Beta-1,6-glucanases and their use

Abstract

The application belongs to the field of bioengineering, and particularly relates to a beta-1,6-glucanase and application thereof, more particularly to a beta-1,6-glucanase, a composition containing the same, and a method for preparing soluble yeast glucan by using the enzyme or the composition. The amino acid sequence of the beta-1,6-glucanase provided by the application comprises the sequence shown in SEQ ID NO. 1. The beta-1,6-glucanase provided by the application has a wide working temperature range and pH range, good thermal stability and pH stability, and a wide use range, and solves the problems of complex environment, poor pH and temperature tolerance in the industrial application of the beta-1,6-glucanase. Meanwhile, the combination of the beta-1,6-glucanase and beta-1,3-glucanase provided by the application is used to efficiently produce soluble yeast glucan, the reaction condition is mild, and an effective solution is provided for the degradation of natural complex beta-1,3 / 1,6-glucan.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to β-1,6-glucanase and its applications, more specifically to β-1,6-glucanase, compositions containing the same, and a method for preparing soluble yeast glucan using the enzyme or composition. Background Technology

[0002] Yeast beta-glucan, a core functional component of yeast cell walls, is a glucan formed by a mixture of β-1,3 / 1,6-glycosidic bonds. Its unique structure endows it with excellent physiological activity, making it a natural and safe functional factor with high value in the food, pharmaceutical, and health product industries. As an immunomodulator, yeast beta-glucan can activate immune cells such as macrophages and neutrophils by recognizing pattern recognition receptors on the surface of immune cells, enhancing cellular and humoral immune responses and improving the body's resistance. Simultaneously, yeast beta-glucan can act as a prebiotic to regulate the balance of intestinal flora, promote the proliferation of beneficial bacteria such as Bifidobacteria and Lactobacillus, repair the intestinal mucosal barrier, and improve intestinal health. Furthermore, yeast beta-glucan also has antioxidant, free radical scavenging, serum cholesterol-lowering, and tumor cell proliferation-inhibiting effects. In terms of applications, yeast beta-glucan is widely used in functional foods (such as dairy products, baked goods, and beverages), health products (immune enhancers), pharmaceutical preparations (intestinal mucosal protectants), and cosmetics (antioxidant and anti-inflammatory ingredients), with market demand continuing to grow.

[0003] However, natural yeast glucan suffers from poor water solubility, easily agglomerates and precipitates in product systems, and is difficult to absorb by the human intestine, resulting in significantly insufficient dispersion uniformity and bioavailability during application. Its dosage form compatibility is limited, failing to meet the needs of water-soluble product development. Furthermore, additional processing techniques or excipients are required to improve applicability, increasing production costs and potentially affecting the product's naturalness and safety. Therefore, utilizing enzymatic hydrolysis of the branched or main chain portions of insoluble glucan to achieve structural modification and soluble conversion offers significant advantages for preparing soluble yeast glucan. For example, reference 1 discloses a method for preparing soluble yeast glucan oligosaccharides using a two-enzyme approach. This method uses water-insoluble yeast glucan as a substrate and utilizes a glucan hydrolase to catalyze a reaction to prepare soluble yeast glucan. The amount of glucan hydrolase added is 10–20 U glucan hydrolase / mg glucan. The glucan hydrolase includes β-1,3-glucan hydrolase (GenBank No. ABD82280.1) and β-1,6-glucan hydrolase (GenBank No. AAO78418.1).

[0004] Enzymes involved in the hydrolysis of yeast glucan mainly include two categories: β-1,6-glucanases and β-1,3-glucanases. One of the core challenges in the enzymatic preparation of soluble yeast glucan is the screening of specific enzyme preparations and cost control. Due to the dense structure of the yeast cell wall and the uneven distribution of glucan branches, existing β-1,6 / 1,3-glucanases generally have low hydrolysis efficiency and the reaction process is difficult to precisely control. Furthermore, due to the synergistic optimization of product molecular weight and solubility, uneven product distribution and insufficient enzyme stability are common problems in production. Balancing the degree of hydrolysis with the retention of physiological activity, and achieving both production efficiency and cost-effectiveness, is also challenging. Currently, there are few β-1,6-glucanases with application value, and they generally suffer from low enzyme activity, poor stability, and low hydrolysis efficiency, limiting the large-scale preparation of soluble yeast glucan. Therefore, it is urgent to explore more β-1,6-glucanases with application potential and establish methods for the large-scale preparation of soluble yeast glucan.

[0005] References:

[0006] Reference 1: CN109852647A Summary of the Invention

[0007] The problem the invention aims to solve

[0008] To address the problems existing in the prior art, such as the scarcity of valuable β-1,6-glucanases and their generally low enzyme activity, poor stability, and poor enzymatic hydrolysis efficiency, this invention provides a β-1,6-glucanase and its preparation method, and also provides the application of this β-1,6-glucanase in the preparation of soluble yeast glucan, thus facilitating the large-scale preparation of soluble yeast glucan.

[0009] Solution for solving the problem

[0010] [1]. A β-1,6-glucanase, characterized in that the amino acid sequence of the β-1,6-glucanase comprises the sequence shown in any one of (a)-(c) below and the amino acid sequence of the protein tag:

[0011] (a) A sequence as shown in SEQ ID NO.1;

[0012] (b) A sequence in which one or more amino acid residues are added, substituted, and / or deleted in a sequence as shown in SEQ ID NO.1 while retaining endo-β-1,6-glucanase activity;

[0013] (c) A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO.1 and retaining endo-β-1,6-glucanase activity;

[0014] The protein tag includes any one or more of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc, and the amino acid sequence of the protein tag is located upstream or downstream of the sequence shown in any one of (a)-(c) and fused with it in the same frame.

[0015] [2]. A polynucleotide, characterized in that the polynucleotide encodes β-1,6-glucanase according to [1];

[0016] Optionally, the nucleotide sequence of the polynucleotide comprises the sequence shown in SEQ ID NO.2 or, under stringent conditions, hybridizes to the sequence shown in SEQ ID NO.2 and encodes a protein having endonuclease β-1,6-glucanase activity.

[0017] [3]. An expression cassette, characterized in that the expression cassette comprises a polynucleotide according to [2];

[0018] Optionally, the expression cassette includes a control element operatively connected to the polynucleotide;

[0019] Optionally, the control element is a starter, a terminator, and / or an enhancer.

[0020] [4]. A recombinant vector, characterized in that the recombinant vector comprises the polynucleotide according to [2] or the expression cassette according to [3];

[0021] Optionally, the recombinant vector is pET-28a(+) containing the polynucleotide.

[0022] [5]. A recombinant host cell, characterized in that the recombinant host cell comprises the polynucleotide according to [2], or the expression cassette according to [3], or the recombinant vector according to [4];

[0023] Optionally, the recombinant host cell is a bacterium or a fungus.

[0024] [6]. The method for preparing β-1,6-glucanase according to [1] is characterized in that the method comprises the following steps:

[0025] S1: Fermentation culture according to the recombinant host cells described in [5] to induce expression;

[0026] S2: Collect and purify the β-1,6-glucanase;

[0027] Optionally, in S2, the β-1,6-glucanase is purified using a Ni affinity chromatography column.

[0028] [7]. A composition characterized in that the composition comprises β-1,6-glucanase and β-1,3-glucanase according to [1];

[0029] Optionally, the β-1,3-glucanase comprises any one or more proteins whose amino acid sequence includes the sequence shown in GenBank numbers AIC93282.1, WP_186966490.1 or AJQ96631.1;

[0030] Optionally, in the composition, the β-1,6-glucanase and β-1,3-glucanase are combined in an enzyme activity ratio of (0.5~5):(0~5).

[0031] [8]. Use of the β-1,6-glucanase according to [1], the polynucleotide according to [2], the expression cassette according to [3], the recombinant vector according to [4], the recombinant host cell according to [5], or the composition according to [7] in the preparation of soluble yeast glucan.

[0032] [9]. A method for preparing soluble yeast glucan, characterized in that the method includes the step of hydrolyzing a substrate containing yeast glucan using β-1,6-glucanase according to [1] or a composition according to [7].

[0033]

[10] . The preparation method according to [9] is characterized in that the substrate containing yeast glucan includes any one or more of yeast cell walls, yeast cells and yeast β-glucan.

[0034] The effects of the invention

[0035] First, through extensive screening studies, this invention provides a β-1,6-glucanase with a wide operating temperature and pH range. This enzyme also exhibits good thermostability and pH stability, making it suitable for a wide range of applications. Even under appropriate high temperatures and extreme pH conditions, it retains over 50% of its residual enzyme activity, solving the problem of complex environments and poor pH and temperature tolerance in the industrial application of β-1,6-glucanase. Furthermore, this enzyme demonstrates strong substrate specificity, belonging to the endonuclease type. Experimental data show that under optimal conditions, it exhibits the highest specific activity for *Auricularia auricula-judae* polysaccharide, which is linked by β-1,6-glycosidic bonds and has an unbranched structure.

[0036] Furthermore, this invention also provides a composition comprising the aforementioned β-1,6-glucanase and β-1,3-glucanase. Combining these two enzymes can efficiently produce soluble yeast glucan. Experimental data shows that the prepared soluble yeast glucan contains oligosaccharides with a degree of polymerization of 2-15, and the reaction conditions are mild, providing an effective solution for the degradation of natural complex β-1,3 / 1,6-glucan. This invention provides key technical support for the development of high-value-added yeast glucan products and is of great significance for promoting its industrial application in functional foods, biomedicine, and other fields. Attached Figure Description

[0037] Figure 1 SDS-PAGE image of β-1,6-glucanase BxGlu30a; where M: standard protein marker; 1: recombinant cell lysate; 2: target protein purified by Ni-IDA affinity chromatography.

[0038] Figure 2 The figures show the results of the enzymatic properties detection of β-1,6-glucanase BxGlu30a; where A: Detection results of the optimal reaction temperature of β-1,6-glucanase BxGlu30a; B: Detection results of the optimal reaction pH of β-1,6-glucanase BxGlu30a; C: Detection results of the thermal stability of β-1,6-glucanase BxGlu30a; D: Detection results of the pH stability of β-1,6-glucanase BxGlu30a.

[0039] Figure 3 TLC images of the hydrolysates of *Opuntia ficus-indica* polysaccharide and gentiobiose produced by β-1,6-glucanase BxGlu30a.

[0040] Figure 4 The graph shows the results of detecting the optimal reaction conditions for the synergistic preparation of soluble yeast glucan by β-1,6-glucanase BxGlu30a and β-1,3-glucanase SlGlc16a; where A: the optimal reaction temperature of the two enzymes; B: the optimal reaction pH of the two enzymes; C: the optimal enzyme dosage of the two enzymes; and D: the optimal enzyme ratio of the two enzymes.

[0041] Figure 5TLC images of the hydrolysates of yeast glucan synthesized by the synergistic hydrolysis of yeast glucan by β-1,6-glucanase BxGlu30a and β-1,3-glucanase SlGlc16a; where A: comparison of hydrolysates of SlGlc16a and BxGlu30a; B: optimized synergistic hydrolysates of SlGlc16a and BxGlu30a; lane 1: hydrolysates of SlGlc16a; lane 2: hydrolysates of BxGlu30a; lane 3: synergistic hydrolysates of SlGlc16a and BxGlu30a; M1 is the β-1,3-glucose oligosaccharide standard (G: glucose; L2-L5 are laminarin-laminarin pentose, respectively), and M2 is the β-1,6-glucose oligosaccharide standard (G: glucose; G2: gentiobiose).

[0042] Figure 6 MALDI-TOF-MS image of the hydrolysis products of yeast glucan by β-1,6-glucanase BxGlu30a and β-1,3-glucanase SlGlc16a. Detailed Implementation

[0043] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0044] In this invention, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0045] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0046] In this invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions, etc., are used or not used.

[0047] In this invention, the numerical range represented by "value A ~ value B", "value A - value B", and "value A above / below" refers to the range that includes the endpoint values ​​A and B.

[0048] In this invention, the term "about" is used to define that the numerical ranges and parameters of this invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. Unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified by "about". Here, "about" generally means that the actual value is within ±5%, ±3%, ±1%, or ±0.5% of a specific value or range. Furthermore, the values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0049] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0050] In this invention, "polynucleotide" refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or similar compounds. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Nucleic acid molecules can be linear or circular.

[0051] In this invention, "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to, plasmids, viruses, bacteriophages, Coase plasmids, transposons, and artificial chromosomes.

[0052] In this invention, "host cell" refers to a cell into which the aforementioned carrier can be introduced. Host cells may include prokaryotic or eukaryotic cells. Prokaryotic cells may be any Gram-positive or Gram-negative bacteria, including but not limited to: cells of Bacillus, Clostridium, Lactobacillus, Streptomyces, Staphylococcus, Escherichia coli, Pseudomonas, and Bacillus-like bacteria. Eukaryotic cells may be cells of mammals, insects, plants, or fungi, including but not limited to: cells of filamentous fungi (Aspergillus, Mucor, Rhizopus, Penicillium, etc.) and yeasts (Pichia pastoris, Candida, Hansenula polymorpha, etc.).

[0053] In this invention, "codon optimization" refers to configuring the nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and translation efficiency in the host cell or organism.

[0054] In this invention, a "connecting peptide" refers to a short peptide used to connect two molecules (e.g., protein, polypeptide), and such a short peptide consists of one or more amino acid residues.

[0055] In this invention, a "tag" refers to a short peptide that is fused or linked to a target protein, thereby facilitating the soluble expression, detection, and / or purification of the recombinant protein. The tag may be fused to or linked to the N-terminus and / or C-terminus of the target protein (optionally via a linker peptide or protease cleavage site).

[0056] In this invention, the term "identity" refers to the percentage of identical amino acids among two or more polypeptides. Sequence identity between two or more polypeptides can be determined by aligning the amino acid sequences of the polypeptides and scoring the number of positions containing identical amino acid residues in the aligned polypeptides, comparing this to the number of positions containing different amino acid residues in the aligned polypeptides. Sequence identity can be calculated by dividing the number of positions containing identical amino acid residues by the total number of amino acid residues in the polypeptide.

[0057] In this invention, amino acid addition can refer to adding 1, 2, 3 or more amino acids at any position at the C-terminus, N-terminus or between the C-terminus and N-terminus of the amino acid sequence, as long as the modified sequence completely or partially retains the activity of the original amino acid sequence.

[0058] In this invention, amino acid deletion can refer to the deletion of 1, 2, 3 or more amino acids from the amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0059] In this invention, amino acid substitution can refer to the replacement of amino acids at positions 1, 2, 3 or more in an amino acid sequence with other amino acids, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0060] Amino acid substitution can be conserved amino acid substitution, referring to the substitution of several amino acids with amino acids of similar or related properties compared to the original amino acid sequence, forming a peptide (conserved variant peptide). For example, these conserved variant peptides can be generated based on the following amino acid substitutions: substitution of Ala with Val, Leu, or Ile; substitution of Arg with Lys, Gln, Asn, or His; substitution of Asn with Gln, His, Lys, or Arg; substitution of Asp with Glu or Asn; substitution of Cys with Ser or Ala; substitution of Gln with Asn or Glu; substitution of Glu with Asp or Gln; substitution of Gly with Ala; substitution of His with Asn, Lys, Gln, or Arg; substitution of Cys with Leu, Met, Ala, Val, Phe, or leucine. Substitutions include: Ile substitutions; substitutions of Leu with Ile, Met, Ala, Val, Phe, or leucine; substitutions of Lys with Asn, Gln, or Arg; substitutions of Met with Ile, Leu, or Phe; substitutions of Phe with Leu, Val, Ile, Ala, or Tyr; substitutions of Pro with Ala; substitutions of Ser with Thr; substitutions of Thr with Ser or Val; substitutions of Trp with Phe or Tyr; substitutions of Tyr with Trp, Phe, Thr, or Ser; and substitutions of Val with Phe, Ala, Met, Ile, Leu, or leucine. Amino acid substitutions can also be non-conserved amino acid substitutions.

[0061] In this invention, "hybridization" refers to the ability of a polynucleotide to bind to a substantially complementary sequence under stringent conditions, without non-specific binding to non-complementary sequences. The stringent conditions described in this invention are selected from moderately stringent, moderately-highly stringent, highly stringent, and very highly stringent conditions. The terms "moderately stringent," "moderately-highly stringent," "highly stringent," or "very highly stringent" as used in this invention describe the conditions for nucleic acid hybridization and washing. For example, the specific hybridization conditions are as follows: (1) Low stringency hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low stringency conditions, the washing temperature can be increased to 55°C); (2) Medium stringency hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High stringency hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high stringency hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0062] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0063] I. β-1,6-glucanase

[0064] This invention provides a β-1,6-glucanase, wherein the amino acid sequence of the β-1,6-glucanase comprises any one of the following (a)-(c):

[0065] (a) A sequence as shown in SEQ ID NO.1;

[0066] (b) A sequence in which one or more amino acid residues are added, substituted, and / or deleted in a sequence as shown in SEQ ID NO.1 while retaining β-1,6-glucanase activity;

[0067] (c) A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO.1 and retaining β-1,6-glucanase activity.

[0068] In some specific embodiments, the amino acid sequence of the β-1,6-glucanase comprises the sequence shown in SEQ ID NO. 1.

[0069] SEQ ID NO.1: HMCSNSDDAEKPVTPVPTGDVTIYATTSSLTRDLTRDAVNFSSKDNLAPTSITLNPTEQYQTMDGFGAAITGATCFNLLQMKPEDRHAFLTETFSDDKGFGFSYIRISIGCSDFSL SEYTCCDTKGIEHFALQSEEKDYILPILKEILSINPSIKVIAAPWTCPKWMKVKSLTDLTPLDSWTNGQLNPAYYQDYATYFVKWVQAFNAEGIDIYAVTPQNEPLNRGNSASLYMSWEEQ RDFVKTALGPKFKAAGLATKIYAYDHNYDYSDIATEKNYPGKMYEDAAASQYLAGAAYHNYGGNREELLNIHKAYPEKELLFTETSIGTWNSGRDLSKRLLEDMKEVALGTINNWCRGVIV WNLMLDNDRAPNREGGCQTCYGAVDISNSDYKTIIRNSHYYIIAHLSSVVKPGAVRIGASGYADSNIMYSAFENPDGTYAFVLMNNNEKTKKITLSDGKRHFAYDVPGKSVTSYRWAKSE.

[0070] In some embodiments, the amino acid sequence of the β-1,6-glucanase further includes the amino acid sequence of a protein tag, the protein tag including any one or more of Poly-Arg, Poly-His, FLAG, Strep-tag II and c-myc, and the amino acid sequence of the protein tag is located upstream or downstream of the sequence shown in any one of (a)-(c) and fused with it in the same frame.

[0071] In some implementations, the protein tag is Poly-Arg, Poly-His, FLAG, Strep-tag II, or c-myc.

[0072] In some embodiments, the amino acid sequence of the protein tag Poly-Arg is RRRRR (SEQ ID NO. 5). In some embodiments, the amino acid sequence of the protein tag Poly-His is HHHHHH (SEQ ID NO. 6). In some embodiments, the amino acid sequence of the protein tag FLAG is DYKDDDDK (SEQ ID NO. 7). In some embodiments, the amino acid sequence of the protein tag Strep-tag II is WSHPQFEK (SEQ ID NO. 8). In some embodiments, the amino acid sequence of the protein tag c-myc is EQKLISEEDL (SEQ ID NO. 9).

[0073] In some specific embodiments, the amino acid sequence of the β-1,6-glucanase comprises the sequence shown in SEQ ID NO.1 and the amino acid sequence of the protein tag Poly-His, wherein the amino acid sequence of the protein tag Poly-His is located upstream or downstream of the sequence shown in SEQ ID NO.1 and is fused with it in the same frame.

[0074] In some specific embodiments, the amino acid sequence of the β-1,6-glucanase comprises the sequence shown in SEQ ID NO. 10.

[0075] In some specific embodiments, the amino acid sequence of the β-1,6-glucanase is as shown in SEQ ID NO.10.

[0076] SEQ ID NO.10: MGSSHHHHHHSSGLVPRGSHMCSNSDDAEKPVTPVPTGDVTIYATTSSLTRDLTRDAVNFSSKDNLAPTSITLNPTEQYQTMDGFGAAITGATCFNLLQMKPEDRHAFLTETFSDDKGFG FSYIRISIGCSDFSLSEYTCCDTKGIEHFALQSEEKDYILPILKEILSINPSIKVIAAPWTCPKWMKVKSLTDLTPLDSWTNGQLNPAYYQDYATYFVKWVQAFNAEGIDIYAVTPQNEPLNRGNS ASLYMSWEEQRDFVKTALGPKFKAAGLATKIYAYDHNYDYSDIATEKNYPGKMYEDAAASQYLAGAAYHNYGGNREELLNIHKAYPEKELLFTETSIGTWNSGRDLSKRLLEDMKEVALGTINNWC RGVIVWNLMLDNDRAPNREGGCQTCYGAVDISNSDYKTIIRNSHYYIIAHLSSVVKPGAVRIGASGYADSNIMYSAFENPDGTYAFVLMNNNEKTKKITLSDGKRHFAYDVPGKSVTSYRWAKSE.

[0077] In some embodiments, the β-1,6-glucanase, as a highly specific endo-β-1,6-glucanase, operates at a temperature of 30°C-70°C, preferably 40°C-55°C, more preferably 50°C, and at a pH range of 4.5-9.0, preferably 6.0-7.5, more preferably 6.5.

[0078] II. Polynucleotides

[0079] The present invention provides a polynucleotide encoding the β-1,6-glucanase described in Part I.

[0080] In some implementations, the nucleotide sequence of the polynucleotide is a codon-optimized sequence based on the target host cell, such as Escherichia coli.

[0081] In some embodiments, the nucleotide sequence of the polynucleotide comprises the sequence shown in SEQ ID NO.2 or, under stringent conditions, hybridizes to the sequence shown in SEQ ID NO.2 and encodes a protein having β-1,6-glucanase activity.

[0082] In some specific embodiments, the nucleotide sequence of the polynucleotide includes the sequence shown in SEQ ID NO.2.

[0083]

[0084] In some embodiments, the nucleotide sequence of the polynucleotide comprises the sequence shown in SEQ ID NO.11.

[0085]

[0086] III. Expression Box

[0087] The present invention provides an expression cassette comprising the polynucleotides described in Part II.

[0088] In some embodiments, the expression cassette includes a control element operatively linked to the polynucleotide.

[0089] In some implementations, the control element is a promoter, a terminator, and / or an enhancer.

[0090] IV. Recombinant Vector

[0091] The present invention provides a recombinant vector comprising the polynucleotide described in Part II or the expression cassette described in Part III.

[0092] In some implementations, the recombinant vector is a recombinant plasmid vector.

[0093] In some implementations, the recombinant vector is a recombinant expression vector.

[0094] In some embodiments, the recombinant vector is pET-28a(+) containing the polynucleotide.

[0095] V. Recombinant host cells

[0096] The present invention provides a recombinant host cell comprising the polynucleotide described in Part II, or the expression cassette described in Part III, or the recombinant vector described in Part IV.

[0097] In some implementations, the recombinant host cell is a bacterium or a fungus.

[0098] In some implementations, the recombinant host cell is Escherichia coli, yeast, or Bacillus subtilis.

[0099] VI. Preparation method of β-1,6-glucanase

[0100] This invention provides a method for preparing the β-1,6-glucanase described in Part I, the method comprising the following steps:

[0101] S1: Fermentation culture of the recombinant host cells described in Part V to induce expression;

[0102] S2: Collect and purify the β-1,6-glucanase.

[0103] In some implementations, the method includes the following steps:

[0104] Construction of recombinant Escherichia coli: A polynucleotide encoding the β-1,6-glucanase described in Part I was inserted into the vector pET-28a(+) to obtain a recombinant expression vector, which was then introduced into Escherichia coli BL21(DE3) to obtain recombinant host cells;

[0105] Fermentation culture and induced expression: Recombinant host cells were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm until the OD600 was between 0.6 and 0.8. Then, lactose, IPTG or methanol or other inducing agents were added to a final concentration of 0.2-2 mM and induced at 15-30℃ for 6-20 hours. Finally, the cells were collected by centrifugation and the crude enzyme solution was obtained after cell disruption, which is the initial protein product.

[0106] Collection and purification: The initial protein product was purified by nickel ion metal chelate affinity chromatography (Ni-IDA affinity column) to obtain the protein as a highly specific endo-type β-1,6-glucanase BxGlu30a.

[0107] VII. Composition

[0108] The present invention provides a composition comprising the β-1,6-glucanase and β-1,3-glucanase described in Part I.

[0109] In some embodiments, the β-1,3-glucanase comprises any one or more proteins whose amino acid sequence comprises the sequence shown in GenBank numbers AIC93282.1, WP_186966490.1, or AJQ96631.1.

[0110] In some embodiments, the β-1,3-glucanase comprises any one or more proteins shown in GenBank No. AIC93282.1, GenBank No. WP_186966490.1, or GenBank No. AJQ96631.1.

[0111] In some embodiments, the β-1,6-glucanase and β-1,3-glucanase are combined in an enzyme activity ratio of (0.5~5):(0~5), preferably in an enzyme activity ratio of (0.1~2):(2~5), and more preferably in an enzyme activity ratio of (0.1~1):(4~5).

[0112] VIII. Uses

[0113] The present invention provides the use of the β-1,6-glucanase described in Part I, the polynucleotide described in Part II, the expression cassette described in Part III, the recombinant vector described in Part IV, the recombinant host cell described in Part V, or the composition described in Part VII in the preparation of soluble yeast glucan.

[0114] IX. Preparation method of soluble yeast glucan

[0115] The present invention provides a method for preparing soluble yeast glucan, the method comprising the step of hydrolyzing a substrate containing yeast glucan using the β-1,6-glucanase described in Part I or the composition described in Part VII.

[0116] In some embodiments, the substrate containing yeast glucan includes any one or more of yeast cell walls, yeast cells, and yeast β-glucan.

[0117] In some embodiments, in the hydrolysis system, the β-1,6-glucanase and β-1,3-glucanase are combined in an enzyme activity ratio of (0.5~5):(0~5), preferably in an enzyme activity ratio of (0.1~2):(2~5), and more preferably in an enzyme activity ratio of (0.1~1):(4~5).

[0118] In some embodiments, the hydrolysis temperature is 30-70°C, preferably 45-65°C, and more preferably 50-60°C.

[0119] In some embodiments, the pH of the hydrolysis is 4.0-7.0, preferably 4.5-6.5, and more preferably 5.0-6.5.

[0120] In some embodiments, the amounts of β-1,6-glucanase and β-1,3-glucanase in the hydrolysis system are each independently 0.5-5 U / mL; the total amount of β-1,6-glucanase and β-1,3-glucanase is 1-10 U / mL, preferably 1-8 U / mL.

[0121] In some implementations, the concentration of the substrate containing yeast glucan in the hydrolysis system is 0.5% (w / v) to 5% (w / v).

[0122] In some embodiments, the hydrolysis occurs in a disodium hydrogen phosphate-citrate buffer solution.

[0123] In some specific implementations, the method includes the following steps: Weigh a certain amount of insoluble yeast dextran, add disodium hydrogen phosphate-citric acid buffer to prepare a yeast dextran suspension with a substrate concentration of 0.5% (w / v)-5% (w / v). Add β-1,6-glucanase and β-1,3-glucanase to the yeast dextran suspension at an enzyme activity unit ratio of (0.5~5):(0~5) for reaction, with a total enzyme dosage of 1-10 U / mL, at a temperature of 30-70℃ and a pH of 4.0-7.0, and inactivate the enzymes by boiling in a water bath for 10 min after 1 h of enzymatic hydrolysis.

[0124] In some embodiments, the soluble yeast dextran produced by the above method comprises soluble oligosaccharides with a degree of polymerization distribution between DP2 and 15.

[0125] Example

[0126] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0127] Example 1: Obtaining the β-1,6-glucanase BxGlu30a gene and constructing its expression vector

[0128] Potential β-1,6-glucanases were screened using the GeneBank and CAZY databases, combined with previously reported β-1,6-glucanase sequence information. A potential β-1,6-glucanase (WP_271703861.1) derived from *Bacteroides xylanisolvens* was obtained from glycoside hydrolase family 30 (GH30 family). The gene sequence was synthesized in its entirety and named the β-1,6-glucanase BxGlu30a gene. The expression vector pET-28a(+) was selected, and the target gene was inserted at NdeI and XhoI restriction sites.

[0129] The specific construction process of recombinant vectors and recombinant host cells is as follows.

[0130] First, bioinformatics analysis was performed on the target gene sequence to remove the N-terminal signal peptide coding region. Based on the gene sequence, an upstream primer, BxGlu30a up: 5'-GGCAGC, was designed. CATATG TGCAGTAACAGTGATGATGCCGAAA-3' (SEQ ID NO. 3) and downstream primer BxGlu30a-down: 5'-TGGTG CTCGAG PCR amplification was performed using TTATTCGCTTTTTGCCCAACGATAA-3' (SEQ ID NO.4). The upstream and downstream primers included NdeI and XhoI restriction sites, respectively (as shown in the underlined parts below).

[0131] The PCR amplification program for this gene was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 12 s, for 30 cycles; final extension at 72℃ for 10 min, and storage at 4℃. The PCR product was recovered by 1% agarose gel electrophoresis and sequenced for verification.

[0132] The amplified product and the pET-28a(+) vector were double-digested with restriction endonucleases NdeI and XhoI, respectively. After electrophoresis and gel recovery, the double-digested product was ligated with the double-digested prokaryotic expression vector pET-28a(+) (Novagen, USA, product number: 69864-3) fragment using T4 DNA ligase to obtain the recombinant plasmid pET28a(+)-BxGlu30a. This plasmid was then transformed into competent E. coli DH5α cells and screened on LB agar plates containing 50 μg / mL kanamycin. Single clones were selected for PCR verification and sequencing (the primers and amplification procedures used for PCR were the same as those described in the previous PCR example).

[0133] Sequencing results showed that a nucleotide fragment from positions 1 to 1437 of SEQ ID NO.2 was inserted between the NdeI and XhoI sites of the vector pET-28a(+).

[0134] Example 2: Expression and purification preparation of β-1,6-glucanase BxGlu30a

[0135] The recombinant plasmid pET28a(+)-BxGlu30a from Example 1 was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain. This strain was then inoculated into 300 mL of LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C and 180 rpm until the OD600 reached between 0.6 and 0.8. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 1 mM, the culture speed was increased to 200 rpm, and the strain was induced overnight at 30°C. After centrifuging to collect the bacterial cells, the cells were resuspended in buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.9) at a ratio of 1:10 (v / v). The cells were then sonicated in an ice-water bath (300 W, sonication for 2.2 s, interval of 3.3 s, 15 min). The supernatant was collected by centrifugation to obtain the crude enzyme solution, which contained β-1,6-glucanase BxGlu30a.

[0136] Based on the sequence encoding a His-Tag protein in the pET28a(+) plasmid, β-1,6-glucanase BxGlu30a (i.e., the recombinant protein with the amino acid sequence shown in SEQ ID NO.10) was purified using an agarose Ni-IDA affinity column. The specific purification steps are as follows.

[0137] The crude enzyme solution was loaded onto a pre-equilibrated Ni-IDA column for purification at a flow rate of 0.5 mL / min. Impurities were washed away with 5 column volumes of 20 mM buffer A (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, pH 7.9), followed by elution with 20 mM imidazole and 200 mM imidazole sequentially at a flow rate of 1 mL / min. The solution was then dialyzed overnight at 4°C in buffer B (20 mM imidazole, 0.1 M NaCl, pH 7.9) to obtain a pure β-1,6-glucanase solution. The crude enzyme solution (i.e., recombinant cell lysate) and the finally purified recombinant protein were analyzed by SDS-PAGE. The results are shown below. Figure 1 A single target band was observed on Coomassie Brilliant Blue stained gel, meeting the electrophoretic purification standard and consistent with the predicted molecular weight of 52 kDa.

[0138] Example 3: Enzymatic Properties Analysis of β-1,6-glucanase BxGlu30a

[0139] (1) Determination of β-1,6-glucanase BxGlu30a enzyme activity

[0140] Preparation method of substrate raw material (rock tripe polysaccharide preparative solution): Weigh a certain amount of β-1,6-glucan (rock tripe polysaccharide). Due to the high substrate concentration, incubate it in a water bath shaker at 50-55℃ and 160 rpm for 4 h to completely dissolve it in water and obtain a 2% (w / v) rock tripe polysaccharide preparative solution.

[0141] Determination of enzyme activity using the DNS (3,5-dinitrosalicylic acid) method: Mix 15 μL of *Auricularia auricula-judae* polysaccharide preparation solution with 85 μL of disodium hydrogen phosphate-citric acid buffer. Add 50 μL of appropriately diluted enzyme solution to 100 μL of *Auricularia auricula-judae* polysaccharide preparation solution. At this point, the final concentration of substrate *Auricularia auricula-judae* polysaccharide in the reaction system is 0.2% (w / v). React at 50℃ for 10 min, then add 800 μL of DNS reagent to terminate the hydrolysis reaction. Immediately transfer to a boiling water bath for 10 min, and finally rapidly cool to room temperature with cold water. Centrifuge at 10,000 rpm for 5 min, and aspirate the supernatant of the above reaction mixture. Measure the absorbance at 540 nm using a UV spectrophotometer.

[0142] Method for determining protein concentration: Bovine serum albumin (BSA) was used as a standard, and the Bradford method was used to determine the protein concentration.

[0143] The definition of an enzyme activity unit (U): the amount of enzyme required to produce 1 μmol of reducing sugar per minute under optimal reaction conditions.

[0144] Method for determining the standard curve of reducing sugar: Take 400 μL of glucose solutions with concentrations of 0.08 g / mL, 0.12 g / mL, 0.16 g / mL, 0.20 g / mL, 0.24 g / mL, 0.28 g / mL and 0.32 g / mL respectively, add 800 μL of DNS reagent and mix well, then measure the absorbance at a wavelength of 540 nm.

[0145] (2) Determination of optimal reaction conditions and stability of β-1,6-glucanase BxGlu30a

[0146] The optimal reaction conditions and stability determination of β-1,6-glucanase BxGlu30a were both performed using a 0.2% (w / v) solution of *Opuntia ficus-indica* polysaccharide as the reaction substrate.

[0147] Methods for determining optimal temperature and thermal stability: The *Auricularia auricula-judae* polysaccharide substrate was dissolved in a 50 mM disodium hydrogen phosphate-citrate buffer solution (pH 4.5). The enzyme solution and substrate were mixed, and enzyme activity was measured at 5°C intervals along a temperature gradient of 30-70°C. Blank controls consisted of a mixture of water and substrate solution. Results are as follows: Figure 2As shown in A, the maximum enzyme activity is set to 100%, and the temperature operating range of β-1,6-glucanase BxGlu30a is 30-70℃, with the optimum temperature being 50℃.

[0148] The thermal stability test involved incubating the enzyme solution at 30-60℃ for 30 min, followed by determining enzyme residual viability at the optimal pH and temperature according to method (1). The results are as follows: Figure 2 As shown in C, the enzyme activity remains above 80% within the temperature range of 30℃-45℃.

[0149] Methods for determining optimal pH and pH stability: *Auricularia auricula-judae* polysaccharide solutions were prepared using 50 mM buffers of different pH values: acetate-sodium acetate buffer (pH 4.5-5.5), disodium hydrogen phosphate-citric acid buffer (pH 4.5-7), and Tris-HCl buffer (pH 7.0-9.0). The enzyme solution was mixed with the substrates of the above different pH values, and the changes in enzyme activity at different pH values ​​were determined according to method (1). The results are as follows: Figure 2 As shown in B, with the highest enzyme activity set to 100%, the pH operating range of β-1,6-glucanase BxGlu30a can be 4.5-9.0, with the optimal pH being 6.5.

[0150] The pH stability test involved diluting the enzyme solution with buffer solutions of different pH values, incubating at 30°C for 30 min, and then determining the enzyme residual viability at the optimal pH and temperature according to method (1). The results are as follows: Figure 2 As shown in D, the enzyme exhibits good stability in buffer solutions with a pH range of 7.0-10.0. After 30 min of incubation, the enzyme activity remains above 80%, indicating that the enzyme has high stability in neutral and weakly alkaline pH ranges, and that the residual enzyme activity can still remain above 50% even under extreme pH conditions.

[0151] (3) Substrate specificity of β-1,6-glucanase BxGlu30a

[0152] The substrate specificity of β-1,6-glucanase BxGlu30a was determined by adding different polysaccharide substrates—Pustulan, Curdlan, Laminarin, Yeastglucan, Lichenin, and sodium carboxymethyl cellulose (CMC-Na)—to 50 mM disodium hydrogen phosphate-citrate buffer at pH 6.5 to prepare 0.2% (w / v) substrate solutions or suspensions. 100 μL of the substrate solution was mixed with 50 μL of appropriately diluted enzyme solution, and the mixture was reacted at 50 °C for 10 min. The catalytic activity of BxGlu30a for different substrates was then determined using the DNS method.

[0153] The results are shown in Table 1. β-1,6-glucanase BxGlu30a exhibits strong substrate specificity, showing the highest specific activity (111.52 U / mg) for rock triptoli polysaccharide with unbranched structure linked by β-1,6-glycosidic bonds. It also has some hydrolytic ability for yeast glucan with β-1,6-glycosidic bond branches. Its hydrolytic activity for laminarin and kelp polysaccharide is relatively low, and it has no catalytic activity for lichen polysaccharide and sodium carboxymethyl cellulose (CMC-Na).

[0154] Table 1. Substrate specificity of BxGlu30a

[0155]

[0156] (4) Determination of the hydrolytic characteristics of β-1,6-glucanase BxGlu30a

[0157] To analyze the hydrolytic characteristics of the protein BxGlu30a, a β-1,6-glucanase, we used rock tripe polysaccharide and gentiobiose as substrates and analyzed the products generated after hydrolysis using 50 mM disodium hydrogen phosphate-citrate buffer at pH 6.5.

[0158] The steps for the β-1,6-glucan hydrolysis reaction are as follows: 100 μL of 0.2% (w / v) *Auricularia auricula-judae* polysaccharide suspension is mixed with 50 μL of enzyme solution and reacted at 50 °C for 2 h. 3 μL of 1% (w / v) gentiobiose solution is mixed with 7 μL of pH 6.5 buffer, and 5 μL of diluted enzyme solution is added, bringing the final concentration of the gentiobiose solution to 0.2% (w / v). The reaction is then carried out at 50 °C for 2 h.

[0159] Thin-layer chromatography (TLC) for the detection of hydrolysis products: To observe the hydrolysis products at different time points and the reaction process, samples were taken at different time points (5 min, 15 min, 30 min, 60 min, 120 min), and the reaction was terminated by boiling in a water bath for 5 min. 2 μL of *Auricularia auricula-judae* polysaccharide hydrolysate and 1 μL of gentiobiose hydrolysate were spotted onto silica gel plates. The silica gel plates were then developed twice in a developing agent system of n-butanol:acetic acid:water (2:1:1, v / v / v), with each development time controlled within 60 minutes. After development, the thin-layer plates were dried with hot air, then uniformly immersed in a colorimetric reagent solution of sulfuric acid and methanol (5:95, v / v), dried again with hot air, and then baked in an electric oven until color development was achieved.

[0160] The results are as follows Figure 3 As shown, when rock tripe polysaccharide is used as a substrate, the hydrolysis products are only monosaccharides (G) and gentiobiose (L2), with no other oligosaccharides with higher degree of polymerization. Furthermore, it has no hydrolytic ability for gentiobiose, proving that BxGlu30a is an endo-type β-1,6-glucanase.

[0161] Example 4: Application of β-1,6-glucanase BxGlu30a or its composition in the preparation of soluble yeast glucan

[0162] Natural β-1,3 / 1,6-glucans are complex polysaccharides with β-1,3-glycosidic bonds as the backbone and β-1,6-glycosidic bonds as branches. Yeast cell wall polysaccharides, as typical natural β-1,3 / 1,6-glucans, exhibit a variety of biological functions, including regulating the body's immune response, inhibiting tumor cell proliferation, scavenging free radicals, and regulating blood glucose levels. However, due to their large molecular weight, dense spatial structure, and frequent formation of complexes with biomolecules such as proteins, natural yeast glucans have poor water solubility, which limits their bioavailability and ultimately significantly restricts their practical application. Compared with chemical modification and physical treatment, enzymatic modification to improve the water solubility of yeast glucans has advantages such as high specificity, mild reaction conditions, high product purity, and environmental friendliness, and has gradually become an important strategy for polysaccharide structure modification.

[0163] This example demonstrates the preparation of soluble yeast glucan through a combination of β-1,6-glucanase BxGlu30a and a known β-1,3-glucanase via hydrolysis.

[0164] (1) Preparation of soluble yeast glucan by hydrolysis with a combination of two enzymes

[0165] A certain amount of insoluble yeast glucan G90 was weighed and added to 10 mL of 50 mM disodium hydrogen phosphate-citric acid buffer to prepare a yeast glucan suspension with a substrate concentration of 4% (w / v). β-1,3-glucanase SlGlc16a (GeneBank: AIC93282.1) and β-1,6-glucanase BxGlu30a were added to the yeast glucan suspension at a 1:1 ratio of enzyme activity units (U) for reaction. The initial experimental conditions were: substrate concentration 4% (w / v), total enzyme volume 1 U / mL, temperature 55℃, pH 4.5. After 1 h of enzymatic hydrolysis, the reaction was terminated by boiling in a water bath for 10 min. The mixture was centrifuged at 10000 r / min for 10 min, and the soluble polysaccharide content of the supernatant was determined using the phenol-sulfuric acid method. A standard curve was prepared using glucose as a standard. The control group consisted of yeast dextran suspension hydrolyzed with single enzymes SlGlc16a and BxGlu30a. The yield of soluble polysaccharides was calculated using the following formula:

[0166]

[0167] (2) Effect of different conditions on the yield of soluble yeast glucan

[0168] The optimal reaction conditions for the preparation of soluble yeast glucan by hydrolysis with a dual-enzyme combination were determined using a yeast glucan suspension at a concentration of 4% (w / v) as the reaction substrate.

[0169] Method for determining the effect of temperature on compound hydrolyzed yeast glucan: Prepare 10 mL of yeast glucan suspension at pH 6.5. At different temperatures of 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃, add SlGlc16a and BxGlu30a in a compound ratio of 1:1 with an enzyme activity unit (U) ratio of 1:1 and react in a water bath shaker at 120 rpm for 1 h. The total enzyme dosage is 1 U / mL. Terminate the reaction by boiling in a water bath for 10 min. Centrifuge at 10000 r / min for 10 min, take the supernatant to determine the polysaccharide content, and calculate the yield of soluble polysaccharides.

[0170] Method for determining the effect of pH on compound hydrolyzed yeast dextran: 10 mL yeast dextran suspensions were prepared using disodium hydrogen phosphate-citric acid buffer solutions with different pH values ​​of 4, 4.5, 5, 5.5, 6, 6.5, and 7. SlGlc16a and BxGlu30a were added at a ratio of 1:1 (enzyme activity units (U)) at 55 °C and reacted on a water bath at 120 rpm for 1 h. The total enzyme dosage was 1 U / mL. The mixture was then placed in a boiling water bath for 10 min to inactivate the enzymes. After centrifugation at 10000 r / min for 10 min, the supernatant was collected for polysaccharide quantification and the yield of soluble sugars was calculated.

[0171] Method for determining the effect of enzyme dosage on compound hydrolyzed yeast glucan: Prepare 10 mL of yeast glucan suspension at pH 6.5. Add SlGlc16a and BxGlu30a at a compound ratio of 1:1 at 55℃ and react in a water bath shaker at 120 rpm for 1 h. The total enzyme dosage is 0.5, 1, 2, 4 and 8 U / mL, respectively. Terminate the reaction by boiling in a water bath for 10 min. Centrifuge at 10000 r / min for 10 min, take the supernatant to determine the polysaccharide content, and calculate the yield of soluble polysaccharides.

[0172] Method for determining the effect of enzyme ratio on the compound hydrolyzed yeast glucan: 10 mL of yeast glucan suspension with pH 6.5 was prepared. SlGlc16a and BxGlu30a were added at 55℃ and reacted in a water bath at 120 rpm for 1 h. The ratios of enzyme activity units (U) were 10:0, 9:1, 7:3, 5:5, 3:7, 1:9, and 0:10 (SlGlc16a:BxGlu30a), with a total enzyme amount of 1 U / mL. The mixture was placed in a boiling water bath for 10 min to inactivate the enzyme. After centrifugation at 10000 r / min for 10 min, the supernatant was collected for polysaccharide quantification analysis, and the yield of soluble polysaccharides was calculated.

[0173] The results are as follows Figure 4 As shown in Figures A and B, the soluble polysaccharide yield reached its highest level at a temperature of 55℃ and a pH of 6.5. This coincides with the optimal enzyme activity conditions for SlGlc16a and BxGlu30a, and the dual-enzyme combination showed better results under neutral conditions. Figure 4 The result in cell C shows that when the total amount of the two enzymes was 1 U / mL, the yield of soluble polysaccharides reached a stable level and did not change significantly with increasing enzyme concentration. Figure 4 As shown in Figure D, the yield of soluble polysaccharides obtained from the dual-enzyme combination was higher than that obtained from the single-enzyme combination. This indicates that the dual-enzyme combination can improve the preparation of soluble yeast glucan. Furthermore, the results show that the optimal enzyme ratio of SlGlc16a:BxGlu30a (9:1) yielded the best results, with a yield of 31.3%. Compared with previous studies, the dual-enzyme combination significantly improved the yield of soluble sugars and has great application potential in the hydrolysis of natural polysaccharides.

[0174] (3) Determination of the hydrolytic characteristics of the dual-enzyme combination

[0175] To compare the differences in hydrolysis products of yeast glucan by different glucanases, two control groups were first used: one using a single enzyme, SlGlc16a (1 U / mL), and the other using BxGlu30a (1 U / mL). Figure 5In lanes A, lanes 1 and 2), a dual-enzyme combination (total enzyme amount 1 U / mL, enzyme activity ratio SlGlc16a: BxGlu30a = 9:1) was used to hydrolyze yeast glucan. Figure 5 Lane A (lane 3) was used. 10 mL of 4% (w / v) yeast dextran suspension at pH 6.5 was mixed with the above three enzyme solutions, and the mixture was reacted at 55°C for 2 h, followed by a boiling water bath for 5 min to terminate the reaction.

[0176] Thin-layer chromatography (TLC) for the detection of hydrolysis products: 1 μL of the above yeast dextran hydrolysate was spotted onto a silica gel plate. The silica gel plate was then developed twice in a developing agent system of n-butanol:acetic acid:water (2:1:1, v / v / v), with each development time controlled within 60 minutes. After development, the thin-layer plate was dried with hot air and then uniformly immersed in a colorimetric reagent of sulfuric acid and methanol solution (5:95, v / v). After drying with hot air again, the plate was baked in an electric oven until color development was achieved.

[0177] from Figure 5 As shown in A, the products of SlGlc16a single-enzyme hydrolysis of yeast glucan include monosaccharides (G), laminarin (L2) to laminarin (L5), while the products of BxGlu30a single-enzyme hydrolysis of yeast glucan include monosaccharides (G) and gentiobiose (G2). The hydrolysis products of dual-enzyme combination hydrolysis of yeast glucan include... Figure 5 As shown in lane A3 of the diagram, the product contains monosaccharides to pentasaccharides and other oligosaccharides with higher degrees of polymerization. It can be seen that the product after the combined hydrolysis is consistent with the sum of the products after hydrolysis by the two single enzymes, but the concentration of the product after the combined hydrolysis is higher than that after hydrolysis by the single enzymes.

[0178] Furthermore, to accurately determine the oligosaccharide distribution in the yeast dextran hydrolysate under the optimized conditions, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to detect the hydrolysate. Sample preparation involved adding two dextran enzyme solutions to 10 mL of pH 6.5, 4% (w / v) yeast dextran suspension. The total enzyme concentration was 1 U / mL, and the enzyme activity units (U) ratio was 7:3 (SlGlc16a: BxGlu30a). The reaction was carried out at 55℃ for 8 h, and the reaction was terminated by heating in a boiling water bath for 5 min. The supernatant was then collected. The reaction solution was first analyzed by thin-layer chromatography (TLC). Figure 5 (B) After confirming that the hydrolysis products were normal, 1 μL of 2,5-dihydroxybenzoic acid (DHB) at a concentration of 20 mg / mL and 1 μL of hydrolysis sample were mixed and dried under airflow. The mass spectrometer was operated in positive acquisition mode and linear low resolution mode, with a scanning range of 150 to 2000 Hz.

[0179] The results are as follows Figure 6As shown, the product of double-enzyme hydrolysis of yeast glucan for 8 hours contains monosaccharides and oligosaccharides with a degree of polymerization of 2-15, with a large number of components, and a high content of tetrasaccharides.

[0180] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A β-1,6-glucanase, characterized in that, The amino acid sequence of the β-1,6-glucanase comprises any one of the sequences shown in (a)-(c) below and the amino acid sequence of the protein tag: (a) A sequence as shown in SEQ ID NO.1; (b) A sequence in which one or more amino acid residues are added, substituted, and / or deleted in a sequence as shown in SEQ ID NO.1 while retaining endo-β-1,6-glucanase activity; (c) A sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence shown in SEQ ID NO.1 and retaining endo-β-1,6-glucanase activity; The protein tag includes any one or more of Poly-Arg, Poly-His, FLAG, Strep-tag II, and c-myc, and the amino acid sequence of the protein tag is located upstream or downstream of the sequence shown in any one of (a)-(c) and fused with it in the same frame.

2. A polynucleotide, characterized in that, The polynucleotide encodes the β-1,6-glucanase according to claim 1; Optionally, the nucleotide sequence of the polynucleotide comprises the sequence shown in SEQ ID NO.2 or, under stringent conditions, hybridizes to the sequence shown in SEQ ID NO.2 and encodes a protein having endonuclease β-1,6-glucanase activity.

3. An expression box, characterized in that, The expression cassette comprises the polynucleotide according to claim 2; Optionally, the expression cassette includes a control element operatively connected to the polynucleotide; Optionally, the control element is a starter, a terminator, and / or an enhancer.

4. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide according to claim 2 or the expression cassette according to claim 3; Optionally, the recombinant vector is pET-28a(+) containing the polynucleotide.

5. A recombinant host cell, characterized in that, The recombinant host cell comprises the polynucleotide according to claim 2, or the expression cassette according to claim 3, or the recombinant vector according to claim 4; Optionally, the recombinant host cell is a bacterium or a fungus.

6. The method for preparing β-1,6-glucanase according to claim 1, characterized in that, The method includes the following steps: S1: Fermentation culture of the recombinant host cell according to claim 5 to induce expression; S2: Collect and purify the β-1,6-glucanase; Optionally, in S2, the β-1,6-glucanase is purified using a Ni affinity chromatography column.

7. A composition, characterized in that, The composition comprises the β-1,6-glucanase and β-1,3-glucanase according to claim 1; Optionally, the β-1,3-glucanase comprises any one or more proteins whose amino acid sequence includes the sequence shown in GenBank numbers AIC93282.1, WP_186966490.1 or AJQ96631.1; Optionally, in the composition, the β-1,6-glucanase and β-1,3-glucanase are combined in an enzyme activity ratio of (0.5~5):(0~5).

8. Use of the β-1,6-glucanase according to claim 1, the polynucleotide according to claim 2, the expression cassette according to claim 3, the recombinant vector according to claim 4, the recombinant host cell according to claim 5, or the composition according to claim 7 in the preparation of soluble yeast glucan.

9. A method for preparing soluble yeast glucan, characterized in that, The method includes the step of hydrolyzing a substrate containing yeast glucan using the β-1,6-glucanase according to claim 1 or the composition according to claim 7.

10. The preparation method according to claim 9, characterized in that, The substrate containing yeast glucan includes any one or more of yeast cell walls, yeast cells, and yeast β-glucan.

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