Cellobiose phosphorylase mutant capable of synthesizing laminobiose

Abstract

The invention provides a cellobiose phosphorylase mutant capable of synthesizing laminobiose, which is characterized in that substrate specificity of cellobiose phosphorylase from thermophilic archaea is changed, wild cellobiose phosphorylase with an amino acid sequence as shown in SEQ ID NO: 2 is used as starting protein, multiple rounds of site-directed mutagenesis are carried out on the starting protein through rational design, and the cellobiose phosphorylase mutant capable of synthesizing laminobiose is obtained. The mutant and a wild type CBP amino acid sequence SEQ ID NO: 2 have at least 90% identity, the mutant has the activity of catalyzing glucose and glucose 1-phosphoric acid to synthesize laminobiose, the mutant is super-heat-resistant laminobiose phosphorylase, the dominant mutant is applied to an in-vitro multi-enzyme system for catalytically producing laminobiose by taking starch as a substrate, and the yield of the laminobiose is improved. And the laminobiose can be synthesized at high temperature, high conversion rate and high efficiency.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a cellobiose phosphorylase mutant capable of synthesizing laminarin. Background Technology

[0002] Laminaribiose is a β-1,3-glycosidic linked reducing disaccharide, a versatile and high-value oligosaccharide. It can be used as a precursor for hyaluronic acid synthesis in the pharmaceutical and cosmetic industries, as a germination promoter and natural preservative in agriculture, as a food additive in the health food industry, and to regulate protein expression in thermophilic bacteria such as Clostridium thermophilum. Traditional methods for preparing laminabiose involve acid hydrolysis or enzymatic hydrolysis of β-1,3 polysaccharides, such as kelp polysaccharides, Poria cocos polysaccharides, lichen polysaccharides, and gel polysaccharides. However, the supply of these natural polysaccharide raw materials is limited, the hydrolysis process is difficult to control, the product separation steps are complex, and the production cost of laminabiose is high. With the development of industrial enzyme biotechnology, scientists have begun to explore enzymatic synthesis of laminabiose. Laminaribiose phosphorylase (LBP, EC2.4.1.31) is a phosphorylase belonging to the glycoside hydrolase (GH)94 family, which reversibly catalyzes the reaction of glucose-1-phosphate and glucose to produce laminabiose. However, there are currently few reported enzymes with LBP activity, and the highest temperature they can tolerate is below 50°C, making them unsuitable for industrial-scale production of laminarin in in vitro biosynthesis systems.

[0003] Among the GH94 family enzymes that catalyze the same substrate as LBP and are frequently reported, cellobiose phosphorylase (CBP, EC 2.4.1.20) is the most commonly reported. CBP reversibly catalyzes the reaction of glucose-1-phosphate and glucose to produce cellobiose. Cellobiose and laminarin are β-1,4 and β-1,3 glycosidic bonds-linked glucosinolates, respectively, showing little difference. Currently, many CBPs derived from thermophilic archaea have been characterized. If the substrate specificity of thermophilic archaea-derived CBPs can be altered to eliminate their activity for cellobiose synthesis and gain activity for laminarin synthesis, then the goal of synthesizing laminarin at high temperatures can be achieved. Therefore, it is urgent to modify the substrate specificity of cellobiose phosphorylases from thermophilic archaea to enable them to prepare laminarin at high temperatures, thereby promoting the industrial production of laminarin. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a cellobiose phosphorylase mutant.

[0005] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned cellobiose phosphorylase mutant.

[0006] A cellobiose phosphorylase mutant, said mutant being selected from any one of the following groups (I)-(V):

[0007] (I) The mutant contains a mutation at at least one of the positions 164, 166, 167, 345, 397, 500, 638, and 701 corresponding to the sequence shown in SEQ ID NO:2, compared to the sequence shown in SEQ ID NO:2;

[0008] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:2;

[0009] (III) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very stringent conditions:

[0010] (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I);

[0011] (b)(a) full-length complementary polynucleotides;

[0012] (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still possesses laminarin phosphorylase activity;

[0013] (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

[0014] Preferably, the above-mentioned cellobiose phosphorylase mutant, the mutant corresponding to the sequence shown in SEQ ID NO:2, has any of the following mutations (d1) to (d36):

[0015] (d1) The mutation at position 164;

[0016] (d2) Mutation at position 166

[0017] (d3) Mutation at position 167

[0018] (d4) Mutation at position 345

[0019] (d5) Mutation at position 397

[0020] (d6) Mutation at position 500

[0021] (d7) Mutation at position 638

[0022] (d8) Mutation at position 701

[0023] (d9) A combined mutation at positions 345 and 500;

[0024] (d10) is a combined mutation at positions 345 and 164;

[0025] (d11) A combined mutation at positions 345 and 166;

[0026] (d12) A combined mutation at positions 345 and 167;

[0027] (d13) A combined mutation at positions 345 and 397;

[0028] (d14) A combined mutation at positions 345 and 638;

[0029] (d15) is a combined mutation at positions 345 and 701;

[0030] (d16) Combination mutations at positions 345, 500, and 701;

[0031] (d17) Mutations at positions 345 and 500 and 164;

[0032] (d18) Mutations at positions 345, 500, and 166;

[0033] (d19) Mutations at positions 345, 500, and 167;

[0034] (d20) mutations at positions 345, 500, and 638;

[0035] (d21) Mutations at positions 345, 500, and 397;

[0036] (d22) Mutations at positions 345 and 500 and positions 701 and 164;

[0037] (d23) Mutations at positions 345 and 500 and positions 701 and 166;

[0038] (d24) Mutations at positions 345 and 500 and positions 701 and 167;

[0039] (d25) mutations at positions 345 and 500 and positions 701 and 638;

[0040] (d26) Mutations at positions 345 and 500 and positions 701 and 397;

[0041] (d27) Combination mutations at positions 345 and 500 and 701 and positions 164 and 166;

[0042] (d28) Combination mutations at positions 345 and 500 and 701 and positions 164 and 167;

[0043] (d29) Mutations at positions 345 and 500 and 701 and positions 164 and 397;

[0044] (d30) is a combination mutation at positions 345 and 500 and 701 and positions 164 and 638;

[0045] (d31) Mutations in combinations of positions 345 and 500 and 701 and 164 and 397 and 166;

[0046] (d32) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167;

[0047] (d33) Combination mutations at positions 345 and 500 and 701 and 164 and 397 and 638;

[0048] (d34) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 166;

[0049] (d35) combines mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638;

[0050] (d36) Combination mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638 and 166;

[0051] (d37) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638 and 166; Optionally, the cellobiose phosphorylase mutant corresponding to the sequence shown in SEQ ID NO:2 has a mutated amino acid at at least one of the following positions:

[0052] N164V, Q166Y, R167F, R345N, H397G, Q500V, E638Y, Q701E.

[0053] Preferably, the above-mentioned cellobiose phosphorylase mutant, wherein the mutant with laminarin phosphorylase activity corresponds to the sequence shown in SEQ ID NO:2, has any of the mutations shown in (m1) to (m36) below:

[0054] (m1)N164V;

[0055] (m2)Q166Y;

[0056] (m3)R167F;

[0057] (m4)R345N;

[0058] (m5)H397G;

[0059] (m6)Q500V;

[0060] (m7)E638Y;

[0061] (m8)Q701E;

[0062] (m9)R345N、Q500V;

[0063] (m10)R345N、N164V;

[0064] (m11)R345N、Q166Y;

[0065] (m12)R345N、R167F;

[0066] (m13)R345N、H397G;

[0067] (m14)R345N、E638Y;

[0068] (m15)R345N、Q701E;

[0069] (m16)R345N、Q500V、Q701E;

[0070] (m17)R345N、Q500V、N164V;

[0071] (m18)R345N、Q500V、Q166Y;

[0072] (m19)R345N、Q500V、R167F;

[0073] (m20)R345N、Q500V、E638Y;

[0074] (m21)R345N、Q500V、H397G;

[0075] (m22)R345N、Q500V、Q701E、N164V;

[0076] (m23)R345N、Q500V、Q701E、Q166Y;

[0077] (m24)R345N、Q500V、Q701E、R167F;

[0078] (m25)R345N, Q500V, Q701E, E638Y;

[0079] (m26)R345N, Q500V, Q701E, H397G;

[0080] (m27)R345N, Q500V, Q701E, N164V, Q166Y;

[0081] (m28)R345N, Q500V, Q701E, N164V, R167F;

[0082] (m29)R345N, Q500V, Q701E, N164V, H397G;

[0083] (m30)R345N, Q500V, Q701E, N164V, E638Y;

[0084] (m31)R345N, Q500V, Q701E, N164V, H397G, Q166Y;

[0085] (m32)R345N, Q500V, Q701E, N164V, H397G, R167F;

[0086] (m33)R345N, Q500V, Q701E, N164V, H397G, E638Y;

[0087] (m34)R345N, Q500V, Q701E, N164V, H397G, R167F, Q166Y;

[0088] (m35)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y;

[0089] (m36)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y, Q166Y;

[0090] More preferably, the cellobiose phosphorylase mutant corresponding to the sequence shown in SEQ ID NO:2 has any of the following mutations:

[0091] (m4)R345N;

[0092] (m9)R345N、Q500V;

[0093] (m16)R345N, Q500V, Q701E;

[0094] (m22)R345N, Q500V, Q701E, N164V;

[0095] (m27)R345N, Q500V, Q701E, N164V, H397G;

[0096] (m32)R345N, Q500V, Q701E, N164V, H397G, R167F;

[0097] (m35)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y;

[0098] (m36)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y, Q166Y.

[0099] An isolated polynucleotide, wherein the polynucleotide encodes the aforementioned cellobiose phosphorylase mutant.

[0100] A recombinant expression vector comprising the aforementioned polynucleotides.

[0101] A recombinant host cell comprising the above-mentioned cellobiose phosphorylase mutant, polynucleotide, or recombinant expression vector.

[0102] A cell culture containing the above-mentioned recombinant host cells.

[0103] A product for preparing laminarin or its derivatives, comprising the above-mentioned cellobiose phosphorylase mutant, polynucleotide, recombinant expression vector, recombinant host cell or cell culture.

[0104] A method for preparing laminarin or a derivative thereof, the method comprising providing the above-mentioned cellobiose phosphorylase mutant, polynucleotide, recombinant expression vector, recombinant host cell, cell culture or product to a reaction system to catalyze the conversion of the substrate into laminarin or a derivative thereof.

[0105] Optionally, the substrate includes starch and its derivatives;

[0106] Optionally, the reaction system is a "one-pot enzymatic method", that is, adding isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase mutant to prepare a four-enzyme reaction system. The enzymatic catalytic pathway includes: isoamylase hydrolyzing the branched chains in starch; starch phosphorylase and glucosidase converting glucose units in debranched starch into glucose-1-phosphate and glucose, respectively; and cellobiose phosphorylase mutant converting glucose and glucose-1-phosphate into laminarin.

[0107] Optionally, the reaction system contains phosphorus; preferably, the amino donor is an inorganic phosphate salt; optionally, the concentration of the substrate is 1–100 mM, preferably 2–20 mM; since inorganic phosphorus is cyclic during the reaction, only a small amount of phosphate buffer needs to be added to start the reaction.

[0108] Preferably, the pH of the reaction system is around 7, and more preferably 6.5 ± 0.5;

[0109] Preferably, the reaction temperature of the reaction system is 40–80°C, and more preferably 50–70°C.

[0110] The above-mentioned cellobiose phosphorylase mutants, polynucleotides, recombinant expression vectors, recombinant host cells or cell cultures are used in the preparation of laminarin or its derivatives.

[0111] Optionally, in the reaction system for preparing laminarin or its derivatives, the substrate includes starch; and / or, in the reaction system for preparing laminarin or its derivatives, the phosphorus donor includes inorganic phosphorus.

[0112] The beneficial effects of this invention are:

[0113] The aforementioned cellobiose phosphorylase mutant capable of synthesizing laminarin, by altering the substrate specificity of cellobiose phosphorylase derived from thermophilic archaea, uses wild-type cellobiose phosphorylase (CBP) with the amino acid sequence shown in SEQ ID NO: 2 as the starting protein. Through rational design, multiple rounds of site-directed mutagenesis are performed on it. The mutant has at least 90% identity with the wild-type CBP amino acid sequence SEQ ID NO: 2, and the mutant has the activity of catalyzing the synthesis of laminarin from glucose and glucose-1-phosphate. It is an ultrathermally resistant laminarin phosphorylase. When the superior mutant is applied to an in vitro multi-enzyme system for the catalytic production of laminarin using starch as a substrate, laminarin can be synthesized at high temperature, with high conversion rate and high efficiency.

[0114] Compared with the prior art, the positive and progressive effects of the present invention are as follows:

[0115] 1. By constructing a series of substrate-specific mutants, a cellobiose phosphorylase mutant that can synthesize laminarin from glucose and glucose-1-phosphate under high temperature conditions was screened. It can maintain good activity under high temperature conditions and is suitable for the production of laminarin in various scenarios.

[0116] 2. The method provided by this invention is a one-pot catalytic process. The reaction begins immediately after the substrate and enzyme are added, without the need for any expensive cofactors. The final product, laminarin, is directly obtained after the reaction. Compared with other existing production methods, the bio-enzymatic preparation method of laminarin provided by this invention has advantages such as suitability for industrial applications, inexpensive raw materials, low production costs, and environmental friendliness, making it suitable for widespread application. Attached Figure Description

[0117] Figure 1 This is the TmCBP mutation process.

[0118] Figure 2 This is a schematic diagram of the in vitro multi-enzyme catalytic pathway for the conversion of starch into laminarin; where: IA is isoamylase, αGP is starch phosphorylase, αG is glucosidase, and CBPm is a cellobiose phosphorylase mutant.

[0119] Figure 3 This describes the in vitro high-temperature synthesis of laminarin from starch treated with 10 g / L IA using multiple enzymes.

[0120] Figure 4 This describes the in vitro multi-enzyme catalysis of high-temperature synthesis of laminarin from starch treated with 50 g / L IA.

[0121] Figure 5 This describes the in vitro multi-enzyme catalysis of high-temperature synthesis of laminarin from starch treated with 100 g / L IA. Detailed Implementation

[0122] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0123] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0124] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0125] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0126] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0127] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which 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 these elements may be combined in any suitable manner in various embodiments.

[0128] In this specification, "optional" and "optionally" mean that the events or circumstances described below may or may not occur, and the description includes both cases where the events or circumstances occur and cases where the events or circumstances do not occur.

[0129] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0130] In this manual, “cellobiose phosphorylase mutant”, “CBPm”, “laminarin phosphorylase”, and “LBP” can be used interchangeably, as they can catalyze the conversion of glucose and glucose-1-phosphate into laminarin.

[0131] In this specification, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., through disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).

[0132] In this specification, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. As used in this invention, "naturally occurring" and "wild-type" are synonyms.

[0133] In this specification, the term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to the "wild type" or "comparative" position. Substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to removing a nucleotide or amino acid occupying a position. Insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0134] In this specification, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In this invention, the term "mutation" refers to an alteration of the nucleotide or amino acid sequence. In one specific embodiment, the term "mutation" refers to "deletion."

[0135] In some embodiments, the "mutation" of this invention may be selected from "conservative mutations." In this invention, the term "conservative mutation" refers to a mutation that maintains the normal function of a protein. A representative example of a conservative mutation is a conserved substitution.

[0136] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0137] In this specification, the terms "sequence identity" or "percentage of identity" in comparisons of two nucleic acids or peptides refer to the percentage of identical sequences or identical sequences when compared and aligned using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection to achieve the highest possible correspondence. In other words, the identity of a nucleotide or amino acid sequence can be defined using a ratio that represents the proportion of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion, assuming the maximum number of identical nucleotides or amino acids and omitting gaps as needed.

[0138] In this specification, the term "recombinant polynucleotide" refers to a polynucleotide having a sequence that is not linked together in nature. Recombinant polynucleotides can be contained in a suitable vector, which can be used for transformation into a suitable host cell. A host cell containing the recombinant polynucleotide is referred to as a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide."

[0139] In this specification, the term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0140] In this specification, the term "expression vector" refers to a DNA construct containing a DNA sequence operatively linked to a suitable control sequence for the expression of a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. Recombinant expression vectors may include, for example, i)

[0141] A collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) structural or coding sequences transcribed into mRNA and translated into proteins; and iii) transcriptional subunits with appropriate transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector, including plasmids, viruses, bacteriophages, and transposons, can be used. Possible vectors used in this invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmids and bacteriophage DNA, from viral DNA such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. Exemplarily, the expression vectors include, but are not limited to, vectors that can be replicated and expressed in prokaryotic cells, such as the pET series, Duet series, pGEX series, pHY300, pHY300PLK, or pQlink series.

[0142] In this specification, the term "host cell" means any cell type that is readily transformed, transfected, transduced, etc., using a mutant polypeptide containing the present invention, a polynucleotide encoding the mutant polypeptide, or a recombinant expression vector. The term "recombinant host cell" is also used.

[0143] The present invention relates to a host cell that differs from the parent cell after the introduction of a multinucleotide or recombinant expression vector encoding a mutant polypeptide, wherein the recombinant host cell is specifically achieved through transformation. The host cell of the present invention can be a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell refers to a prokaryotic cell, specifically, the host cell is derived from microorganisms of the genera *Escherichia*, *Bacillus*, or *Corynebacterium*. In some preferred embodiments, the host cell is derived from *Escherichia*, more preferably *Escherichia coli*, including *Escherichia coli* DH5α, *Escherichia coli* Top10, *Escherichia coli* Trans T1, *Escherichia coli* MC1061, *Escherichia coli* BL21, etc.

[0144] In this specification, the term "cell culture" refers to a combination of cells and cell culture medium, wherein the cells are cultured in a cell culture medium outside the organism.

[0145] The terms "transformation," "transfection," and "transduction" in this invention have the meanings commonly understood by those skilled in the art, referring to the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method of introducing nucleic acids into cells, including but not limited to electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0146] In this specification, "conversion" may also refer to the enzymatic conversion (or bioconversion) of a substrate (one or more) to a corresponding product (one or more). "Conversion percentage" refers to the percentage of substrate converted into a product over a specified period of time under specified conditions. Therefore, the "enzyme activity" or "activity" of a cellobiose phosphorylase mutant polypeptide can be expressed as the "conversion percentage" of substrate to product over a specific time period.

[0147] In this specification, "culture" refers to the growth of a microbial cell population under any suitable conditions (e.g., using liquid, gel, or solid culture media), including but not limited to plate culture, shake flask culture, batch culture, continuous culture, and fed-batch culture, and various culture conditions such as temperature, time, and pH of the culture medium can be appropriately adjusted according to actual conditions.

[0148] In this specification, the terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components removed from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is intended to be defined relatively.

[0149] Unless otherwise defined or clearly indicated by the context, all 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.

[0150] The technical solution of the present invention will be described in detail below:

[0151] The inventors discovered in their previous research that laminarin phosphorylase is not very stable at high temperatures, and there are currently no reports of laminarin phosphorylases that can withstand ultra-high temperatures.

[0152] <First Aspect>

[0153] Based on the above research, in a first aspect of the present invention, a series of heat-resistant cellobiose phosphorylase mutants catalyzing the preparation of laminarin from glucose and glucose-1-phosphate are provided, wherein the mutants are selected from any one of the following groups (I)-(V):

[0154] (I) The mutant contains a mutation at at least one of the positions 164, 166, 167, 345, 397, 500, 638, and 701 corresponding to the sequence shown in SEQ ID NO:2, compared to the sequence shown in SEQ ID NO:2;

[0155] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO:2;

[0156] (III) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very stringent conditions:

[0157] (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I);

[0158] (b)(a) full-length complementary polynucleotides;

[0159] (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still possesses laminarin phosphorylase activity;

[0160] (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

[0161] In some embodiments, the cellobiose phosphorylase mutant corresponds to the sequence shown in SEQ ID NO:2 and has any of the mutations shown in (d1) to (d36):

[0162] (d1) The mutation at position 164;

[0163] (d2) Mutation at position 166

[0164] (d3) Mutation at position 167

[0165] (d4) Mutation at position 345

[0166] (d5) Mutation at position 397

[0167] (d6) Mutation at position 500

[0168] (d7) Mutation at position 638

[0169] (d8) Mutation at position 701

[0170] (d9) A combined mutation at positions 345 and 500;

[0171] (d10) is a combined mutation at positions 345 and 164;

[0172] (d11) A combined mutation at positions 345 and 166;

[0173] (d12) A combined mutation at positions 345 and 167;

[0174] (d13) A combined mutation at positions 345 and 397;

[0175] (d14) A combined mutation at positions 345 and 638;

[0176] (d15) is a combined mutation at positions 345 and 701;

[0177] (d16) Combination mutations at positions 345, 500, and 701;

[0178] (d17) Mutations at positions 345 and 500 and 164;

[0179] (d18) Mutations at positions 345, 500, and 166;

[0180] (d19) Mutations at positions 345, 500, and 167;

[0181] (d20) mutations at positions 345, 500, and 638;

[0182] (d21) Mutations at positions 345, 500, and 397;

[0183] (d22) Mutations at positions 345 and 500 and positions 701 and 164;

[0184] (d23) Mutations at positions 345 and 500 and positions 701 and 166;

[0185] (d24) Mutations at positions 345 and 500 and positions 701 and 167;

[0186] (d25) mutations at positions 345 and 500 and positions 701 and 638;

[0187] (d26) Mutations at positions 345 and 500 and positions 701 and 397;

[0188] (d27) Mutations in combinations of positions 345 and 500 and 701 and positions 164 and 166;

[0189] (d28) Combination mutations at positions 345 and 500 and 701 and positions 164 and 167;

[0190] (d29) Mutations at positions 345 and 500 and 701 and positions 164 and 397;

[0191] (d30) is a combination mutation at positions 345 and 500 and 701 and positions 164 and 638;

[0192] (d31) Mutations in combinations of positions 345 and 500 and 701 and 164 and 397 and 166;

[0193] (d32) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167;

[0194] (d33) Combination mutations at positions 345 and 500 and 701 and 164 and 397 and 638;

[0195] (d34) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 166;

[0196] (d35) combines mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638;

[0197] (d36) Combination mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638 and 166;

[0198] (d37) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638 and 166; Optionally, the cellobiose phosphorylase mutant corresponding to the sequence shown in SEQ ID NO:2 has a mutated amino acid at at least one of the following positions:

[0199] N164V, Q166Y, R167F, R345N, H397G, Q500V, E638Y, Q710E.

[0200] In some specific embodiments, the cellobiose phosphorylase mutant corresponds to the sequence shown in SEQ ID NO:2 and has any of the mutations shown in (m1) to (m36):

[0201] (m1)N164V;

[0202] (m2)Q166Y;

[0203] (m3)R167F;

[0204] (m4)R345N (M1 in this specification);

[0205] (m5)H397G;

[0206] (m6)Q500V;

[0207] (m7)E638Y;

[0208] (m8)Q701E;

[0209] (m9)R345N, Q500V (M2 in this manual);

[0210] (m10)R345N, N164V;

[0211] (m11)R345N、Q166Y;

[0212] (m12)R345N, R167F;

[0213] (m13)R345N, H397G;

[0214] (m14)R345N, E638Y;

[0215] (m15)R345N, Q701E;

[0216] (m16)R345N, Q500V, Q701E (M3 in this manual);

[0217] (m17)R345N, Q500V, N164V;

[0218] (m18)R345N, Q500V, Q166Y;

[0219] (m19)R345N, Q500V, R167F;

[0220] (m20)R345N, Q500V, E638Y;

[0221] (m21)R345N, Q500V, H397G;

[0222] (m22)R345N, Q500V, Q701E, N164V (M4 in this manual);

[0223] (m23)R345N, Q500V, Q701E, Q166Y;

[0224] (m24)R345N, Q500V, Q701E, R167F;

[0225] (m25)R345N, Q500V, Q701E, E638Y;

[0226] (m26)R345N, Q500V, Q701E, H397G;

[0227] (m27)R345N, Q500V, Q701E, N164V, Q166Y;

[0228] (m28)R345N, Q500V, Q701E, N164V, R167F;

[0229] (m29)R345N, Q500V, Q701E, N164V, H397G (M5 in this manual);

[0230] (m30)R345N, Q500V, Q701E, N164V, E638Y(;

[0231] (m31)R345N, Q500V, Q701E, N164V, H397G, Q166Y;

[0232] (m32)R345N, Q500V, Q701E, N164V, H397G, R167F (M6 in this manual);

[0233] (m33)R345N, Q500V, Q701E, N164V, H397G, E638Y;

[0234] (m34)R345N, Q500V, Q701E, N164V, H397G, R167F, Q166Y;

[0235] (m35)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y (M7 in this manual);

[0236] (m36)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y, Q166Y (M8 in this manual);

[0237] In some preferred embodiments, the cellobiose phosphorylase mutant corresponds to the sequence shown in SEQ ID NO:2 and has any of the following mutations:

[0238] (m4)R345N (M1 in this specification);

[0239] (m9)R345N, Q500V (M2 in this manual);

[0240] (m16)R345N, Q500V, Q701E (M3 in this manual);

[0241] (m22)R345N, Q500V, Q701E, N164V (M4 in this manual);

[0242] (m27)R345N, Q500V, Q701E, N164V, H397G (M5 in this manual);

[0243] (m32)R345N, Q500V, Q701E, N164V, H397G, R167F (M6 in this manual);

[0244] (m35)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y (M7 in this manual);

[0245] (m36)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y, Q166Y (M8 in this manual).

[0246] In some embodiments, the cellobiose phosphorylase mutants exhibit a half-life exceeding 10 hours at 70°C, demonstrating good heat resistance and enabling stable, long-term conversion to laminarin. In some specific embodiments, the cellobiose phosphorylase mutants are prepared and used in the form of cells expressing the enzyme, as a crude extract, or as a formulation for isolation or purification. In some exemplary embodiments, the cellobiose phosphorylase mutants are prepared as lyophilized powder, powder form (e.g., acetone powder), or as an enzyme solution. In some preferred embodiments, the cellobiose phosphorylase mutants are in the form of a substantially pure formulation.

[0247] <Second aspect>

[0248] In a second aspect of the invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes a cellobiose phosphorylase mutant as described in the first aspect of this disclosure.

[0249] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0250] The polynucleotide encoding the mutants of the present invention includes: a coding sequence that encodes only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and a non-coding sequence.

[0251] <Third aspect>

[0252] In a third aspect of this disclosure, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the polynucleotide described in the second aspect of this invention.

[0253] In some embodiments, the polynucleotide described in the second aspect is operatively linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide.

[0254] <Fourth Aspect>

[0255] In a fourth aspect of the invention, a recombinant host cell is provided, wherein the recombinant host cell comprises the cellobiose phosphorylase mutant described in the first aspect of the invention, the isolated polynucleotide described in the second aspect of the invention, or the recombinant expression vector described in the third aspect of the invention.

[0256] In some implementations, an expression vector containing a heterologous polynucleotide encoding a cellobiose phosphorylase mutant peptide is introduced into a suitable host cell to express the corresponding cellobiose phosphorylase mutant peptide.

[0257] In some alternative embodiments, the recombinant host cell is derived from microorganisms of the genera Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus, or Corynebacterium.

[0258] In some preferred embodiments, the recombinant host cell is derived from Escherichia coli, Corynebacterium glutamicum, or Bacillus subtilis.

[0259] In some preferred embodiments, the recombinant host cell is derived from *Escherichia coli*.

[0260] <Fifth Aspect>

[0261] In a fifth aspect of the invention, a cell culture comprising the recombinant host cells described in the fourth aspect of the invention is provided.

[0262] <Sixth Aspect>

[0263] In a sixth aspect of the invention, a product is provided for preparing laminarin or a derivative thereof, comprising the cellobiose phosphorylase mutant of the first aspect, the polynucleotide of the second aspect, the recombinant expression vector of the third aspect, the recombinant host cell of the fourth aspect, and the cell culture of the fifth aspect.

[0264] In some alternative implementations, the product may include enzyme preparations, microbial agents, reagent kits, etc.

[0265] <Seventh Aspect>

[0266] In a seventh aspect of the invention, a method for preparing laminarin or a derivative thereof is provided, comprising providing to a reaction system the cellobiose phosphorylase mutant of the first aspect, the polynucleotide of the second aspect, the recombinant expression vector of the third aspect, the recombinant host cell of the fourth aspect, the cell culture of the fifth aspect, or the product of the sixth aspect, to catalyze the conversion of a substrate into laminarin or a derivative thereof.

[0267] This invention provides a method for preparing laminarin using an enzyme-catalyzed reaction. The method is characterized by using starch as a substrate and constructing a multi-enzyme catalytic reaction system by adding isoamylase (EC 3.2.1.68, IA), α-glucanphosphorylase (EC 2.4.1.1, αGP), α-glucosidase (EC 3.2.1.20, αG), and a cellobiose phosphorylase mutant. In this invention, starch is used as the substrate. Starch is catalyzed by isoamylase to obtain debranched starch. The debranched starch is then catalyzed by starch phosphorylase and glucosidase to convert it into glucose-1-phosphate and glucose, respectively. Glucose-1-phosphate and glucose are then catalyzed by the cellobiose phosphorylase mutant to generate laminarin.

[0268] Preferably, the starch is any one or a mixture in any proportion of soluble starch, soluble amylose, soluble amylopectin, starch dextrin, maltodextrin, maltose polysaccharide, and maltose.

[0269] Preferably, the concentration of starch in the enzyme-catalyzed reaction system is 1-200 g / L.

[0270] In a preferred embodiment, when the starch contains α-1,6 glycosidic bonds (e.g., soluble starch, soluble amylopectin, starch dextrin, maltodextrin, maltopolysaccharide), isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase mutants are added to the reaction system.

[0271] Preferably, the starch concentration is 1-500 g / L, more preferably 10-300 g / L, even more preferably 50-250 g / L, and most preferably 200 g / L.

[0272] Preferably, buffer solution, phosphate, and metal ions are also added to the reaction system.

[0273] Those skilled in the art will understand that various buffer solutions can be used in this invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer (e.g., sodium citrate buffer), etc. Preferably, the buffer solution is HEPES buffer. Preferably, the pH of the buffer solution is 5.0-8.0, more preferably 6.0-7.5, and most preferably 6.5. Preferably, the concentration of the buffer solution in the reaction system is 10-500 mM, further preferably 20-150 mM, more preferably 50-120 mM, and most preferably 100 mM.

[0274] Those skilled in the art will understand that various phosphates can be used in this invention, such as potassium phosphate, sodium phosphate, etc. Preferably, the phosphate is potassium phosphate. Preferably, the concentration of phosphate in the reaction system is 1-50 mM, more preferably 2-30 mM, more preferably 5-15 mM, and most preferably 20 mM.

[0275] Those skilled in the art will understand that various metal ions can be used in this invention, such as zinc ions, magnesium ions, manganese ions, etc. Preferably, the metal ion is zinc ion. Preferably, the concentration of zinc ions in the reaction system is 1-20 mM, more preferably 2-15 mM, more preferably 3-10 mM, and most preferably 5 mM.

[0276] In this invention, the mutants of isoamylase, starch phosphorylase, glucosidase, and cellobiose phosphorylase added to the enzyme catalytic reaction system can be in any proportion.

[0277] Preferably, the ratio of the mutant isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase is 0.2:1.5-3:0.5:1-3.

[0278] More preferably, the ratio of the mutants of isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase is 0.2:2.5:0.5:1.8.

[0279] In this invention, various sources of starch phosphorylase, cellobiose phosphorylase, isoamylase, and glucosidase can be used. For example, starch phosphorylase can be derived from *Thermotoga maritima*, *Clostridium thermocellum*, *Thermus thermophilus*, etc., preferably from *Thermotoga maritima*; cellobiose phosphorylase can be derived from *Clostridium thermocellum*, *Thermotoga maritima*, *Acholeplasmalaidlawii*, etc., preferably from *Thermotoga maritima*; isoamylase can be derived from *Sulfolobus tokodaii*, *Clostridium thermocellum*, *Flavobacterium sp.*, etc., preferably from *Sulfolobus tokodaii*; glucosidase can be derived from *Geobacillus stearothermophilus*, *Sulfolobus tokodaii*, *Paecilomyces violaceum*. The glucosidase is preferably derived from *Bacillus thermophilus* (e.g., *lilacinus*). The present invention can also use amylase, cellobiose phosphorylase mutants, isoamylases, and glucosidases with an amino acid sequence having at least 60%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% identity with the various enzymes from the above sources.

[0280] This invention uses starch as a substrate and adds isoamylase, starch phosphorylase, glucosidase, and cellobiose phosphorylase mutant to prepare a four-enzyme reaction system. The enzyme catalytic pathway includes: isoamylase hydrolyzing the branched chains in starch; starch phosphorylase and glucosidase converting glucose units in debranched starch into glucose-1-phosphate and glucose, respectively; and cellobiose phosphorylase mutant converting glucose and glucose-1-phosphate into laminarin.

[0281] Since inorganic phosphorus is cyclical during the reaction, only a small amount of phosphate buffer needs to be added to start the reaction and keep it running. Therefore, the use of phosphate in actual production does not cause environmental stress.

[0282] <Eighth Aspect>

[0283] In an eighth aspect of the invention, the use of the cellobiose phosphorylase mutant of the first aspect, the polynucleotide of the second aspect, the recombinant expression vector of the third aspect, the recombinant host cell of the fourth aspect, the cell culture of the fifth aspect, or the product of the sixth aspect in the preparation of laminarin or its derivatives is provided.

[0284] In some alternative embodiments, the substrate in the reaction system for preparing laminarin or its derivatives includes starch and its derivatives.

[0285] In some alternative embodiments, phosphorus is included in the reaction system for preparing laminarin or its derivatives. Preferably, the amino donor comprises an inorganic phosphate salt, further comprising sodium phosphate, potassium phosphate, magnesium phosphate, zinc phosphate, manganese phosphate, etc.

[0286] 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 in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0287] The following materials are used in the embodiments of this invention:

[0288] Soluble starch, ACROS product, product number: 424490020;

[0289] pET28a vector, Novagen, Madison, WI;

[0290] Escherichia coli expression strain BL21(DE3), Invitrogen, Carlsbad, CA;

[0291] All enzymes in this invention are available from Sigma-Aldrich and can be obtained by prokaryotic expression using genetic engineering methods.

[0292] CBP mutant vector construction method: Primers were designed using the Phusion site-directed mutagenesis method. PCR amplification was performed using either the vector pET20b-cbp containing wild-type CBP or the vector containing the optimal mutant for each round as templates. The PCR reaction mixture consisted of: 25 μL of 2×PrimerStar mixture, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 1 μL of template DNA, and 22 μL of double-distilled water. PCR amplification conditions were: 98℃ pre-denaturation for 45 s; followed by 30 cycles of 98℃ for 15 s, 55℃ for 15 s, and 72℃ for 45 s, with a final incubation at 72℃ for 10 min. The PCR product was digested with Dpn I for 30 min and then purified using a PCR product purification kit. 3 μL of purified PCR product was mixed with 1 μL of T4 PNK, 1 μL of 10×T4 PNK buffer, 1 μL of 100 mM ATP, and 4 μL of double-distilled water, and phosphorylated at 37°C for 2 h. 10 μL of the phosphorylated PCR product solution was then mixed with 1 μL of rapid ligase and 10 μL of rapid ligase buffer, and incubated at 25°C for 10 min for blunt-end ligation. The ligation products were then transformed into competent *E. coli* TOP10 cells, plated on LB agar plates containing antibiotics, and incubated overnight. Single colonies were picked, incubated overnight in LB liquid medium, and plasmids were extracted and sequenced for verification.

[0293] Expression of CBP mutants: BL21(DE3) strains containing the correctly sequenced mutant gene vector were inoculated into LB liquid medium and cultured at 37℃ and 200 rpm until the OD600nm reached 0.6-0.8. IPTG was added to a final concentration of 0.1 mM for induction, and the culture was continued at 25℃ for 12 h. The bacterial cells were collected, and the mutant protein crude enzyme solution was obtained by sonication. The solution was purified by nickel column chromatography, ultrafiltration concentration, and buffer replacement to obtain electrophoretically pure mutant protein.

[0294] Method for determining laminarin:

[0295] High performance liquid chromatography (HPLC) detection method: RID detector, HPX-87H column (Bio-Rad Hercules, CA), mobile phase: 5mM H2SO4, flow rate: 0.6mL / min, column temperature: 55℃, injection volume: 20μL.

[0296] Culture medium (g / L): tryptone 10, yeast extract 5, NaCl 10.

[0297] Example 1: Rational analysis to determine the mutation site

[0298] Using the vector pET20b-tmcbp containing the codon-optimized wild-type CBP encoding gene (as shown in SEQ ID NO:1) as a template, PCR amplification was performed using degenerate primers to construct an expression vector for the CBP mutant. The constructed mutant library was transformed, and bacterial cells were cultured in 250 mL Erlenmeyer flasks to obtain the protein expression supernatant. The supernatant was purified by heat treatment, and the enzyme activity catalyzing the synthesis of cellobiose was determined to be 2.7 U / mg, while the enzyme activity catalyzing the synthesis of laminarin was 0 U / mg. Its thermostability was also determined, showing a half-life exceeding 12 h at 80 °C. The crystal structure of wild-type CBP from Thermotoga maritima was predicted using AlphaFold2. Molecular docking was performed using AutoDock Vina to determine the amino acid sites contained in 21 substrate pockets. Multiple sequence alignment analysis was then performed to identify conserved amino acids. The above 8 non-conserved sites were selected as candidate mutation sites. Through molecular dynamics simulations, FoldX calculations, and substrate pocket analysis of laminarin and cellobiose phosphorylase, the amino acids to be mutated at each site were determined to be N164V, Q166Y, R167F, R345N, H397G, Q500V, E638Y, and Q701E.

[0299] Example 2: First Round of Site-Directed Mutation

[0300] Using the vector pET20b-CBP containing the codon-optimized wild-type CBP encoding gene (as shown in SEQ ID NO:1) as a template, a single-point mutant expression vector for CBP was constructed by PCR amplification using specific primers. The constructed vector was transformed, and bacterial cells were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80°C. As shown in SEQ ID NO:2, the wild-type CBP exhibited no catalytic activity in the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70°C. Mutants were constructed sequentially: CBP-N164V, CBP-Q166Y, CBP-R167F, CBP-R345N, CBP-H397G, CBP-Q500V, CBP-E638Y, and CBP-Q701E. Their activities in catalyzing the synthesis of glucose and glucona-1-phosphate from laminaribose and inorganic phosphorus at 70℃ were 0.1, 0.6, 0.3, 0.9, 0.2, 0.1, 0.2, and 0.2 mU / mg, respectively, essentially losing their native activity for synthesizing cellobiose. Among them, the CBP-R345N mutant exhibited the highest enzyme activity in catalyzing the synthesis of glucose and glucona-1-phosphate from laminaribose and inorganic phosphorus at 70℃, and its stability was comparable to the wild type. The amino acid sequence of mutant CBP-N164V is shown in SEQ ID NO:3; the amino acid sequence of mutant CBP-Q166Y is shown in SEQ ID NO:4; the amino acid sequence of mutant CBP-R167F is shown in SEQ ID NO:5; the amino acid sequence of mutant CBP-R345N is shown in SEQ ID NO:6; the amino acid sequence of mutant CBP-H397G is shown in SEQ ID NO:7; the amino acid sequence of mutant CBP-Q500V is shown in SEQ ID NO:8; the amino acid sequence of mutant CBP-E638Y is shown in SEQ ID NO:9; the amino acid sequence of mutant CBP-Q701E is shown in SEQ ID NO:10; the amino acid sequence of the optimal mutant CBP-R345N obtained in this round is shown in SEQ ID NO:6, and it is named M1.

[0301] Example 3: Combinatorial Mutation-Iterative Process

[0302] Starting with M1, single-point mutations were performed at seven other sites, and PCR amplification was carried out using specific primers to construct expression vectors for other single-point mutants. The constructed vectors were transformed, and bacterial cells were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80°C. The mutant CBP-M1, as shown in SEQ ID NO:6, exhibited an activity of 0.9 mU / mg at 70°C catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona 1-phosphate. Mutants M1-N164V, M1-Q166Y, M1-R167F, M1-H397G, M1-Q500V, M1-E638Y, and M1-Q701E were constructed sequentially. Their activities in catalyzing the synthesis of glucose and glucona-1-phosphate from laminaribose and inorganic phosphorus at 70℃ were 1.1, 1.3, 1.7, 4.2, 2.1, 1.2, and 2.2 mU / mg, respectively, completely losing their native activity for synthesizing cellobiose. Among them, the M1-Q500V mutant exhibited the highest enzyme activity in catalyzing the synthesis of glucose and glucona-1-phosphate from laminaribose and inorganic phosphorus at 70℃, and its stability was comparable to the wild type. The amino acid sequence of mutant M1-N164V is shown in SEQ ID NO:11;

[0303] The amino acid sequence of M1-Q166Y is shown in SEQ ID NO:12; the amino acid sequence of mutant M1-R167F is shown in SEQ ID NO:13; the amino acid sequence of mutant M1-H397G is shown in SEQ ID NO:14; the amino acid sequence of mutant M1-Q500V is shown in SEQ ID NO:15; the amino acid sequence of mutant M1-E638Y is shown in SEQ ID NO:16; the amino acid sequence of mutant M1-Q701E is shown in SEQ ID NO:17; the amino acid sequence of the optimal mutant M1-Q500V obtained in this round is shown in SEQ ID NO:15, and it is named M2.

[0304] Then, starting with M2, single-point mutations were performed at six other sites, and PCR amplification was carried out using specific primers to construct expression vectors for other single-point mutants. The constructed vectors were transformed, and bacteria were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80℃. The mutant CBP-M2, as shown in SEQ ID NO:15, exhibited an activity of 4.2 mU / mg in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃. Mutants M2-N164V, M2-Q166Y, M2-R167F, M2-H397G, M2-E638Y, and M2-Q701E were constructed sequentially. Their activities in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃ were 7.1, 6.3, 5.7, 5.2, 9.2, and 12.2 mU / mg, respectively, completely losing their native activity in synthesizing cellobiose. Among them, the M2-Q701E mutant exhibited the highest enzyme activity at 70℃ in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate, and its stability was comparable to that of the wild type. The amino acid sequence of mutant M2-N164V is shown in SEQ ID NO:18; the amino acid sequence of mutant M2-Q166Y is shown in SEQ ID NO:19; the amino acid sequence of mutant M2-R167F is shown in SEQ ID NO:20; the amino acid sequence of mutant M2-H397G is shown in SEQ ID NO:21; the amino acid sequence of mutant M2-E638Y is shown in SEQ ID NO:22; the amino acid sequence of mutant M2-Q701E is shown in SEQ ID NO:23; the optimal mutant obtained in this round, M2-Q701E, has the amino acid sequence shown in SEQ ID NO:23 and is named M3.

[0305] Then, starting with M3, single-point mutations were performed at five other sites, and PCR amplification was carried out using specific primers to construct expression vectors for other single-point mutants. The constructed vectors were transformed, and bacteria were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80℃. The mutant CBP-M3, as shown in SEQ ID NO:23, exhibited an activity of 12.2 mU / mg in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃. Mutants M3-N164V, M3-Q166Y, M3-R167F, M3-H397G, and M3-E638Y were constructed sequentially. Their activities in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃ were 37.1, 26.3, 15.7, 19.2, and 22.2 mU / mg, respectively, completely losing their native activity in synthesizing cellobiose. Among them, the M3-N164V mutant exhibited the highest enzyme activity at 70℃ in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate, with stability comparable to the wild type. The amino acid sequences of mutant M3-N164V are shown in SEQ ID NO:24; mutant M3-Q166Y in SEQ ID NO:25; mutant M3-R167F in SEQ ID NO:26; mutant M3-H397G in SEQ ID NO:27; and mutant M3-E638Y in SEQ ID NO:28. The optimal mutant obtained in this round, M3-N164V, has the amino acid sequence shown in SEQ ID NO:24 and is named M4.

[0306] Then, starting with M4, single-point mutations were performed at the other four sites, and PCR amplification was carried out using specific primers to construct expression vectors for other single-point mutants. The constructed vectors were transformed, and bacteria were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80℃. The mutant CBP-M4, as shown in SEQ ID NO:24, exhibited an activity of 37.1 mU / mg in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃. Mutants M4-Q166Y, M4-R167F, M4-H397G, and M4-E638Y were constructed sequentially. Their activities in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃ were 77.1, 46.3, 109.2, and 62.2 mU / mg, respectively, completely losing their native activity in synthesizing cellobiose. Among them, the M4-H397G mutant exhibited the highest enzyme activity at 70℃ in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate, and its stability was comparable to that of the wild type. The amino acid sequence of mutant M4-Q166Y is shown in SEQ ID NO:29; the amino acid sequence of mutant M4-R167F is shown in SEQ ID NO:30; the amino acid sequence of mutant M4-H397G is shown in SEQ ID NO:31; the amino acid sequence of mutant M4-E638Y is shown in SEQ ID NO:32. The optimal mutant obtained in this round, M4-H397G, has the amino acid sequence shown in SEQ ID NO:31 and is named M5.

[0307] Then, starting with M5, single-point mutations were performed on the other three sites, and PCR amplification was carried out using specific primers to construct expression vectors for other single-point mutants. The constructed vectors were transformed, and bacteria were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80℃. The mutant CBP-M5, as shown in SEQ ID NO:31, exhibited an activity of 37.1 mU / mg in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃. Mutants M5-Q166Y, M5-R167F, and M5-E638Y were constructed sequentially, exhibiting activities of 116.3, 317.2, and 122.2 mU / mg, respectively, in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃, completely losing their native activity in synthesizing cellobiose. Among them, the M5-R167F mutant exhibited the highest enzyme activity at 70℃ in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate, and its stability was comparable to that of the wild type. The amino acid sequence of mutant M5-Q166Y is shown in SEQ ID NO:33; the amino acid sequence of mutant M5-R167F is shown in SEQ ID NO:34; the amino acid sequence of mutant M5-E638Y is shown in SEQ ID NO:35, and the optimal mutant obtained in this round, M5-R167F, has the amino acid sequence shown in SEQ ID NO:34 and is named M6.

[0308] Then, starting with M6, single-point mutations were performed on the other two sites, and PCR amplification was carried out using specific primers to construct expression vectors for other single-point mutants. The constructed vectors were transformed, and bacteria were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80℃. The mutant CBP-M6, as shown in SEQ ID NO:34, exhibited an activity of 37.1 mU / mg at 70℃ in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate. Mutants M6-Q166Y and M6-E638Y were constructed sequentially, exhibiting activities of 617.2 and 1024.2 mU / mg, respectively, in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃, completely losing their native activity in synthesizing cellobiose. Among them, the M6-E638Y mutant showed the highest enzyme activity in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃, and its stability was comparable to the wild type. The amino acid sequence of mutant M6-Q166Y is shown in SEQ ID NO:36; the amino acid sequence of mutant M6-E638Y is shown in SEQ ID NO:37; and the amino acid sequence of the optimal mutant M6-E638Y obtained in this round is shown in SEQ ID NO:37, and it is named M7.

[0309] Then, starting with M7, single-point mutations were performed at one other site, followed by PCR amplification using specific primers to construct expression vectors for other single-point mutants. The constructed vectors were transformed, and bacteria were cultured in disposable culture tubes to obtain the mutant protein expression supernatant, which was then purified by heat treatment at 80℃. The mutant CBP-M7, as shown in SEQ ID NO:37, exhibited an activity of 1024.1 mU / mg in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃. The mutant M7-Q166Y was constructed, exhibiting an activity of 2119.8 mU / mg in catalyzing the conversion of laminarin and inorganic phosphorus to glucose and glucona-1-phosphate at 70℃, completely losing its native activity in synthesizing cellobiose, and exhibiting stability comparable to the wild type. The amino acid sequence of the mutant M7-Q166Y is shown in SEQ ID NO:38, and it is named M8. The combinatorial mutagenesis process is as follows... Figure 1 As shown.

[0310] Example 4: Preparation of laminarin from 10 g / L starch at 70 °C using multi-enzyme catalysis

[0311] The catalytic pathway for converting starch to laminarin via an in vitro multi-enzyme catalytic system is described in [link to relevant documentation]. Figure 2 The key enzymes and steps involved include: (1) starch phosphorylase (αGP, EC 2.4.1.1), used to release glucose-1-phosphate from starch; (2) glucosidase (αG, EC 3.2.1.20), used to release glucose from starch; and (3) cellobiose phosphorylase mutant (CBPm), used to catalyze the reaction of glucose-1-phosphate and glucose to produce laminarin. Isoamylase can assist in the hydrolysis of starch, that is, adding isoamylase (IA, EC 3.2.1.68), which can help hydrolyze starch, to the reaction system can increase the yield of laminarin.

[0312] In this embodiment, the reaction system contained 100 mM HEPES buffer (pH 6.5), 5 mM divalent zinc ions, 20 mM potassium phosphate (pH 6.5), 0.2 U / mL isoamylase, 2.5 U / mL starch phosphorylase, 0.5 U / mL glucosidase, 1.2 U / mL cellobiose phosphorylase mutant M8, and 10 g / L starch. The reaction was carried out at 70°C for 12 hours.

[0313] The concentration of laminarin was determined by high-performance liquid chromatography (HPLC). 94.5 μL of the reaction sample was taken, and 5.5 μL of 10% sulfuric acid was added to terminate the reaction. The supernatant was collected by centrifugation, and the concentration of laminarin was calculated by determining the peak area and peak height using HPLC.

[0314] After the reaction was completed, the final concentration of laminarin produced by the CBP mutant M8 catalysis was measured. Figure 3 The concentration is 22.4 mM, and the conversion rate relative to starch (10 g / L, approximately 55.5 mM glucose equivalent, 2 glucose equivalents to synthesize 1 molecule of laminabiose) is 80.7%.

[0315] Example 5: Preparation of laminarin from 50 g / L starch at 70 °C using multi-enzyme catalysis

[0316] In this embodiment, the reaction system contained 100 mM HEPES buffer (pH 6.5), 5 mM divalent zinc ions, 20 mM potassium phosphate (pH 6.5), 1 U / mL isoamylase, 12.5 U / mL starch phosphorylase, 2.5 U / mL glucosidase, 6 U / mL cellobiose phosphorylase mutant M8, and 50 g / L starch. The reaction was carried out at 70°C for 12 hours.

[0317] The concentration of laminarin was determined by high-performance liquid chromatography (HPLC). 94.5 μL of the reaction sample was taken, and 5.5 μL of 10% sulfuric acid was added to terminate the reaction. The supernatant was collected by centrifugation, and the concentration of laminarin was calculated by determining the peak area and peak height using HPLC.

[0318] After the reaction was completed, the final concentration of laminarin produced by the CBP mutant M8 catalysis was measured. Figure 4 The concentration is 111.5 mM, and the conversion rate relative to starch (50 g / L, approximately 277.5 mM glucose equivalent, 2 glucose equivalents to synthesize 1 molecule of laminabiose) is 80.4%.

[0319] Example 6: Preparation of laminarin from 100 g / L starch at 70 °C using multi-enzyme catalysis

[0320] In this embodiment, the reaction system contained 100 mM HEPES buffer (pH 6.5), 5 mM divalent zinc ions, 20 mM potassium phosphate (pH 6.5), 2 U / mL isoamylase, 25 U / mL starch phosphorylase, 5 U / mL glucosidase, 12 U / mL cellobiose phosphorylase mutant M8, and 100 g / L starch. The reaction was carried out at 70°C for 12 hours.

[0321] The concentration of laminarin was determined by high-performance liquid chromatography (HPLC). 94.5 μL of the reaction sample was taken, and 5.5 μL of 10% sulfuric acid was added to terminate the reaction. The supernatant was collected by centrifugation, and the concentration of laminarin was calculated by determining the peak area and peak height using HPLC.

[0322] After the reaction was completed, the final concentration of laminarin produced by the CBP mutant M8 catalysis was measured. Figure 5The concentration is 222.3 mM, and the conversion rate of starch (100 g / L, approximately 555 mM glucose equivalent, 2 glucose equivalents to synthesize 1 molecule of laminabiose) is 80.1%.

[0323] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A cellobiose phosphorylase mutant, characterized in that: The mutant is selected from any one of the following groups (I) to (V): (I) The mutant contains a mutation at at least one of the positions 164, 166, 167, 345, 397, 500, 638, and 701 corresponding to the sequence shown in SEQ ID NO: 2, compared to the sequence shown in SEQ ID NO: 2; (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2; (III) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very stringent conditions: (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (I); (b)(a) full-length complementary polynucleotides; (IV) A fragment of a mutant shown in any one of (I), (II) or (III), wherein the fragment still possesses laminarin phosphorylase activity; (V) A polypeptide with an amino acid sequence as shown in (I), (II), (III) or (IV) having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus.

2. The cellobiose phosphorylase mutant according to claim 1, characterized in that: The mutant corresponds to the sequence shown in SEQ ID NO: 2 and has any of the mutations shown in (d1) to (d36) below: (d1) The mutation at position 164; (d2) Mutation at position 166 (d3) Mutation at position 167 (d4) Mutation at position 345 (d5) Mutation at position 397 (d6) Mutation at position 500 (d7) Mutation at position 638 (d8) Mutation at position 701 (d9) A combined mutation at positions 345 and 500; (d10) is a combined mutation at positions 345 and 164; (d11) A combined mutation at positions 345 and 166; (d12) A combined mutation at positions 345 and 167; (d13) A combined mutation at positions 345 and 397; (d14) A combined mutation at positions 345 and 638; (d15) is a combined mutation at positions 345 and 701; (d16) Combination mutations at positions 345, 500, and 701; (d17) Mutations at positions 345 and 500 and 164; (d18) Mutations at positions 345 and 500 and 166; (d19) Mutations at positions 345, 500, and 167; (d20) is a combination mutation at positions 345, 500, and 638; (d21) Mutations at positions 345, 500, and 397; (d22) Mutations at positions 345 and 500 and positions 701 and 164; (d23) Mutations at positions 345 and 500 and positions 701 and 166; (d24) Mutations at positions 345 and 500 and positions 701 and 167; (d25) mutations at positions 345 and 500 and positions 701 and 638; (d26) Mutations at positions 345 and 500 and positions 701 and 397; (d27) Mutations in combinations of positions 345 and 500 and 701 and positions 164 and 166; (d28) Combination mutations at positions 345 and 500 and 701 and positions 164 and 167; (d29) Mutations at positions 345 and 500 and 701 and positions 164 and 397; (d30) is a combination mutation at positions 345 and 500 and 701 and positions 164 and 638; (d31) Mutations in combinations of positions 345 and 500 and 701 and 164 and 397 and 166; (d32) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167; (d33) Combination mutations at positions 345 and 500 and 701 and 164 and 397 and 638; (d34) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 166; (d35) combines mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638; (d36) Combination mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638 and 166; (d37) Combinations of mutations at positions 345 and 500 and 701 and 164 and 397 and 167 and 638 and 166; Optionally, the cellobiose phosphorylase mutant corresponding to the sequence shown in SEQ ID NO: 2 has a mutated amino acid at at least one of the following positions: N164V, Q166Y, R167F, R345N, H397G, Q500V, E638Y, Q701E.

3. The cellobiose phosphorylase mutant according to claim 1 or 2, characterized in that: The mutant with laminarin phosphorylase activity corresponds to the sequence shown in SEQ ID NO: 2 and has any of the mutations shown in (m1) to (m36) below: (m1)N164V; (m2)Q166Y; (m3)R167F; (m4)R345N; (m5)H397G; (m6)Q500V; (m7)E638Y; (m8)Q701E; (m9)R345N、Q500V; (m10)R345N, N164V; (m11)R345N、Q166Y; (m12)R345N, R167F; (m13)R345N, H397G; (m14)R345N, E638Y; (m15)R345N, Q701E; (m16)R345N, Q500V, Q701E; (m17)R345N, Q500V, N164V; (m18)R345N, Q500V, Q166Y; (m19)R345N, Q500V, R167F; (m20)R345N, Q500V, E638Y; (m21)R345N, Q500V, H397G; (m22)R345N, Q500V, Q701E, N164V; (m23)R345N, Q500V, Q701E, Q166Y; (m24)R345N, Q500V, Q701E, R167F; (m25)R345N, Q500V, Q701E, E638Y; (m26)R345N, Q500V, Q701E, H397G; (m27)R345N, Q500V, Q701E, N164V, Q166Y; (m28)R345N, Q500V, Q701E, N164V, R167F; (m29)R345N, Q500V, Q701E, N164V, H397G; (m30)R345N, Q500V, Q701E, N164V, E638Y; (m31)R345N, Q500V, Q701E, N164V, H397G, Q166Y; (m32)R345N, Q500V, Q701E, N164V, H397G, R167F; (m33)R345N, Q500V, Q701E, N164V, H397G, E638Y; (m34)R345N, Q500V, Q701E, N164V, H397G, R167F, Q166Y; (m35)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y; (m36)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y, Q166Y; More preferably, the cellobiose phosphorylase mutant corresponding to the sequence shown in SEQ ID NO: 2 has any of the following mutations: (m4)R345N; (m9)R345N、Q500V; (m16)R345N, Q500V, Q701E; (m22)R345N, Q500V, Q701E, N164V; (m27)R345N, Q500V, Q701E, N164V, H397G; (m32)R345N, Q500V, Q701E, N164V, H397G, R167F; (m35)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y; (m36)R345N, Q500V, Q701E, N164V, H397G, R167F, E638Y, Q166Y.

4. A polynucleotide, characterized in that: The polynucleotide encodes a cellobiose phosphorylase mutant according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide of claim 4.

6. A recombinant host cell, characterized in that: The recombinant host cell comprises the cellobiose phosphorylase mutant of any one of claims 1 to 3, the polynucleotide of claim 4, or the recombinant expression vector of claim 5.

7. A cell culture comprising the recombinant host cell of claim 6.

8. A product for preparing laminarin or its derivatives, characterized in that: It comprises the cellobiose phosphorylase mutant of any one of claims 1 to 3, the polynucleotide of claim 4, the recombinant expression vector of claim 5, the recombinant host cell of claim 6, or the cell culture of claim 7.

9. A method for preparing laminarin or its derivatives, characterized in that: The method includes providing a cellobiose phosphorylase mutant according to any one of claims 1 to 3, a polynucleotide according to claim 4, a recombinant expression vector according to claim 5, a recombinant host cell according to claim 6, a cell culture according to claim 7, or a product according to claim 8 to a reaction system to catalyze the conversion of a substrate into lamina monosaccharide or a derivative thereof. Optionally, the substrate includes starch and its derivatives; Optionally, the reaction system is a "one-pot enzymatic method", that is, adding isoamylase, starch phosphorylase, glucosidase and cellobiose phosphorylase mutant to prepare a four-enzyme reaction system. The enzymatic catalytic pathway includes: isoamylase hydrolyzing the branched chains in starch; starch phosphorylase and glucosidase converting glucose units in debranched starch into glucose-1-phosphate and glucose, respectively; and cellobiose phosphorylase mutant converting glucose and glucose-1-phosphate into laminarin. Optionally, the reaction system contains phosphorus; preferably, the amino donor is an inorganic phosphate salt; optionally, the concentration of the substrate is 1–100 mM, preferably 2–20 mM; since inorganic phosphorus is cyclic during the reaction, only a small amount of phosphate buffer needs to be added to start the reaction. Preferably, the pH of the reaction system is around 7, and more preferably 6.5 ± 0.5; Preferably, the reaction temperature of the reaction system is 40–80°C, and more preferably 50–70°C.

10. The use of the cellobiose phosphorylase mutant of any one of claims 1 to 3, the polynucleotide of claim 4, the recombinant expression vector of claim 5, the recombinant host cell of claim 6, the cell culture of claim 7, or the product of claim 8 in the preparation of laminarin or its derivatives; Optionally, in the reaction system for preparing laminarin or its derivatives, the substrate includes starch; and / or, in the reaction system for preparing laminarin or its derivatives, the phosphorus donor includes inorganic phosphorus.

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