spectrum of β-1,2-glucan produced using the B13βGP enzyme. The boxed portion of the NMR spectrum is shown enlarged in Figure 63. [Figure 63] FIG. 63 is an enlarged view of the boxed portion of the NMR spectrum in FIG. 62. [Figure 64] 13C-NMR spectrum of β-1,2-glucan produced using B13βGP enzyme. [Figure 65] This is an enlarged view of the boxed area of ​​the NMR spectrum in Figure 64. DETAILED DESCRIPTION OF THE INVENTION

[0015] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part 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.

[0016] In this document, the words "a," "an," or "the" are used to include one or more 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 referenced in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of inconsistent usage between this document and those documents so incorporated by reference, the usage in the incorporated references should be construed as supplementary to that of this document. In the case of irreconcilable conflicts, the usage in this document shall control.

[0017] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. 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 stated range. The statement "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the statement "about X, Y, or about Z" has the same meaning as "about X, Y, or about Z" unless otherwise indicated.

[0018] Unless expressly stated, ppm (parts per million), percentages and ratios are by weight. Percentages by weight are also referred to below as % by weight or % (by weight).

[0019] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, each of the following terms has the meaning associated with it as defined below.

[0020] The present disclosure relates to β-1,2-oligoglucan compositions and compositions and methods for producing such β-1,2-oligoglucans. Generally, compositions for producing β-1,2-oligoglucans include α-D-glucose-1-phosphate (G1P, also known as "alpha-D-glucose-1-phosphate"), a primer molecule, and the enzyme β-glucan phosphorylase (βGP). The methods for producing β-1,2-oligoglucans described herein include incubating a composition containing G1P, the primer molecule, and βGP at a temperature and for a time sufficient to produce β-1,2-oligoglucan. The present disclosure also provides a composition for synthesizing G1P from maltodextrin, comprising maltodextrin, phosphate, and α-glucan phosphorylase (αGP). The methods for producing G1P described herein include incubating a composition containing maltodextrin, phosphate, and αGP at a temperature and for a time sufficient to produce G1P. The present disclosure further provides compositions comprising the produced β-1,2-oligoglucans.

[0021] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure required to confer its function and properties on the recited macromolecule. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. Recitation of any particular enzyme is understood to include cofactors, coenzymes, and metals required for the enzyme's proper function, either independently or as part of a biosynthetic pathway. A summary of art-recognized amino acids and their three-letter and one-letter symbols is provided in Table 1. Amino acid names, three-letter symbols, and one-letter symbols are used interchangeably herein.

[0022] [Table 1]

[0023] Variants or sequences having substantial identity or homology to the polypeptides described herein can be utilized in practicing the disclosed dyes, compositions, and methods. Such sequences can be referred to as variant or modified sequences. That is, a polypeptide sequence can be modified while still retaining the ability to exhibit a desired activity. Generally, a variant or modified sequence can comprise or exceed about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with a wild-type, naturally occurring polypeptide sequence, or a variant polypeptide described herein.

[0024] 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 for amino acid and nucleic acid sequence alignment are well known. Sequence alignment and sequence identity generation include global and local alignments, which typically use computational approaches. Alignment can be performed using BLAST (National Center for Biological Information, NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. 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 size: 6; Maximum match in query range: 0; Matrix: BLOSUM62; Gap cost: (Presence: 11, Extension: 1); Composition adjustment: Conditional composition score matrix adjustment; Filter: None; Mask: None. The percent 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 size: 28; Maximum match in query range: 0; Match / Mismatch score: 1, -2; Gap cost: Linear; Filter: Low complexity region; Mask: Mask for lookup table only. A sequence with an identity score of XX% (e.g., 80%) to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical to the reference sequence, or equivalently, to have XX% sequence identity.

[0025] Polypeptide or polynucleotide sequence identity may be measured over the length of the entire defined polypeptide sequence, for example, as defined by a particular SEQ ID NO:, or over a shorter length, for example, over the length of a fragment derived from the larger defined polypeptide sequence, for example, 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 contiguous residues. It is understood that such lengths are merely exemplary, and that any fragment length supported by the sequences shown in the specification, tables, figures, or sequence listing can be used to describe the length over which percent identity can be measured.

[0026] Polypeptides disclosed herein can include "variant" polypeptides, "mutants," and "derivatives thereof." As used herein, the term "wild-type" is a term of art understood by those skilled in the art and refers to the typical form of a polypeptide as it occurs in nature, as distinguished from variant or mutant forms. As used herein, a "variant," "mutant," or "derivative" refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant can have one or more insertions, deletions, or substitutions of amino acid residues compared to the reference molecule.

[0027] The amino acid sequence of a polypeptide variant, mutant, derivative, or fragment contemplated herein may contain conservative amino acid substitutions compared to a reference amino acid sequence. For example, a variant, mutant, derivative, or fragment polypeptide may contain conservative amino acid substitutions compared to a reference molecule. A "conservative amino acid substitution" is a substitution in which an amino acid is substituted with a different amino acid, such that the substitution is predicted to least interfere with the properties of the reference polypeptide. In other words, a conservative amino acid substitution substantially preserves 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, for example, 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 bulkiness of the side chain.

[0028] A composition for the synthesis of β-1,2-oligoglucan comprises G1P, a primer molecule, 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 buffer, sodium phosphate buffer, or preferably, a citrate-NaHPO buffer system, 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, SO, and combinations thereof. The composition may have a pH of 6.5-7.5.

[0029] 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 may be present in the compositions and methods described herein at a concentration of 0.05M to 2.0M, 0.1M to 1.75M, or 0.2M to 1.5M.

[0030] As used herein, "βGP," "beta-GP," "beta-glucan phosphorylase," and "β-glucan phosphorylase" are used interchangeably to refer to an enzyme that reversibly catalyzes the phosphorylation of glycosidic bonds in β-glucans to form glucose-1-phosphate. The reverse reaction catalyzes the synthesis of β-glucans via the phosphorolytic transfer of glucose from glucose-1-phosphate to an acceptor primer molecule (e.g., glucose, sophorose, laminaribiose, cellobiose, etc.). βGP enzymes may be regioselective, e.g., preferentially catalyzing the bond at the β-1,2 position of the glycose in the primer molecule. As used herein, an enzyme that catalyzes the regioselective addition of glucose at the β-1,2 position of a primer molecule derived from G1P is said to have "β-1,2-glucan phosphorylase activity." βGP may be annotated in the Carbohydrate-Active enZYmes database (CAZY) as belonging to glycoside hydrolase family 94 (GH94). βGP with β-1,2-glucan phosphorylase activity may also be referred to in the art as laminaribiose phosphorylase. βGP with β-1,2-glucan phosphorylase activity may have a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 10, 11, 13, 15, or 16.

[0031] A βGP polypeptide having β-1,2-glucan phosphorylase activity may be or may be derived from Paenibacillus sp. laminaribiose phosphorylase (PsLBP) of SEQ ID NO: 10. A 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: 10.

[0032] A βGP polypeptide having β-1,2-glucan phosphorylase activity may be or may be derived from the Rhizobium tropici β-1,2-glucan phosphorylase (RtSOGP) of SEQ ID NO: 11. A 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: 11.

[0033] A βGP polypeptide having β-1,2-glucan phosphorylase activity may be or may be derived from Clostridium saccharoperbutylaceonicum β-1,2-glucan phosphorylase (CsSOGP) of SEQ ID NO: 13. A 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: 13.

[0034] A βGP polypeptide having β-1,2-glucan phosphorylase activity may be or may be derived from the Paenibacillus stellifer β-glucan phosphorylase (PsGP) of SEQ ID NO: 15. A 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.

[0035] A βGP polypeptide having β-1,2-glucan phosphorylase activity may be or may be derived from Beutenbergia cavernae β-1,2-glucan phosphorylase (BcSOGP) of SEQ ID NO: 16. A 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.

[0036] As used herein, "primer molecule" refers to a monosaccharide, disaccharide, or polysaccharide containing D-glucose. The primer molecule may be, but is 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 molecule acts as an initial scaffold upon which additional glucose molecules are added to produce β-1,2-oligoglucans. When the βGP enzyme is provided in the composition as a crude cell extract or lysate, the primer molecule may be the residual glucose in the extract / lysate, and no additional primer molecule is required. The primer molecule may also be referred to in the art as an acceptor molecule. In the compositions and methods described herein, the primer molecule may be present at a concentration of 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 molecule 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 concentrations of the primer molecule and G1P may be adjusted to produce a β-1,2-oligoglycan with a specific degree of polymerization. For example, as demonstrated in Example 11 below, a combination of low concentrations of primer molecules may be used to produce a β-1,2-oligoglycan with a high degree of polymerization.

[0037] The composition for the synthesis of β-1,2-oligoglucans may optionally contain a phosphatase inhibitor. Suitable phosphatase inhibitors are known and described in the art. For example, phosphatase inhibitors include sodium molybdate (Na2MoO4), (NH4)6Mo7O 24 , ATP, Cu 2+ (e.g., CuSO), GDP, GTP, HgCl, iodoacetic acid, NaAsO, NaVO, NaF, sodium citrate, tartaric acid, or a combination thereof. A phosphatase inhibitor such as sodium molybdate may be added to the composition for synthesizing β-1,2-oligoglucan at a concentration of 1 to 500 mM, 50 to 400 mM, or 100 to 300 mM.

[0038] The composition for β-1,2-oligoglucan synthesis may be used in a method for producing β-1,2-oligoglucan as described herein. The method includes incubating a composition containing 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-oligoglucan. Based on the disclosure herein, those skilled in the art will understand the appropriate time and conditions for producing β-1,2-oligoglucan. The composition may be incubated at a temperature of 30°C to 70°C, 35°C to 65°C, 37°C to 60°C, 40°C to 57°C, or 40°C to 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, or about 65°C. The composition may have a pH of 5.5 to 7.5. The composition may have a pH of about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5. 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 with shaking or stirring.

[0039] The present disclosure also provides a composition for synthesizing G1P from maltodextrin. The composition for synthesizing G1P includes a glucan substrate, inorganic phosphate, and α-glucan phosphorylase, as described herein. The composition may further include a buffer and / or a reducing agent. Suitable buffers may include, but are not limited to, phosphate buffers (e.g., KHPO / KHPO or NaHPO / NaHPO). Suitable reducing agents may include, but are not limited to, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), ascorbic acid, cysteine, sodium bisulfite, SO, and combinations thereof. The composition may have a pH of 6.5 to 8.

[0040] The glucan substrate may be maltodextrin, starch (e.g., starch liquefaction), 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 the composition for synthesis of G1P at a concentration of 100 to 2000 mM, 150 to 1800 mM, or 200 to 1000 mM. 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 composition has a dry matter percentage of 1% to 30%, 2% to 25%, or 5% to 20%. The glucan substrate may be added so that the composition has a dry matter percentage of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20%. Generally, glucan substrates for use in compositions for the synthesis of 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 be used as substrates for the αGP enzyme for the synthesis of G1P.

[0041] A glucan substrate (e.g., maltodextrin) may be debranched prior to addition to the composition for synthesis of 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 may be incubated with a pullulanase enzyme (e.g., such as that sold under the trade name OPTIMAX™ L-1000) and / or an isoamylase enzyme to debranch the maltodextrin.

[0042] The inorganic phosphate may be provided in the composition in any suitable form. Suitable forms of inorganic phosphate include, but are not limited to, sodium phosphate (NaHPO), potassium phosphate (KHPO), other salts, and combinations thereof. The inorganic phosphate may be present in the composition at a concentration of 100-2000 mM, 150-1800 mM, or 200-1000 mM. The inorganic phosphate may be present in the composition at a starting concentration of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or about 2000 mM.

[0043] The glucan substrate (e.g., maltodextrin) and inorganic phosphate may be present in any suitable ratio in the composition for the synthesis of G1P. For example, the maltodextrin and inorganic phosphate may be present in the composition at a starting molar ratio of 1:0.25, 1:0.5, or 1:1. The maltodextrin and inorganic phosphate may be present in the composition at a starting molar ratio of 1:0.1 to 1:2, 1:0.2 to 1:1.5, 1:0.25 to 1:1.25, or 1:0.5 to 1:1.

[0044] As used herein, "αGP," "alpha-GP," "alpha-glucan phosphorylase," and "α-glucan phosphorylase" are used interchangeably and refer to an enzyme that reversibly catalyzes the phosphorolytic cleavage of an α-1,4 glycosidic bond to form glucose-1-phosphate. See Scheme 1. The α-1,4 glycosidic bond can be cleaved in substrates such as glycogen, starch, and maltodextrin. αGP may be annotated in the Carbohydrate-Active enZYmes database (CAZY) as belonging to glycosyltransferase family 35 (GT35). 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, 3, 4, 5, or 18. 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. 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.

[0045] [ka]

[0046] The αGP polypeptide may be or may be derived from Anaerolinea thermophila αGP (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.

[0047] The αGP polypeptide may be or may be derived from Thermobaculum terrenum αGP (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.

[0048] The αGP polypeptide may be or may be derived from Thermincola potens αGP (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.

[0049] The αGP polypeptide may be or may be derived from Thermodesulfobacterium geofontis αGP (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.

[0050] The αGP polypeptide may be or may be derived from Thermosipho africanus αGP (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.

[0051] The αGP polypeptide may be or may be derived from Thermosipho melanesiensis αGP (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.

[0052] The composition for synthesizing G1P may be used in a method for producing G1P, as described herein. The method includes incubating a composition containing a glucan substrate, inorganic phosphate, and an αGP polypeptide for a time and under conditions suitable for producing G1P. Based on the disclosure herein, those skilled in the art will understand the time and conditions suitable for producing G1P from a glucan substrate. The composition may be incubated at a temperature of 30°C to 70°C, 35°C to 65°C, 37°C to 60°C, 40°C to 57°C, or 40°C to 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, or about 65°C. The composition may have a pH of 5.5 to 8.0, 6.0 to 7.5, or 7.0 to 7.5. The composition may have a pH of 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 with shaking or stirring. The method may further include a step for debranching a glucan substrate (e.g., maltodextrin) in the same reaction vessel prior to or simultaneously with G1P synthesis.

[0053] Generally, the β-1,2-oligoglucans produced by the compositions and methods described herein are characterized by a polydispersity (Mw / Mn) of 2-40 (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, 150-400, or 200-350), a molecular weight of 20 kDa to 200 kDa (e.g., 25 kDa to 150 kDa, 40 kDa to 125 kDa, or 50 kDa to 100 kDa), and a viscosity of greater than 200 cP (e.g., 200-350 cP or 250-350 cP) when measured at 30° C., 50 rpm agitation, and a concentration of 30% dry matter. Alternatively, the β-1,2-oligoglucans produced by the compositions and methods described herein are characterized by a polydispersity (Mw / Mn) of 2-40 (e.g., 4-30, 6-20, 8-15, or any value or subrange therein), a degree of polymerization (DP) of 3-150 (e.g., 3-150, 6-100, or 10-50), a molecular weight of 20 kDa-200 kDa (e.g., 25 kDa-150 kDa, 40 kDa-125 kDa, or 50 kDa-100 kDa), and a viscosity of greater than 200 cP (e.g., 200-350 cP or 250-350 cP) when measured at 30° C., 50 rpm agitation, and a concentration of 30% dry matter.

[0054] 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 assessed using the in vitro digestion assay 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.

[0055] The β-1,2-oligoglucans produced can be used in β-1,2-oligoglucan compositions. The β-1,2-oligoglucan compositions may be used in the preparation of food, beverage, and / or animal feed products. For example, the β-1,2-oligoglucan compositions may be used to partially or completely replace 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 compositions may be used as a prebiotic or immunostimulant, or may be used in the preparation of 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 may be used to replace other calorie components of a food, beverage, and / or animal feed product while retaining the beneficial bulking or fiber properties of the food, beverage, and / or animal feed product.

[0056] As used herein, the terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence," and "nucleic acid" are used interchangeably and refer to a sequence of nucleotides 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 or antisense strand. A DNA polynucleotide may be a cDNA or genomic DNA sequence.

[0057] A polynucleotide is said to encode a polypeptide if, in its natural state, or when manipulated by methods known to those of skill in the art, it can be transcribed and / or translated to produce a polypeptide or a fragment thereof. The antisense strand of such a polynucleotide is also said to encode a sequence.

[0058] Those skilled in the art understand the degeneracy of the genetic code and that various polynucleotides can encode the same polypeptide. In some embodiments, a polynucleotide (i.e., a polynucleotide encoding an αGP polypeptide or a βGP polypeptide) can be codon-optimized for expression in a particular cell, including, but 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 that encodes a desired form of a polypeptide described herein can be used. Thus, non-naturally occurring sequences can be used. These may be desirable, for example, to enhance expression of the polypeptide or protein in a heterologous expression system. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and the human hand can also be used to generate degenerate coding sequences.

[0059] 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 also be cDNA sequences that 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.

[0060] 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 polynucleotides encoding βGP polypeptides may also be cDNA sequences that 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.

[0061] The polypeptides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide (including, but not limited to, DNA and RNA), which may be single-stranded or double-stranded and may represent the sense or antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources, or they may be synthetic. Thus, constructs may include new modifications to endogenous genes introduced, for example, by genome editing techniques. Constructs may also include recombinant polynucleotides generated, for example, using recombinant DNA methodologies. A construct may be a vector comprising a promoter operably linked to a polynucleotide encoding a thermolabile EforRed polypeptide. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. A vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments can be incorporated.

[0062] Cells containing any of the polynucleotides, constructs, or vectors described herein are also provided. The cells may be prokaryotic or eukaryotic. Suitable prokaryotic cells include bacterial cells, such as 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, Trichoderma reesei, Issatchenkia orientalis, and Aspergillus niger cells. For example, cells containing a polynucleotide encoding at least one of SEQ ID NOs: 1, 2, 3, 4, 5, 10, 11, 13, 15, 16, or 18 may 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 know how to suitably express and purify any of the polypeptides described herein from cell-based or cell-free systems. [Example]

[0063] The present invention will be described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0064] Example 1 - Selection of alpha (α)-glucan phosphorylase (αGP) and beta (β)-glucan phosphorylase (βGP) Approximately 1600 sequences were extracted from the Carbohydrate-Active enZYmes database (CAZY) for the selection of possible αGPs (CAZY-annotated glycosyltransferase family 35 members (GT35)), and approximately 1000 sequences were extracted for possible βGPs (CAZY-annotated glycoside hydrolase family 94 members (GH94)). Sequence extraction and creation of lists with unique sequences were performed computationally. These lists were used for sequence alignment and protein tree construction in the ClustalOmega EMBL-EBI online tool (Multiple Sequence Alignment). Tree visualization was performed in iTOL (Interactive Tree of Life), an online tool for displaying, annotating, and managing phylogenetic trees.

[0065] The selection of candidate sequences was partially focused on genes and enzymes from thermophilic sources, given that these enzymes should exhibit stability and activity at higher temperatures. Candidate βGPs were selected based on the likelihood that the selected enzymes would exhibit the desired activity. Selection was also aided by using previously characterized enzyme sequences. The seven selected αGPs and 12 selected βGPs are outlined in Table 2. Protein sizes were calculated using the ProtParam (ExPASy, SIB Bioinformatics Resource Portal) online tool.

[0066] [Table 2]

[0067] Example 2 - Expression, stability, and activity of αGP The synthetic genes encoding the αGP enzymes listed in Table 2 were codon-optimized for expression in E. coli and subcloned into the pET30a(+) plasmid vector with an N-terminal 6-HIS tag and linker (SEQ ID NO: 20). E. coli BL21(DE3) cells were transformed with the pET30a(+) vector and grown under conditions where expression of the enzymes was chemically induced.

[0068] The SDS-PAGE in Figure 1 shows that three of the five enzyme candidates were highly expressed in the soluble fraction (A1, A3, and A8), with A3 being the most abundant. Similar results were seen for expressions performed at both 20°C and 30°C. Although A5 and A6 did not show distinct bands visible on the gel, it is possible that the low-expressing variants are still active, and these enzymes were tested further.

[0069] Initial stability studies were performed by incubating crude protein extracts at 60°C for 1 hour. After incubation, the extracts were centrifuged to precipitate denatured proteins. The soluble and insoluble fractions (the latter containing the denatured proteins) were examined by SDS-PAGE (Figure 2). The results demonstrated that the A1, A3, and A8 enzymes remained present in the soluble fraction at the correct molecular weight and were therefore stable at 60°C.

[0070] The activity of αGP enzyme was analyzed using a glucose-1-phosphate enzyme assay. Two maltodextrins with different molecular weights (Mw) and polydispersities (Mw / Mn) were used as substrates (Table 3). MD1 was a maltodextrin substrate with a polydispersity of 23.2, and MD2 was a maltodextrin substrate with a polydispersity of 16.3. Initial results demonstrate that all selected enzymes exhibited activity at 37°C and 57°C with both maltodextrin substrates. Activity was higher for all proteins in assays performed at 57°C (Table 4).

[0071] [Table 3]

[0072] [Table 4]

[0073] Example 3 - Characterization of αGP enzyme The thermostable enzyme candidates A1, A3, and A8 were further characterized to determine their specific activity, residual activity after prolonged incubation at elevated temperatures, the effect of pH on activity, and the effect of temperature on activity. Figure 3 shows the results of crude soluble enzyme extracts and His-tag purification of the A1, A3, and A8 enzyme candidates. Approximate enzyme concentrations from the His-tag purification are shown in Table 5.

[0074] [Table 5]

[0075] The purified enzymes were further investigated to determine specific activity (U / mg), temperature range, pH range, and residual activity after prolonged incubation at 55°C. 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, as well as the activity at 55°C and 65°C for each enzyme, are reported in Table 6. All assays reported in Table 6 were performed with 2% maltodextrin substrate in 50 mM phosphate buffer at pH 7 unless otherwise indicated.

[0076] [Table 6]

[0077] The residual specific activity after extended incubation at 55°C was determined for all αGP candidates. Figure 5 shows the specific activity of the enzymes after 0, 24, or 44 hours of incubation at 55°C. The times refer to enzyme incubation, not assay run time. A3, A1, and A8 maintain approximately 80%, 40%, and 20% of their specific activity, respectively, after 44 hours of incubation. Nevertheless, the A8 candidate enzyme still has higher specific activity at all time points. The A8 enzyme lost approximately 70% of its specific activity after 24 hours of incubation at 55°C and approximately 80% of its specific activity after 44 hours of incubation at 55°C.

[0078] Substrate specificity was also tested for the A1, A3, and A8 candidate enzymes 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 activity toward both maltodextrin substrates, while A8 had approximately 60% lower initial activity toward the MDX2 substrate (Figure 6).

[0079] Example 4 - Enzyme kinetic parameters of αGP The αGP enzyme candidates A1, A3, and A8 were further characterized with respect to kinetic parameters. See Table 7 and Figures 7-9. Enzyme A8 was not inhibited by the substrate (maltodextrin), but the inhibitor constant was highest for A1. Similarly, A8 had the lowest Michaelis-Menten constant (K) compared to A1 and A3. m ), the highest turnover number (K cat ), and the highest K cat / K m The ratio has the best kinetic properties.

[0080] [Table 7]

[0081] Enzyme A8 was further tested at maltodextrin substrate concentrations up to 60% (see Figure 10). These results demonstrate that the A8 enzyme is inhibited by the substrate at higher concentrations, likely starting at approximately 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 became evident starting at approximately 30%. Because the 60% graph in Figure 10 considers all substrate concentrations, the specific activity appears to decline more rapidly than the 25% graph. However, the specific activity likely remains fairly constant up to approximately 30% substrate concentration, beyond which it declines sharply. The kinetic characteristics of the assay shown in Figure 10 are reported in Table 8.

[0082] [Table 8]

[0083] Example 5 - Expression, stability, and activity of βGP The synthetic genes encoding the βGP enzymes listed in Table 2 were codon-optimized for expression in E. coli and subcloned into the pET30a(+) plasmid vector with an N-terminal 6-HIS-tag. E. coli BL21(DE3) cells were transformed with the pET30a(+) vector and grown under conditions where enzyme expression was chemically induced. The SDS-PAGE in Figure 11 shows that all enzyme candidates, except for B2, were expressed in the soluble fraction.

[0084] Initial stability studies were performed by incubating crude protein extracts at 60°C for 1 hour. After incubation, the extracts were centrifuged to precipitate denatured proteins. The soluble and insoluble fractions (the insoluble fraction containing the denatured protein) were examined by SDS-PAGE (Figure 12). The results demonstrated that the B4 enzyme remained present in the soluble fraction at the correct molecular weight and was therefore stable at 60°C.

[0085] The activity of βGP enzymes was measured using a combination of the Gawronski phosphate release assay (measuring phosphate concentration) (see Gawronski et al., "Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection," Analytical Biochem. 2004 Apr 1, 327(1):114-8) and the GOD-POD assay (measuring glucose concentration). Absence of βGP activity would result in equal concentrations of phosphate and glucose released over time, whereas a higher concentration of phosphate relative to glucose would indicate that the candidate enzyme had the desired βGP activity. Endpoint measurements for both assays were performed at 2, 6, 15, and 30 minutes of incubation at 37°C using 1 mM laminaribiose as substrate. The results (Figure 13) showed significantly higher concentrations of phosphate released relative to glucose for four of the seven candidate enzymes (B1, B4, B5, and B6), indicating that these enzymes may have the desired activity. The B6 candidate showed the greatest difference in glucose and phosphate release over time.

[0086] The activity of the B4βGP enzyme was tested with three different substrates (laminaribiose, sophorose, and cellobiose) using a combination of the Gawronski phosphate release assay (to measure phosphate concentration) and the GOD-POD assay (to measure 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) for each of the three substrates is reported in Table 9. The different substrates, which can also be called primers, are acceptor molecules for the transfer of glucose from G1P. In other words, they are primers for the resulting oligosaccharides or polysaccharides that are generated using glucose molecules derived from G1P.

[0087] [Table 9]

[0088] 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). Experiments were performed by incubating the enzyme / primer-substrate / G1P mixture at 35°C and 45°C for 20 or 40 minutes, after which a small amount was loaded onto the TLC and the chromatogram developed to detect the formed product. Cellobiose, sophorose, gentiobiose, laminaribiose, trehalose, glucose, maltose, and isomaltulose were used as substrates at a concentration of 1 mM in 50 mM MOPS buffer (pH 7) along with 1 mM glucose-1-phosphate (G1P). As shown in Figure 14, the B6 enzyme has activity toward sophorose. The TLC samples in Figure 14 are summarized in Table 10. The dark spots indicated by the arrows indicate the potential formation of >DP10 glucan formation when sophorose was used as a substrate. The smear on the spot is likely to be smaller DP glucans that are "too heavy" to be pulled onto the slide. This activity by B6 on sophorose was confirmed by anion exchange chromatography, as shown in Figure 15.

[0089] [Table 10]

[0090] Example 6 - Characterization of βGP enzyme The B6 candidate βGP enzyme was further characterized to identify its optimal activity temperature, pH, and kinetic parameters. As shown in Figure 16, B6 has peak activity at approximately 50°C and a pH of 6.5-7. Figure 17 shows the thermal stability of the B6 enzyme. After incubation at 50°C for 15 minutes, the B6 enzyme exhibits a loss of activity, losing more than 80% of its initial activity after 3 hours at 50°C.

[0091] Using the Gawronski phosphate release assay (which measures phosphate concentration), the B6 enzyme exhibited high activity and substrate inhibition, with an inhibition constant of 14.3 mM sophorose. Michaelis-Menten kinetics are reported in Table 11 and Figure 18.

[0092] [Table 11]

[0093] Example 7 - Characterization of βGP enzyme The βGP enzyme candidates B7, B11, B12, B13, and B14 were purified using HIS-tag purification (Figure 19). The B7, B12, B13, and B14 enzymes were present in reasonable amounts in the eluted samples, but purification (and / or expression) of B11 was unsuccessful.

[0094] The activities of B7, B11, B12, B13, and B14 were screened for β-disaccharides (cellobiose, gentiobiose, laminaribiose, and sophorose), α-disaccharides (isomaltose, maltose, maltulose, sucrose, and trehalose), and glucose, focusing on product detection by TLC. The reactions were performed at 30 °C for 20 min, after which 1 μL was loaded and developed by TLC. The final substrate concentration in the reaction mixture was 5 mM, and the G1P concentration was 50 mM. TLC demonstrated that B12 was active toward glucose by forming disaccharides. B7 and B13 were active toward sophorose; B7 was active primarily by forming oligosaccharides, whereas B13 primarily formed polysaccharides. B13 also had activity toward laminaribiose, primarily by producing polysaccharides, whereas neither enzyme was active toward cellobiose or gentiobiose (by TLC analysis) (Figure 20). Samples were subsequently analyzed by anion exchange chromatography. The results confirmed the predicted activity (Figures 21-26) and further demonstrated that B13 was also active toward glucose, forming oligosaccharides and polysaccharides. Neither enzyme was active toward α-disaccharide substrates. Anion exchange chromatography also demonstrated that the disaccharide formed by B12 was laminaribiose.

[0095] Example 8 - Characterization of B6 on mixed substrates A mixed substrate syrup composition was prepared using β-glucosidase from Aspergillus niger, sold under the trade name "Novozyme 188." Anion exchange chromatography showed 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, as shown in Figure 27.

[0096] [Table 12]

[0097] Example 9 - Synthesis of G1P from maltodextrin and phosphate catalyzed by αGP A8 Various maltodextrins (Cargill, Incorporated and Sigma Aldrich) were used as substrates for the synthesis of G1P. The maltodextrins were of various average chain lengths and degrees of branching. The A8αGP enzyme cannot bypass the branches in branched substrates, and therefore, branched substrates are thought to interfere with the synthesis of G1P. To increase G1P synthesis using A8αGP, the maltodextrins were debranched at pH 4.8 and 50°C before performing the assay (the debranching method is described below).

[0098] As reported by Weinhausel 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), α-glucan phosphorylase cannot utilize maltodextrin chains with a degree of polymerization (DP) of 4 or less (DP ≤ 4) as substrates. As a result, only a portion of the total glucose units present in maltodextrin can be utilized by the αGP enzyme.

[0099] The initial assay in this example was performed using Maltodextrin C DRY MD™ 01910 ("Maltodextrin 01910") manufactured by Cargill, Incorporated. Based on calculations using Equation 1 shown below, it is estimated that up to 63% of the Maltodextrin 01910 fed to the A8-catalyzed reaction can be converted to G1P. Equation 1 accounts for the fact that chains with DP=4 (or less) cannot be used as substrates by the A8 enzyme. This calculation is based on the molecular weight distribution obtained using low molecular weight gel permeation chromatography (Table 13).

[0100] [Table 13]

[0101]

number

[0102] The percentage of available glucose units across all DP ranges calculated using Equation 1 is then used in Equation 2 to determine the total percentage of available glucose in the maltodextrin. The DP ranges and percentages of available glucose units are reported in Table 14, and the available glucose (glc) units are reported in Table 15.

[0103] [Table 14]

[0104] [Table 15]

[0105] If the maltodextrin substrate is branched, the above calculation may be an overestimation, as it does not take into account the branching linkages. 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 respective chromatograms are shown in Figures 28 to 33.

[0106] [Table 16]

[0107] [Table 17]

[0108] GPC molecular weight distribution data show that the number average molecular weight (Mn) of debranched maltodextrin types 01910 and 01912 was approximately 1000 Daltons, while the rest of the maltodextrins had larger Mn, varying from 5000 to 200,000 Daltons.

[0109] Screening of dry substrate, reaction temperature, enzyme dosage, and molar ratio Na2HPO4 / MDX for G1P production The production of G1P was carried out in a two-step process: first, a debranching step using two types of debranching enzymes to debranch the substrate maltodextrin, followed by a G1P production step. All tested maltodextrins were incubated with a combination of 0.2% pullulanase and 0.1% isoamylase (based on maltodextrin concentration) at pH 4.8-5 and 50°C for 5 hours.

[0110] The G1P synthesis reaction step was designed to achieve near-complete consumption of phosphate during the reaction by adding excess carbohydrate (i.e., maltodextrin). The reaction was carried out at pH 7 using various ratios of maltodextrin 01910 / NaHPO (1:0.25, 1:0.50, and 1:1).

[0111] The effects of reaction temperature, enzyme dosage, and type of dry matter in the reaction mixture were evaluated. Samples were taken after 24 hours of reaction time. G1P yield was determined based on phosphate consumption (i.e., subtracting the measured residual NaHPO concentration from the initial phosphate concentration provided to the reaction mixture). Additionally, G1P yield was determined using the G1P assay described by Silverstein et al. ("Purification and mechanism of action of sucrose phosphorylase," Journal of Biological Chemistry, 1967, 242(6):1338-1346, herein referred to as the "Silverstein G1P assay") to confirm the data based on NaHPO consumption. Substrate conversion was determined as a function of G1P yield based on the initial maltodextrin substrate concentration.

[0112] Five experiments (Tests 1–5) were conducted using 10 or 20 wt% dry matter (ds) maltodextrin (MDX) solutions. MDX 01910 (6.46 g; 92.81% ds) was dissolved in a total of 20 g of demineralized water and the pH was adjusted to 4.8 with 0.1 M HCl.

[0113] 0.2% OPTIMAX™ L-1000 pullulanase (0.012 g total based on 6 g dry MDX) and 0.1% isoamylase (0.006 g total based on 6 g dry MDX) were added to the MDX solution and incubated at 50 °C for 5 h. No inactivation step was performed between two consecutive steps. Next, the debranched MDX solution was subjected to G1P conversion by adding 250, 500, or 1000 mM / L NaHPO. 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 initiated by adding α-glucan phosphorylase (A8) at concentrations ranging from 64 U to 128 U enzyme / g dry maltodextrin or 12.8 U / mL to 24 U enzyme / mL of the reaction mixture. Incubations were carried out for 24 hours at 50 or 60°C in a 30 mL (32 g) scale thermomixer (shaking speed 950 rpm). 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.

[0114] [Table 18]

[0115] The results show higher activity at 60°C (Tests 9.1 and 9.2), which corresponds to the peak activity temperature of thermostable α-glucan phosphorylase (A8). When the initial NaHPO concentration was doubled (from 250 mM to 500 mM) in reactions performed at 60°C and 20% ds, an increase in G1P yield / substrate conversion (11.8% for reaction 9.4) was observed (Tests 9.1 and 9.4).

[0116] Test 9.3 was performed at 10% dry matter and 60°C using approximately equimolar amounts of NaHPO and MDX and demonstrated a significant increase in maltodextrin conversion (18.5%). To further elucidate the contribution of lower dry substrate percentages to equimolar amounts of NaHPO and MDX, Test 9.5 was performed at 20% dry matter. To accommodate both the increased phosphate concentration and the increased dry matter percentages, the reactions used 24 U / mL of A8 enzyme. To achieve the 24 U / mL concentration, the A8 enzyme was lyophilized and added to the reaction in lyophilized form. The results of Test 9.5 did not show a further increase in maltodextrin conversion, suggesting that the reaction may be limited by the equilibrium conversion of phosphate (approximately 20%).

[0117] In Experiments 9.1-9.4, a good correlation was observed between the analysis of maltodextrin conversion to G1P and the analysis of phosphate consumption. However, Experiment 9.5 showed a significant difference between the two analytical methods. The REFLECTOQUANT® Phosphate Test Kit used to measure residual phosphate appears to be sensitive to contaminants in the enzyme reaction solution. Due to this sensitivity, the remaining experiments and examples used the Silverstein G1P assay as a tool for determining the amount of G1P produced after the reaction.

[0118] To shift the equilibrium of the A8 reaction in favor of G1P synthesis, the addition of excess NaHPO and higher reaction temperatures (70 °C) were investigated. Samples were taken at regular intervals during the reaction (17, 24, and 48 hours) and assayed for G1P concentration. These assays were performed using 10 or 15 wt% dry matter 01912 maltodextrin solutions. MDX 01912 (1.073 g total; 93.16% ds) was dissolved in 5 g total demineralized water, and the pH was adjusted to pH 4.8 with 0.1 M HCl.

[0119] 0.2% OPTIMAX™ L-1000 pullulanase (0.002 g total based on 1 g dry MDX) and 0.1% iso-amylase (0.001 g total based on 1 g dry MDX) were added to the MDX solution and incubated for 5 hours at 50° C. No inactivation step was performed prior to the G1P conversion reaction.

[0120] The debranched 01912 MDX solution was then subjected to G1P conversion by adding 1000–2000 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 initiated by the addition of A8 enzyme (64 U enzyme / g dry maltodextrin or 12.8 U / mL reaction mixture). The reaction was incubated at 50 or 70°C for 17, 24, or 48 hours in a 5 mL (6.6 g) scale thermomixer (950 rpm shaking speed). The enzyme was inactivated by raising the temperature of the reaction mixture to 90°C for 5 minutes. The G1P concentration was measured using the Silverstein G1P assay. A summary of the data (reaction conditions; analysis) is shown in Table 19.

[0121] The results show that increasing the phosphate concentration up to 2000 mmol / L does not enhance the rate of G1P production. This is likely because high concentrations of Na2HPO4 result in a viscous, insoluble reaction mixture. Similarly, excessively high insoluble salt concentrations can cause enzyme inactivation, as in Test 9.7. The optimal concentration of phosphate in this reaction system, while keeping the maltodextrin concentration constant, is 1000 mmol / L. i (Test 9.6). Evaluation of G1P production as a function of time showed no further increase in G1P synthesis. Due to the equilibrium constant of the reaction, it appears that less than 20% of the phosphate was converted to G1P. The results of Test 9.8, with a decrease in the % dry matter combined with an increase in reaction temperature to 70°C, are similar to Test 9.6.

[0122] [Table 19]

[0123] Screening of substrate type, Pi source, and pH for G1P production 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. Similarly, instead of sodium phosphate, the more soluble potassium phosphate substrate may be beneficial.

[0124] To investigate G1P synthesis by the A8 enzyme using soluble starch substrate (5% w / v dry matter) and potassium phosphate, four further experiments were carried out at different soluble starch and KH2PO4 molar ratios (1:2.5, 1:3.5) and two different pH values ​​(pH 7 and 8).

[0125] 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™ L-1000 pullulanase (0.002 g total based on 1 g dry soluble starch) and 0.1% isoamylase (0.001 g total based on 1 g dry soluble starch) were added to the soluble starch solution and incubated at 50° C. for 5 hours. No inactivation step was performed prior to the G1P synthesis reaction.

[0126] The debranched soluble starch solution (1.725 g of debranched soluble starch solution containing 0.1572 g of dry weight starch) was then used in a G1P conversion reaction with 2500–3500 mmol / L KH2PO4. After homogenization (vortexing) of the suspension, the pH was measured and adjusted to pH 7 using 0.1 M NaOH solution. The reaction was initiated by adding A8 enzyme (64 U enzyme / g dry soluble starch) from crude cell lysate. The reaction was incubated in a thermomixer (950 rpm shaking speed) at a temperature of 60°C in a 10 mL (+ / - 10.4 g) scale for reaction times of 6, 17, 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 and reaction conditions are summarized in Table 20.

[0127] The results show that G1P yield was higher in the reaction run at pH 7. Run 9.10 showed high soluble starch conversion (24% after 17 hours) at a soluble starch:KH2PO4 molar ratio of 1:3.5. This represents a 5% increase in G1P yield compared to the previous reaction run using Na2HPO4 and maltodextrin 01912. The cause of the decrease in G1P yield after 24 hours, as observed in Runs 9.9 and 9.10, is not entirely clear. The A8 enzyme lysis debris may contain contaminants (e.g., phosphatases) that cause degradation of G1P, or it may be due to the presence of precipitates in the heterogeneous crude enzyme sample.

[0128] [Table 20]

[0129] [Table 21]

[0130] Based on the results of Test 9.10, additional reactions were performed (Test 9.13) identical to Test 9.10, but with shorter reaction times (5 and 8 hours) and three times the amount of A8αGP enzyme (192 U / g dry starch). Test 9.13 showed a very high conversion rate of up to 44.8% after 8 hours. In Test 9.14, the A8 enzyme concentration was tripled. The reaction conditions for Test 9.14 were identical to Test 9.13, but the incubation time was varied based on the A8 concentration.

[0131] Increasing the reaction time demonstrated equilibrium in the formation of G1P over a 3-24 hour period. Approximately ±30% equilibrium conversion of soluble starch to G1P was achieved over a 24 hour period (see Table 21 and Figure 34).

[0132] In addition to soluble starch, other substrates with longer average chain lengths were 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 varying dextrose equivalents (13.0–17.0 and 4.0–7.0 dextrose equivalents, Sigma-Aldrich) were evaluated using reaction conditions similar to those in Test 9.14. All substrates were debranched prior to the A8-catalyzed reaction. The Zulkowsky starch reaction (Test 9.15) was performed using A8αGP (192 U enzyme / g dry Zulkowsky starch) cell debris lysate. Tests using 13-17 and 4-7 dextrose equivalent maltodextrins (Tests 9.16 and 9.17, respectively) were conducted using cell debris suspensions with A8 inclusion bodies. The Silverstein G1P assay was used to assess G1P concentration in the reaction products, and the GOPOD-FORMAT procedure from Megazyme was used to measure the presence of glucose. The formation of glucose in these assays is likely due to the presence of contaminants in the enzyme preparation. The results and reaction conditions are summarized in Tables 22, 23, 24, 25, and 26. Figures 35-37 show the production of G1P and glucose during the course of the reactions in Tests 9.15, 9.16, and 9.17, respectively.

[0133] Under the reaction conditions of Test 9.15, a 44% G1P yield was achieved and maintained over a 7-10 hour incubation period, again confirming the thermostability of the active A8 enzyme. The use of Zulkowsky starch increases the G1P yield by 1.5-fold compared to Tests 9.16 and 9.17. Given that the amount of glucose formed remained at a minimal level, the crude A8 cell lysate used in the assay of Test 9.15 is likely free of phosphatase contamination.

[0134] However, because glucose levels were elevated throughout the assay time points (see Tables 24, 25, and 26 and Figures 36 and 37), the cell debris suspension with A8 inclusion bodies used in Runs 9.16 and 9.17 may have been contaminated with phosphatase (which converts G1P to glucose), which may have contributed to the overall low yield of G1P.

[0135] [Table 22]

[0136] [Table 23]

[0137] [Table 24]

[0138] [Table 25]

[0139] [Table 26]

[0140] Example 10 - Synthesis of β-glucan from G1P and primer molecules catalyzed by β-glucan phosphorylase B6 This example demonstrates the production of 1,2-β-oligoglucans using the B6βGP enzyme. In this example, the B6 enzyme is supplied to the reaction as a crude cell extract to stabilize the enzyme at the high reaction temperature. This crude cell extract also contains some residual glucose that can act as a primer molecule during β-glucan synthesis. Oligoglucan assembly requires a starting or "primer" molecule onto which the oligoglucan is built and the glucose molecule is transferred. The crude cell extract will contain residual glucose to act as this primer molecule; however, if the B6 enzyme is purified from the cell extract (e.g., His-tag purification), a separate primer molecule (sophorose or glucose) is added to the reaction.

[0141] Effect of substrate concentration on β-glucan synthesis In the first example, the effect of substrate (G1P) concentration in this second reaction of the process is 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 correspond to 14%, 26%, and 40% dry matter, respectively.

[0142] α-D-Glucose-1-phosphate (G1P, 98% purity) was dissolved in approximately 30 mL of 100 mM pH 7 phosphate buffer, the pH of which was adjusted with 1 M HCl. 0.1 mL of 1 M dithiothreitol (DTT) was added to protect the enzyme from oxidation. The reaction mixture was prepared in a 50 mL FALCON test tube and mixed by vortexing to obtain a homogeneous solution.

[0143] The activity and protein content of crude B6 enzyme cell lysates prepared by sonication were measured. The corresponding activity of 30 U / mL enzyme solution was determined by Gawronski phosphate release test. The protein content of 10 mg protein / mL was quantified using the Pierce BCA kit from Thermofisher.

[0144] Three mL (90 U) of β-glucan phosphorylase B6 was added to the 30 mL reaction mixture, resulting in an enzyme concentration of 3 U enzyme per mL of substrate in the reaction solution. Due to the presence of residual glucose in the crude cell lysate, no primer syrup or sophorose (primer molecules) was added. The tube was placed in a preheated thermomixer at 40°C (shaking speed 900 rpm). After a 24-hour incubation period, a sample was taken and heated to 90°C for 10 minutes to inactivate the B6 enzyme. A precipitate (0.6% by weight, based on the starting weight of G1P) was observed during the reaction and after enzyme inactivation. This turbidity, which is not soluble in the HO / DMSO (10% / 90%) solution, likely originates from the enzyme and is not related to β-glucan formation. (See Figure 38.)

[0145] To evaluate the difference between sonication and homogenization applied during cell lysis, a second set of reactions (3) was performed under identical reaction conditions, but with homogenized lysed β-glucan phosphorylase added to the reaction mixture. An enzyme activity of 22.32 U / mL and 8.2 mg protein / mL enzyme was measured.

[0146] The β-glucan concentration obtained for each reaction was determined based on the concentration of residual G1P (phosphoglucomutase / glucose-6-dehydrogenase assay) and glucose (glucose oxidase / peroxidase assay) in the reaction product. The molecular weight distribution of the obtained β-glucan was determined using high-molecular-weight GPC.

[0147] Because the phosphate, G1P, and β-glucan peaks overlap on high-molecular-weight GPC (Table 27), G1P and phosphate must be removed from the reaction product before analysis. The reaction product was treated with 0.5% wheat germ acid phosphatase (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.

[0148] [Table 27]

[0149] The β-glucan yield, glucose concentration, and oligosaccharide molecular weight (MW) distribution of the above reactions are summarized in Tables 28 and 29.

[0150] [Table 28]

[0151] [Table 29]

[0152] A higher β-glucan yield (57.2%) was obtained in the reaction using a lower initial substrate concentration (0.5 M G1P, Test 10.1). However, the glucose concentration in the reaction product was twice as high (9.28%) compared to Test 10.2. Test 10.3 reaction, with an initial G1P concentration of 1.5 M, showed a lower β-glucan conversion rate (5.98%). This may be due to the high dry matter concentration in the reaction mixture, which was too viscous for efficient enzyme activity.

[0153] Comparing reactions performed with enzymes prepared under different lysis methods, sonication resulted in higher β-glucan conversion (52.6% in Test 10.2 compared to 33.4% in Test 10.5). However, reactions with homogenized crude extracts resulted in the production of larger β-glucans with degrees of polymerization (DP) up to 630 (Test 10.5). Overall, the data demonstrate that crude enzyme extracts prepared from either sonication or homogenization produce β-glucan products.

[0154] Effects of temperature and phosphatase inhibitors on β-glucan synthesis To evaluate the effects of phosphatase inhibitors and the absence of glucose as a primer, B6 enzyme was preincubated with SUMIZYME® Glucose Oxidase and Catalase Cocktail and / or sodium molybdate. 200 mM sodium molybdate and / or 10 mg (per mL of B6 enzyme) SUMIZYME® Glucose Oxidase and Catalase Cocktail were incubated with B6 enzyme at room temperature (approximately 22°C) for 5 hours at pH 6.5-7.

[0155] After preincubating B6 enzyme with phosphatase inhibitors, reactions were prepared containing 3 U / mL B6 enzyme, 1 M G1P, 100 mM phosphate buffer pH 7, and 0.1 mL DTT per 30 mL reaction mixture. The reactions also included sodium molybdate and / or SUMIZYME® GOP glucose oxidase and catalase cocktail with B6 enzyme preincubation (i.e., inhibitors were not removed prior to initiating the reaction). Reactions were carried out at either 40°C or 50°C for 24 hours. The β-glucan yield, glucose concentration, and MW distribution of the resulting β-glucans are reported in Tables 30 and 31.

[0156] [Table 30] * Supplemented with 3U / mL B6 enzyme for 17 hours

[0157] [Table 31]

[0158] The results show that the use of SUMIZYME® GOP glucose oxidase and catalase cocktail reduces glucose formation and increases β-glucan yield (by approximately 60%). The β-glucan synthesized in reactions containing the SUMIZYME® GOP glucose oxidase and catalase cocktail inhibitor had a higher molecular weight (Test 10.7) than the equivalent reaction lacking glucose oxidase and catalase cocktail (Test 10.1). Similar results were seen with the use of sodium molybdate inhibitor (see Figure 40 and Tables 30 and 31). In Test 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 approximately 2-4% increase in β-glucan yield. A reaction performed at 50°C (Run 10.9) resulted in a lower β-glucan yield and higher glucose content, but the synthesized β-glucan had a higher molecular weight. (See Figure 39 and Table 31.) Both SUMIZYME® GOP glucose oxidase and catalase cocktail and sodium molybdate inhibitor were added to Run 10.10, but this combination did not further improve the β-glucan yield or increase the molecular weight of the resulting β-glucan.

[0159] To confirm that the synthesized β-glucan was a linear β-1,2-oligoglucan, the isopropanol-precipitated purified product from Test 10.8 was lyophilized for analysis using NMR. The structural identity of the β-1,2-glucan was confirmed by 1H-NMR and 13C-NMR (Figures 41 and 42). 1D 1H and 13C spectra were recorded on an Avance II Bruker spectrometer equipped with a 5 mm 1H / BB BBO probe operating at a 1H frequency of 400 MHz and running Topspin 2.1 in an ICON environment. The sample temperature was set to 25°C and controlled to within ±0.1°C with a Eurotherm 2000 VT controller. A sample was prepared from NMR by weighing 18.3 mg of β-glucan and dissolving it in 598.86 μL of DO and 1.14 μL of tBuOH as an internal standard to a final concentration of 20 mM in a total volume of 600 μL. After adding the solvent, the sample was vortexed and centrifuged multiple 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).

[0160] B6βGP concentration To evaluate the effect of reduced enzyme concentration and activity, tests were performed using homogenized crude enzyme lysates at concentrations of 3, 2, 1, or 0.5 U / mL of the reaction mixture. Additionally, the B6 enzyme used in these tests was from a separate growth and preparation of E. coli cells to test the batch-to-batch reproducibility of B6 enzyme activity. These reactions included SUMIZYME® GOP glucose oxidase and catalase cocktail. The results are summarized in Tables 32 and 33.

[0161] [Table 32]

[0162] [Table 33]

[0163] Comparison of Experiments 10.17 and 10.13 confirms the reproducibility of the B6 enzyme between preparations. Although the molecular weight of the synthesized β-glucan was higher at lower enzyme concentrations, overall β-glucan yield was highest using the 3 U / mL concentration. In Experiment 10.17, both the G1P substrate and the B6 enzyme were separately preincubated with the SUMIZYME® GOP glucose oxidase and catalase cocktail, but this additional step did not further improve β-glucan yield.

[0164] Large-scale β-glucan synthesis using B6βGP Five large-scale experiments were performed using 100 g of G1P, 3 U / mL of pre-incubated B6 enzyme, 15 mg (per mL of B6 enzyme) of SUMIZYME® GOP glucose oxidase and catalase cocktail, and a starting concentration of 1 M DTT in 100 mM phosphate buffer, pH 7. The B6 enzyme was pre-incubated with SUMIZYME® GOP glucose oxidase and catalase cocktail for 5 hours at room temperature, at a pH of 6.5 to 7. Each reaction was carried out at 40°C for 24 hours. After the reaction, the reaction mixture was heated to 90°C for 10 minutes to inactivate the B6 enzyme. The results are summarized in Tables 34 and 35.

[0165] The β-glucan products for all five runs were separated from the reaction mixture by ethanol precipitation, dried, and pooled. Approximately 120 g of dried sample was used for the tests in Example 11.

[0166] [Table 34]

[0167] [Table 35]

[0168] The reaction solution is cooled to 37°C, and the pH is adjusted from 7.23 to 4.8 by adding 19.2% HCl. Further incubation with 0.5 g of acid phosphatase at 37°C for 24 hours resulted in the conversion of unreacted G1P to glucose. At regular intervals, the phosphate content is measured until a constant value is obtained. If no additional phosphate is released, a constant phosphate reading is used as an indicator of reaction completion. The reaction mixture is heated at 70°C for 10 minutes, after which the solution is filtered through a Buchner filter equipped with filter paper. The presence of a kind of precipitate or debris after washing with 50 mL of demineralized water corresponds to only 0.1% of the total G1P (dry product).

[0169] A total of 400 mL of filtered reaction solution was recovered, with a conductivity of 54.6 mS / cm. Four tubes of Spectra / Por® dialysis membrane standard RC tubing, MWCO 6-8 kD, were filled with 100 mL of the reaction mixture each. Dialysis was performed against tap water (running water) for 24 hours, and the conductivity, phosphate, and glucose were measured (test strip method). A final dialysis was performed in milli-Q water, followed by concentration on a rotary evaporator to approximately 50 mL or 50% ds, as measured by an IR balance.

[0170] Example 11 - Characterization of β-1,2-oligoglucans synthesized by B6βGP The pooled β-glucan samples from Runs 10.18-10.22 of Example 10 were further analyzed (Figure 43). Table 36 summarizes the aggregate molecular weight distribution of the combined β-glucan products.

[0171] [Table 36]

[0172] Across runs 10.18-10.22, an average of 56% of G1P was converted to β-glucan, with only 1.7% converted to glucose. The β-glucan had a purity of 98.5% and a DP of 157. 76% of the MW was located in the 2500-200,000 kDa MW range.

[0173] The viscosity and shear-thinning properties of the synthesized β-glucan were also analyzed. β-glucan syrup was prepared at 50% dry matter (prepared from the samples produced in Tests 10.18–10.22) using 20 g of powder and 20 g of low-conductivity water. The solution was stirred and equilibrated at 60°C until optically clear. Samples were stored at 20°C prior to analysis. Isothermal viscosity was measured at 20°C (baseline temperature) and at a series of temperatures ranging from 30 to 80°C (in duplicate; Figures 44 and 45). The cooling water bath was set at 15°C. As shown in Figure 46, the viscosity and shear-thinning properties of the produced β-glucan are higher than those of sucrose or commercially available maltodextrin.

[0174] The digestibility of the β-glucan products was evaluated using an in vitro digestion assay and compared to that of sucromalt, isomaltulose, and Promitor 70 soluble corn fiber. Samples were subjected to in vitro digestion in triplicate for 72 hours and analyzed for glucose release using a glucose oxidase colorimetric assay. As shown in Figure 47, β-glucan was resistant to digestion, with minimal glucose release over the 72-hour incubation. Glucose release (as % total glucose) after 72 hours was 5% from β-1,2-glucan, 97% from isomaltulose, 66% from sucromalt, and 27% from Promitor 70. 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 samples may be due to residual glucose autohydrolyzed from rat intestinal powder (RIP) at the time points shown in Figure 47.

[0175] Example 12 - β-Glucan synthesis starting from G1P and pure B6 using glucose as a primer While previous reactions were performed using crude cell lysates containing B6 enzyme, the tests in this example use His-tagged purified B6 enzyme. Reactions used starting concentrations of either 0.2 M or 0.5 M G1P and 5, 10, 20, 100, 200, or 300 mM glucose as a primer. Neither DTT nor SUMIZYME® GOP glucose oxidase and catalase cocktail was used in these reactions. Reactions were performed in 100 mM phosphate buffer, pH 7. 0.32 mL of purified B6 enzyme (at 5.2 mg / mL and 140 U / mL) was added to the reaction mixture. This resulted in the addition of B6 enzyme at 3 U / mL in the reaction mixture, the same as in previous tests. Reactions were performed at 40°C for 24, 48, and 72 hours, followed by enzyme inactivation at 90°C for 10 minutes. The results are summarized in Figures 48 and 49 and Table 37.

[0176] [Table 37]

[0177] For reactions initiated 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., 100-300 mM) were associated with higher glucose consumption (up to about 91%, Figure 48). However, for reactions initiated with 0.2 M G1P, glucose consumption at all initial glucose concentrations was at least about 50% at 48 h (Figure 49).

[0178] Data collected for the 0.5M G1P reaction suggest that the higher the initial glucose concentration, the faster the reaction reaches its equilibrium. For initial glucose concentrations of 300, 200, and 100 mM at 0.5M G1P, the total G1P concentration decreased until approximately 52% of the initial G1P was converted to β-glucan over the course of 48–72 h. Given the lack of a significant increase in glucose concentration over time (i.e., above the initial glucose primer concentration), there is no side reaction cleaving G1P to glucose in the presence of pure B6. As shown in Figure 49, the initial 0.2M G1P concentration was converted to approximately 35% β-glucan equilibrium, but only with the consumption of approximately 70% of the glucose initially supplied. This suggests that once a glucose molecule is used as a primer by B6 and converted to DP2, this disaccharide becomes the enzyme's preferred acceptor for continuing β-glucan synthesis.

[0179] 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 Table 37 and Figure 50). The largest polymer (DP = 2700) was produced using the lowest concentration of glucose (10 mM) as a primer. These reactions using pure enzyme resulted in longer β-glucans than reactions performed with crude enzyme, and the size of the β-glucan correlated with the primer concentration.

[0180] Example 12 - Synthesis of β-glucan from maltodextrin This example demonstrates an overall process for producing β-glucan from maltodextrin via a G1P intermediate. In this example, two enzymatic processes are carried out in the same reaction vessel to test whether the equilibrium of the first reaction can be shifted to the right (i.e., toward the production of G1P) when G1P is consumed in the second reaction.

[0181] This one-pot approach requires that both enzymes be active at the same pH and temperature. Reactions were performed at 40°C and pH 7. Given that the B6 βGP enzyme has higher activity at 40°C than the A8 αGP enzyme, the concentration of A8 enzyme was increased in the reactions to account for the difference in activity. Enzyme ration, phosphate concentration, maltodextrin type and concentration, glucose concentration, and incubation time were all variables in this two-enzyme β-glucan synthesis.

[0182] Reaction of MDX 01912 with crude A8 and B6 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 αGP enzyme (10 mL at approximately 12-13 U / mL) was added to a pH 7 reaction mixture containing debranched maltodextrin 01912 (6.5% dry matter) and NaHPO and incubated at 60°C for 4 hours to produce G1P before adding B6 βGP enzyme. Approximately 21.1% of G1P (maltodextrin conversion) was produced during this incubation. After cooling to 40°C, 18 U (0.43 mL at 42 U / mL) of B6 enzyme pretreated with Sumizyme® GOP glucose oxidase and catalase cocktail was added, and the reaction was maintained at 40°C. Samples were taken at 8 and 24 hours for analysis. After 24 hours, 18 U of B6 was added to the reaction, and a final sample was taken at 48 hours for analysis. Prior to analysis, the sample was treated with a combination of glucoamylase and pullulanase to hydrolyze residual 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 fingerprinting (Ag+ column). The reaction scheme is shown in Figure 51, and the results are summarized in Tables 38 and 39.

[0183] [Table 38]

[0184] [Table 39]

[0185] Reaction of HIS-tagged purified A8 and B6 with Zulkowsky starch or low-viscosity branched dextrins Prior to reaction, Zulkowsky and maltodextrin DE1 were separately 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% MDX DE1 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. 128 U of His-tagged purified A8αGP enzyme (21 mL at approximately 6 U / mL) was added to each pH 7 reaction mixture and incubated at 60°C for 4 hours to generate G1P before the addition of B6βGP enzyme. After cooling to 40°C, 18 U (0.12 mL at approximately 160 U / mL) of His-tagged purified B6 enzyme was added, and the reaction was maintained at 40°C. Samples were taken at 7.5, 19.5, 24, and 48 hours for analysis. After 24 hours, an additional 18 U of B6 was added to the reaction. Prior to analysis, the sample was treated with a combination of glucoamylase and pullulanase to hydrolyze residual 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 fingerprinting (Ag+ column). The results are summarized in Tables 40 and 41 and Figure 52.

[0186] Reaction of immobilized HIS-tagged purified A8 and B6 with Zulkowsky starch The His-tagged purified A8 and B6 enzymes immobilized on Duolite A-568 support were loaded onto separate, consecutive, glass-jacketed columns (2 cm x 14 cm) to separate the A8-catalyzed synthesis of G1P and the B6-catalyzed synthesis of β-glucan. A circulating water return was used to maintain a reaction temperature of 50°C in both columns. A substrate solution with 7% dry matter, containing 3.2% debranched Zulkowsky starch, 0.8% glucose (as a primer), and 3.2% KH2PO4 at pH 7, was circulated through the columns at 3 BV per hour using a peristaltic pump. Samples were taken after 7, 28, and 52 hours. Prior to oligosaccharide analysis, 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.

[0187] [Table 40]

[0188] [Table 41]

[0189] [Table 42] * Total DP is the sum of DPn to DP2 ** The total DP of the unknown is the total DP that takes into account impurities from enzymes and ions still present in the reaction mixture.

[0190] [Table 43]

[0191] Example 13 - β-Glucan Viscosity 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 high-speed viscoelastic analyzer while stirring at 50 rpm. For comparison, the viscosity of various maltodextrin samples was also measured. As reported in Figure 53, the DP 150 β-1,2-oligoglucan had a significantly higher viscosity than any of the assayed maltodextrin samples at all temperatures.

[0192] Similarly, the viscosity of the β-glucan samples was compared to a mixture of 50% 30 DE syrup and 50% DP 150 β-glucan, in addition to various maltodextrin samples (see Figure 54). While the mixture has a slightly higher viscosity than the maltodextrin samples, the pure β-glucan sample has a higher viscosity than all the samples tested. The 50 / 50 mixture sample mimics the β-glucan composition immediately after synthesis with the B6 enzyme and before purification. This sample represents the presence of residual starch and low molecular weight oligosaccharides that would be present in the reaction product of the synthesized β-1,2-oligoglucan before purification.

[0193] Example 14 - Characterization of αGP The enzymes from Thermosipho melanesiensis (TmGP) and Thermosynechococcus sp. (TsGP) were recombinantly expressed in Escherichia coli and analyzed for their biochemical and kinetic characteristics. At the time of analysis, TmGP and TsGP were most identical to TaGP compared to all predicted α-GP enzyme sequences in the NCBI database (available on the World Wide Web at ncbi.nlm.nih.gov), with 84% and 56%, respectively (Table 44 and Figure 55).

[0194] [Table 44]

[0195] For detailed biochemical and kinetic characterization, TsGP and TmGP were purified by His-tag affinity chromatography (Figure 56D). As expected, the enzymes showed a single protein band on SDS-PAGE gel, with estimated molecular weights (MW) of 99 and 96 kDa, respectively (Table 45). The optimum temperature of TmGP (60°C) was found to be similar to that of TaGP. TmGP has very low tolerance to temperature changes, preserving more than 50% of its specific activity at temperatures between 55 and 60°C (Table 45). TsGP had the lowest T among all α-GPs analyzed in this study. opt = 50°C (Table 45). In contrast to TmGP, thermal stability evaluation of TsGP by SDS-PAGE did not yield a strong protein band after 1 hour of incubation at 60°C (Figure 56C). The pH optimum of TmGP was significantly higher than that of TaGP (pH opt It is found that the activity of TmGP is lower at pH 6.5 than at pH 8 (Table 45). However, TmGP retained more than 50% of its activity at pH 8 (Table 45). In Table 45, the pH and temperature profiles were determined using 50 mM phosphate buffer and a 2% maltodextrin mixture as substrates, the theoretical molecular weights were calculated using the ProtParam tool in the ExPSAY server, and the concentrations of the enzymes purified by affinity chromatography were determined by the protein A280 method.

[0196] [Table 45]

[0197] The specific activity of TmGP was 2.5-fold lower than that of TaGP (Table 45). Furthermore, TmGP was obtained in a yield that was approximately 2-fold lower than that of TaGP (Table 45). The affinity of TmGP for maltodextrin was equal to that of TaGP, but the catalytic efficiency was 1.7-fold lower (Table 46). In contrast to TaGP, TmGP was slightly inhibited by high maltodextrin concentrations, with a K of approximately 53% of that of maltodextrin. iTsGP has the lowest Michaelis-Menten constant of 0.03 mM maltodextrin compared to all α-GPs analyzed in this study, but 2 mg L -1 is expressed as an insufficient yield of soluble protein of 5 U mg -1 It has a low specific activity of 26 times lower than that of TmGP (Table 45).

[0198] [Table 46]

[0199] Example 15 - Structural analysis of β-glucan 1,2-β-glucan compositions were prepared by adding 0.7 mg of either 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 to 100°C for 5 minutes. After heat inactivation of the enzyme, the enzyme and residual salts were removed by centrifugation and mixed-bed resin treatment. The β-glucan compositions were further purified by isopropanol precipitation and vacuum oven drying. Glucose and fructose were dissolved in water and then removed from the dried composition by dialysis. The dialysis-purified β-glucan compositions were then lyophilized, and the oligosaccharide composition of the lyophilized product was analyzed using high-performance liquid chromatography (HPLC) and low molecular weight gas phase chromatography (LMW GPC). LMW GPC results for the β-glucan compositions produced by Enzyme B7 are reported in Table 47, and results for the β-glucan compositions produced by Enzyme B13 are reported in Table 48. HPLC data are reported in Table 49.

[0200] [Table 47]

[0201] [Table 48]

[0202] [Table 49]

[0203] Additionally, both the B7 and B13 β-glucan products were analyzed by NMR using the methods outlined in Example 10. The structural identity of the β-1,2-glucans produced from either the B7 or B13 enzymes was confirmed by H-NMR and C-NMR (Figures 58-65).

Claims

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

2. The method according to claim 1, further comprising 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.

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

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

5. The method according to claim 1, wherein the βGP G1P contact step is performed at a pH of 6.0 to 7.5, and / or the substrate αGP contact step is performed at a pH of 6.5 to 8.

0.

6. i) Beta (β)-glucan-phosphorylase (βGP), ii) α-D-glucose-1-phosphate (G1P), iii) A composition comprising a primer molecule.

7. The composition according to claim 6, further comprising a 1,2-beta-oligoglucan, a phosphatase inhibitor (e.g., sodium molybdate), a buffer, and / or a reducing agent.

8. The composition according to claim 6 or 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), an inorganic phosphate, and a substrate (e.g., maltodextrin, starch, liquefied starch, trehalose, sucrose, cellulose, cellodextrin, cellobiose, and combinations thereof).

10. The αGP is the glycoside hydrolase 94 enzyme, and / or The composition according to claim 9 or the method according to claim 2, 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 NOs: 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 NOs: 1, 2, or 5, or most preferably a sequence that is at least 90% identical to SEQ ID NO:

5.

11. The βGP is the glycosyltransferase 35 enzyme, and / or The composition according to claim 6 or 9 or the method according to claim 1, wherein the βGP has an amino acid sequence identical to at least one of SEQ ID NOs: 10, 11, 13, 15, and 16 by at least 80%, at least 85%, at least 90%, or at least 95%, preferably having a sequence identical to at least 90% of SEQ ID NOs: 11, 13, or 16.

12. A 1,2-beta-oligoglucan composition produced by the method described in claim 1, wherein the composition has a polydispersity of 2 to 40, a degree of polymerization (DP) of about 6 to 150, and a viscosity of 800 to 1200 mPas at 50°C.

13. The 1,2-beta-oligoglucan composition according to claim 12, wherein the composition is not digestible.

14. Use of the composition according to claim 6 to produce a 1,2-beta-oligoglucan composition having a polydispersity of 2 to 40, a degree of polymerization (DP) of about 6 to 150, and a viscosity of 800 to 1200 mPas at 50°C.

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 Sequence 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 sequence numbers 11, 13, and 16.

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

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