Sucrose phosphorylase mutant and application thereof

Abstract

The invention discloses a sucrose phosphorylase mutant, the amino acid sequence of the sucrose phosphorylase mutant is SEQ ID NO: 3 or SEQ ID NO: 5, and the sucrose phosphorylase mutant is obtained by site-directed mutagenesis on the basis of sucrose phosphorylase from microorganism Bifidobacterium thermophilum. Compared with sucrose phosphorylase from other sources, the sucrose phosphorylase mutant provided by the invention can catalyze a high-concentration substrate, has higher catalytic activity, wider substrate range, higher yield and higher-purity product 2-alpha-GG, and can be used for industrial production of glycerol glucoside.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically, it relates to a sucrose phosphorylase mutant and its application, especially in the synthesis of 2-α-glyceroglucoside. Background Technology

[0002] Glucosylglycerol (GG) is a class of substances composed of glycerol and glucose molecules linked by glycosidic bonds. It is the main active ingredient of the miraculous desert plant *Myrothamnus fruticosa*. Due to differences in the configuration of the glucose molecule and the position of the glycerol molecule, there are many isomers of glutoside. Among them, 2-O-α-D-glutoside (2-α-glutoside, or 2-αGG) is the most studied, and its structural formula is shown below:

[0003]

[0004] 2-αGG is a product of glucosylation of the second hydroxyl group on a glycerol molecule, i.e., coupling a D-glucopyranose to the second hydroxyl group of glycerol via a glycosidic bond. Its chemical name is α-D-glucopyranosyl-α-(1-2,2)-propanetriol. It is an osmotic protectant spontaneously synthesized by microorganisms under stress conditions, protecting cells from harsh environments such as high osmotic pressure, high temperature, drought, and ultraviolet radiation. Like many osmotic agents, 2-αGG has strong stabilizing effects and can be used as an additive to protect proteins from inactivation at high temperatures or during freeze-drying, or to enhance the soluble expression of easily aggregated proteins. 2-αGG is a multifunctional active cosmetic ingredient with powerful moisturizing, anti-aging, soothing, repairing, and cell-activating properties. It is suitable not only for daily skincare but also for anti-fatigue, environmental stress relief (such as UV radiation and PM2.5 pollution mitigation), anti-aging, anti-sensitivity, and anti-eczema effects. It is also non-cariogenic and can be added to food as a non-cariogenic sweetener. Furthermore, it is a stabilizer for macromolecules, used for the long-term preservation of protein drugs. In addition, research has found it to have health benefits such as treating allergic respiratory diseases, protecting the conjunctiva, lowering blood sugar, and controlling visceral fat accumulation. Therefore, the properties and physiological functions of 2-αGG, such as high moisturizing properties, low water absorption, low sweetness, and anti-tumor activity, show great application potential in cosmetics, health care products, food, enzyme production, and pharmaceuticals, possessing significant industrial value and market prospects. However, due to low production efficiency and high production costs, the market price of 2-αGG is high and its supply is limited.

[0005] Currently, the main methods for synthesizing 2-αGG include chemical synthesis, fermentation synthesis, and enzymatic synthesis. Chemical synthesis produces a mixture of different isomers with extremely low 2-αGG content (below 18%), requiring complex multi-step purification processes and thus having low industrial value. Fermentation methods obtain glycerol glucoside through bacterial or cyanobacterial secretion, yielding high-purity 2-αGG but with low yields (typically not exceeding 2 g / L). In contrast, enzymatic synthesis is low-cost and highly efficient, suitable for rapid large-scale production and market promotion. Currently, sucrose phosphorylase (Spase) is widely used in the enzymatic synthesis of glycerol glucoside. The substrates are generally sucrose and glycerol, and a one-step enzymatic catalysis can efficiently synthesize 2αGG, making it the most promising method for industrial production.

[0006] Sucrose phosphorylase belongs to the GH13 family of glycoside hydrolases and catalyzes the specific transfer of glucose groups, enabling the glycosylation of small molecule compounds. Compared to other enzymes capable of transglycosylation, sucrose phosphorylase has advantages such as readily available and inexpensive glycosyl donor substrates, a wide range of glycosyl acceptor substrates, high regioselectivity, and non-hydrolysis of glycosylated products. Currently, various sucrose phosphorylases from different sources have been disclosed for use in the production of glycerol glucosides. For example, patents CN111172127A and CN110358750B disclose sucrose phosphorylase and its mutants from *Lactobacillus reuteri*; patent CN109988799B discloses sucrose phosphorylase and its mutants from *L. pseudodomesenteroides*; patents CN107858335B and CN109423485B disclose sucrose phosphorylase and its mutants from *Bifidobacterium adolescentis*; and patent CN110438100B discloses sucrose phosphorylase derived from *Streptococcus*. Sucrose phosphorylase derived from *Mutans UA159*, *Leuconostocmesenteroides*, *Zhongshania aliphaticivorans*, *Marinobacter psychrophilus*, or *Marinobacter adhaerens HP15*.

[0007] However, current common biocatalytic methods for producing glycerol glucoside suffer from incomplete sucrose conversion and difficulty in removing it later, resulting in low product content. Furthermore, the enzyme's spatial selectivity is poor during the reaction, generating not only 2-α-GG but also a large amount of 1-α-GG, leading to low product purity. Therefore, finding a novel source of sucrose phosphorylase with high activity and improving its stability and other properties for the catalytic synthesis of 2-α-glycerol glucoside in aqueous solution has extremely high industrial application value. Summary of the Invention

[0008] To develop a method capable of catalyzing the one-step enzymatic reaction of glycerol and sucrose to produce glyceroglucoside, significantly reducing the content of the byproduct 1-αGG or significantly increasing the purity of 2-αGG, the inventors conducted extensive screening of sucrose phosphorylases and unexpectedly discovered a sucrose phosphorylase derived from *Bifidobacterium thermophilum* (Uniport ID accession number A0A2N3QNR8, named Spase5 in this paper), which can catalyze the production of glyceroglucoside using glycerol and sucrose as substrates. Based on this enzyme, this invention, through codon optimization and further directed evolution, obtained two sucrose phosphorylase mutants with promising industrial applications.

[0009] Specifically, the present invention includes the following technical solutions.

[0010] The first objective of this invention is to provide a sucrose phosphorylase mutant with the following amino acid sequence:

[0011] SEQ ID NO:3 is a mutant of SEQ ID NO:1 in which P is replaced by Q at position 134; or

[0012] SEQ ID NO:5 is a mutant of SEQ ID NO:1 in which L is replaced with W at position 341.

[0013] The sequence of SEQ ID NO:3 is as follows:

[0014] MKNKVQLITYADRLGEGTIKSLTDVLRTRFDGVYEGVHILPFFTPFDGADAGFDPVDHTKVDPRLGTWDDIAELSKTHDIMVDTIVNHMSWESKQFQDVMKRGEDSPYYPMFLTMSSVFPDGATEEDLAGIYRQRPGLPFTHYTWGGKTRLVWTTFTPQQVDIDTDSKEGWDYLLSILDQLSRSHVSYIRLDAVGYGAKQAKTSCFMTPKTFDLIGRIKAEAESRGLETLIEVHSYYKKQVAIAGKVDRVYDFAIPGLLLHALTTGKTGPIAKWVEVRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEEVDQLVETIHENTHGESRAATGAAASNLDLYQVNSTYYSALGCNDQHYLAARAVQFFLPGVPQVYYVGALAGVNDMELLHRTNVGRDINRHYYSVEEIDRNLERPVVKALNALCRMRNQLDAFDGEFTFSHEGDTLTFDWKGETTSASLTFEPKRGLGVDNQASVCTLRWSDTAGEHETDDLLANPPQVA。

[0015] Among them, the sequence of SEQ ID NO:5 is:

[0016] MKNKVQLITYADRLGEGTIKSLTDVLRTRFDGVYEGVHILPFFTPFDGADAGFDPVDHTKVDPRLGTWDDIAELSKTHDIMVDTIVNHMSWESKQFQDVMKRGEDSPYYPMFLTMSSSVFPDGATEE DLAGIYRPRPGLPFTHYTWGGKTRLVWTTFTPQQVDIDTDSKEGWDYLLSILDQLSRSHVSYIRLDAVGYGAKQAKTSCFMTPKTFDLIGRIKAEAESRGLETLIEVHSYYKKQVAIAGKVDRVYD FAIPGLLLHALTTGKTGPIAKWVEVRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEEVDQLVETIHENTHGESRAATGAAASNWDLYQVNSTYYSALGCNDQHYLAARAVQFFLPGVPQVY YVGALAGVNDMELLHRTNVGRDINRHYYSVEEIDRNLERPVVKALNALCRMRNQLDAFDGEFTFSHEGDTLTFDWKGETTSASLTFEPKRGLGVDNQASVCTLRWSDTAGEHETDDLLANPPQVA.

[0017] A second objective of this invention is to provide a gene encoding the aforementioned sucrose phosphorylase mutant.

[0018] Preferably, the gene encoding the above-mentioned sucrose phosphorylase mutant has the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.6.

[0019] The sequence of SEQ ID NO:4 is as follows:

[0020]

[0021] The sequence of SEQ ID NO:6 is as follows:

[0022]

[0023] A third objective of this invention is to provide an expression vector containing the aforementioned genes.

[0024] In one or more embodiments, the expression vector is a plasmid. Preferably, the vector is selected from pET-28a(+) plasmid, pET-28b(+) plasmid, or pET-20b(+) plasmid.

[0025] A fourth object of the present invention is to provide a host cell comprising the above-described sucrose phosphorylase mutant.

[0026] In one or more embodiments, the host cell is a microorganism. Preferably, the host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yersinia lipolytica, Bacillus subtilis, etc.

[0027] The fifth aspect of this invention aims to provide the application of the above-mentioned sucrose phosphorylase mutant or the above-mentioned host cell in the enzymatic synthesis of glycerol glucoside, especially in the enzymatic synthesis of 2-α-glycerol glucoside.

[0028] The sixth aspect of this invention aims to provide a method for preparing glycerol glucoside, the method comprising the step of adding the above-mentioned sucrose phosphorylase mutant or an expression system containing the enzyme to a reaction system containing the substrate glycerol and sucrose for reaction.

[0029] In one or more embodiments, the glycerol concentration in the reaction system is 1.5–3.5 mol / L; the sucrose concentration is 0.8–1.6 mol / L; the pH is 5.5–7.5; and the reaction temperature is 25–37°C.

[0030] This invention, based on wild-type sucrose phosphorylase from the microorganism *Bifidobacterium thermophilum*, modifies the molecular structure of sucrose phosphorylase through site-directed mutagenesis, ultimately obtaining a sucrose phosphorylase mutant with significantly increased catalytic activity. Compared to sucrose phosphorylases from other sources, the sucrose phosphorylase mutant provided by this invention can catalyze high concentrations of substrates, exhibiting higher catalytic activity, a broader substrate range, higher yield, and higher purity of the product 2-α-GG, which can be used for the industrial production of glycerol glucoside. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of plasmid pET28a-Spase5, constructed in this invention for expressing the wild-type enzyme sucrose phosphorylase Spase5.

[0032] Figure 2This is an HPLC chromatogram of the reaction process catalyzed by wild-type sucrose phosphorylase to produce glycerol glucoside from sucrose and glycerol.

[0033] Figure 3 This is an HPLC chromatogram of the reaction process in which the sucrose phosphorylase mutant, as shown in SEQ ID NO:3, catalyzes the reaction of sucrose and glycerol to produce glycerol glucoside.

[0034] Figure 4 This is an HPLC chromatogram of the reaction process in which the sucrose phosphorylase mutant catalyzes the formation of glycerol glucoside from sucrose and glycerol, as shown in SEQ ID NO:5. Detailed Implementation

[0035] The inventors conducted extensive screening of sucrose phosphorylases and unexpectedly discovered a sucrose phosphorylase (Uniport ID accession number A0A2N3QNR8, named Spase5 in this paper) derived from *Bifidobacterium thermophilum*, which can catalyze the production of glycerol glucoside from glycerol and sucrose as substrates. The amino acid sequence of this wild-type Spase5 (SEQ ID NO:1) is shown below:

[0036] MKNKVQLITYADRLGEGTIKSLTDVLRTRFDGVYEGVHILPFFTPFDGADAGFDPVDHTKVDPRLGTWDDIAELSKTHDIMVDTIVNHMSWESKQFQDVMKRGEDSPYYPMFLTMSSSVFPDGATEE DLAGIYRPRPGLPFTHYTWGGKTRLVWTTFTPQQVDIDTDSKEGWDYLLSILDQLSRSHVSYIRLDAVGYGAKQAKTSCFMTPKTFDLIGRIKAEAESRGLETLIEVHSYYKKQVAIAGKVDRVYD FAIPGLLLHALTTGKTGPIAKWVEVRPNNAVTVLDTHDGIGVIDIGSDQLDRSLKGLVPDEEVDQLVETIHENTHGESRAATGAAASNLDLYQVNSTYYSALGCNDQHYLAARAVQFFLPGVPQVY YVGALAGVNDMELLHRTNVGRDINRHYYSVEEIDRNLERPVVKALNALCRMRNQLDAFDGEFTFSHEGDTLTFDWKGETTSASLTFEPKRGLGVDNQASVCTLRWSDTAGEHETDDLLANPPQVA.

[0037] Although the wild-type Spase5 can catalyze the conversion of glycerol and sucrose to glycerol glucoside, its catalytic activity is still low, and the purity of 2-α-GG in the product is low, making it unsuitable for industrial production. The inventors improved the enzyme-substrate binding... We rationally designed amino acids within the range and determined mutation points with higher affinity for substrate binding after mutation through molecular dynamics simulation. Further screening finally identified two Spase5 mutants with significantly improved enzyme activity, namely P134Q and L341W.

[0038] In this paper, the terms "wild-type," "wild-type (sucrose phosphorylase) enzyme," and "wild-type Spase5" have the same meaning, referring to the sucrose phosphorylase with the amino acid sequence SEQ ID NO:1 (Uniport ID A0A2N3QNR8). Correspondingly, the terms "mutant," "mutant enzyme," and "sucrose phosphorylase mutant" have the same meaning, referring to enzymes that retain the same catalytic characteristics after amino acid sequence modification of the wild-type enzyme Spase5, especially enzymes with increased activity and / or enhanced activity stability, such as mutants with amino acid sequences SEQ ID NO:3 and 5. For ease of expression, wild-type sucrose phosphorylase and its mutants are collectively referred to as "sucrose phosphorylase" in this paper.

[0039] In this article, the term “(enzyme activity) increase” as used above means an increase of at least 100% compared to a reference level (e.g., the enzyme activity of Spase5), such as an increase of at least 1, at least 2, or at least 3, or at least 5, or at least 10, or at least 20 times compared to a reference level.

[0040] It should be understood that the term "mutation" as used herein includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A "conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in polypeptides with amino acids from the same or similar amino acid definition class. However, as used herein, a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue-to-small residue substitutions if a conserved mutation can be alternatively expressed as aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue-to-restriction residue substitutions. As is known in the art, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated to another aliphatic residue or another nonpolar residue. Exemplary conservative substitutions can be performed according to the table below, wherein amino acids belonging to the same partition in the second column can be substituted for each other, and preferably, amino acids in the same row in the third column can be substituted for each other:

[0041]

[0042] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0043] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion may include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of the engineered maleic acid hydratase. Deletion may target the interior and / or ends of the peptide. In several embodiments, the deletion may comprise a continuous segment or may be discontinuous.

[0044] "Insertion" refers to a modification of a peptide by adding one or more amino acids to a reference peptide. In some embodiments, modified engineered maleate hydratases include inserting one or more amino acids into naturally occurring maleate hydratases and inserting one or more amino acids into other modified maleate hydratase peptides. Insertion can be internal to the peptide, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring peptide.

[0045] The amino acid sequence of the sucrose phosphorylase mutant of the present invention is well known, so those skilled in the art can easily obtain its encoding gene, expression cassette and plasmid containing these genes, and transformants containing the plasmid.

[0046] In this paper, the product glycerol glucoside catalyzed by the sucrose phosphorylase or its mutant described in this invention has two configurations: 2-α-GG (or 2-αGG) and 1-α-GG (1-αGG). 2-αGG has higher application value, therefore, those skilled in the art desire products with higher purity of 2-αGG. This invention obtains two mutants by modifying wild-type Spase5. Using these mutants to catalyze the reaction of sucrose and glycerol, the purity of 2-αGG in the obtained product is significantly higher than that obtained by catalyzing the reaction using wild-type Spase5.

[0047] To optimally express sucrose phosphorylase or its mutants in E. coli, the most commonly used gene in genetic engineering, codon optimization of the expression genes for these enzymes can be performed.

[0048] Codon optimization is a technique used to maximize protein expression in an organism by increasing the translation efficiency of genes of interest. Different organisms often exhibit a particular preference for one of a set of codons encoding the same amino acid due to mutational predisposition and natural selection. For example, in fast-growing microorganisms such as *E. coli*, optimized codons reflect the composition of their respective genomic tRNA repertoires. Thus, in fast-growing microorganisms, low-frequency codons for amino acids can be used for high-frequency codon substitutions of the same amino acid. Consequently, the expression of optimized DNA sequences is improved in fast-growing microorganisms.

[0049] For example, to express sucrose phosphorylase in E. coli, the gene encoding the codon-optimized amino acid sequence SEQ ID NO:1 could be SEQ ID NO:2, whose sequence is shown below:

[0050]

[0051] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0052] The transformant host can be any microorganism suitable for expressing sucrose phosphorylase, including bacteria and fungi. Preferred microorganisms are Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, with Escherichia coli being preferred, and Escherichia coli BL21(DE3) being more preferred.

[0053] When used as a biocatalyst, the sucrose phosphorylase of the present invention can be added to the reaction system either in the form of an enzyme or in the form of bacterial cells. The enzyme form includes free enzymes and immobilized enzymes, including purified enzymes, crude enzymes, fermentation broth, and enzymes immobilized on a carrier; the bacterial cell form includes surviving cells, dead cells, and immobilized cells.

[0054] When microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli cease fermentation and proliferation and are instead used for enzyme-catalyzed reactions, they themselves become naturally immobilized enzymes. Furthermore, they do not require disruption or even extraction and purification; they can be directly used as enzyme preparations for catalytic reactions. Since the reaction substrates and products are small molecules, they can easily cross the cell membrane—the biological barrier of the microorganisms—thus eliminating the need for cell disruption, which is economically advantageous.

[0055] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0056] Example

[0057] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0058] Materials and methods

[0059] The whole gene synthesis, primer synthesis and sequencing in the examples were outsourced to Nanjing GenScript Biotech Co., Ltd.

[0060] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual," 3rd edition (J. Sambrook and DW Russell, eds., translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions could be determined through simple experiments if necessary.

[0061] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0062] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium with an additional 20 g / L agar powder.)

[0063] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K₂HPO₄·3H₂O, 2.31 g / L KH₂PO₄, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium with an additional 20 g / L agar powder.)

[0064] The HPLC detection conditions for the substrate glycerol, sucrose, and the product glycerol glucoside are as follows:

[0065] 1.1 Instruments

[0066] Agilent HPLC 1260 (or similar) and electronic balance (accurate to 0.0001g).

[0067] 1.2 Reagents

[0068] Acetonitrile (HPLC), ultrapure water.

[0069] 1.3 Chromatographic conditions

[0070] Column: Asahipak NH2P-50 4E

[0071] Mobile phase: ultrapure water: acetonitrile = 25:75

[0072] Flow rate: 1.0 mL / min

[0073] Column temperature: 30℃

[0074] RID temperature: 30℃

[0075] Injection volume: 10 μL

[0076] Running time: 15min

[0077] Diluent: 75% acetonitrile

[0078] 1.4 Curve Range:

[0079] Glycerol 0.5 g / L~5.0 g / L, fructose 0.5 g / L~5.0 g / L, glycerol glucoside 0.5 g / L~5.0 g / L, glucose 0.5 g / L~5.0 g / L, sucrose 0.5 g / L~5.0 g / L.

[0080] 1.5 Typical Atlas:

[0081] Retention times: glycerol 4.77 min, fructose 7.38 min, 2-α-glyceroglucoside 7.73 min, 1-α-glyceroglucoside 8.24 min, glucose 8.91 min, sucrose 11.97 min.

[0082] It should be noted that, for the sake of convenience, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share the same number. This is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0083] Example 1: Construction of the wild-type enzyme Spase5 engineered strain

[0084] Based on the sucrose phosphorylase Spase5 (Uniport ID accession number A0A2N3QNR8, amino acid sequence SEQ ID NO:1) derived from *Bifidobacterium thermophilum*, its coding gene SEQ ID NO:2 was designed through codon expression optimization based on *E. coli* preferences. The complete gene sequence was synthesized by Nanjing GenScript Biotech Co., Ltd., and cloned into plasmid pET28a. The 3' end was inserted into the XhoI site, and the 5' end was seamlessly cloned to obtain plasmid pET28a-Spase5. (See [link to relevant documentation]). Figure 1 .

[0085] Nanjing Genscript Biotechnology Co., Ltd. transformed the recombinant plasmid pET28a-Spase5 into Escherichia coli BL21(DE3) competent cells (Invitrogen) expressing wild-type sucrose phosphorylase by electroporation, resulting in recombinant Escherichia coli expressing wild-type sucrose phosphorylase, named SH2392.

[0086] The recombinant plasmid pET28a-Spase5 can also be transformed into other hosts such as Pichia pastoris and Bacillus subtilis to express wild-type sucrose phosphorylase Spase5.

[0087] Example 2: Construction of Spase5 mutant strains P134Q and L341W

[0088] The coding genes for Spase5 mutants P134Q and L341W were designed, with sequences SEQ ID NO:4 and SEQ ID NO:6, respectively. The whole gene sequences were synthesized by Nanjing Genscript Biotech Co., Ltd., and cloned into plasmid pET28a according to the method in Example 1 to obtain plasmids pET28a-Spase5-P134Q and pET28a-Spase5-L341W. Then, the two recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent cells (Invitrogen) by electroporation to obtain recombinant Escherichia coli expressing two mutant sucrose phosphorylases, named SH2452 and SH2453, respectively.

[0089] The recombinant plasmids pET28a-Spase5-P134Q and pET28a-Spase5-L341W can also be transformed into other hosts such as Pichia pastoris and Bacillus subtilis to express the sucrose phosphorylase Spase5 mutants P134Q and L341W.

[0090] Example 3: Fermentation and Catalytic Reaction of Strains

[0091] Wild-type Spase5 and two mutant strains were inoculated into LB medium (test tubes) at a 1% inoculum and cultured at 37°C and 220 rpm for 16 h to obtain seed culture. The seed culture was then inoculated into 400 mL of LB medium at a 1% inoculum and cultured at 37°C and 180 rpm until the logarithmic growth phase was reached. 0.5 mM of IPTG inducer was added and cultured at 18°C ​​and 120 rpm for 16 h to induce enzyme production. The cells were then centrifuged at 4°C and 4000 rpm for 10 min to collect the wet cells.

[0092] 1L reaction system: 1.5M sucrose, 1.7M glycerol, 55℃, pH controlled at 5.5 during reaction, 20g / L wet bacterial cells, with 3 different types of wet bacterial cells added.

[0093] The experimental results after 12 h and 20 h of reaction are shown in Table 1:

[0094] Table 1 Results of wet cell reaction

[0095]

[0096] The results above indicate that the purity of 2-αGG in the products of the two mutant strains was significantly improved compared with that of the wild-type strain.

[0097] Example 4: Pure enzyme-catalyzed reaction

[0098] Wild-type Spase5 and two mutants were inoculated and induced, and the bacterial cells were collected for purification to prepare pure enzymes. The specific steps are as follows: The collected bacterial cells were resuspended in 20 mL of equilibration buffer (50 mM HEPES, 50 mM NaCl, 10 mM imidazole, pH 7.5) to obtain a bacterial suspension. The suspension was then sonicated under ice bath conditions to obtain a crude enzyme solution. The crude enzyme solution was centrifuged at 12000 rpm for 30 min, and the supernatant was collected. The purified enzyme was extracted using a nickel column. First, the nickel column was rinsed with water, then rinsed and equilibrated with 10 column volumes of equilibration buffer. The supernatant was loaded onto the column and allowed to bind with the packing material for a period of time. Elute the impurities with 20 mL of washing buffer (50 mM HEPES, 50 mM NaCl, 25 mM imidazole, pH 7.5), then elute the target protein with 4 mL of elution buffer (50 mM HEPES, 50 mM NaCl, 250 mM imidazole, pH 7.5). Aliquot 1 mL into 1.5 mL EP tubes to obtain the pure enzyme solution.

[0099] The catalytic reaction was carried out using pure enzymes, and the conditions are shown in Table 2:

[0100] Table 2 Reaction conditions

[0101]

[0102] Following the above reaction conditions, three replicates were performed for both the wild type and the two mutants, and the reaction was carried out overnight in a shaker at 50°C and 250 rpm. Samples were taken for liquid chromatography analysis, and the results are shown in Table 3.

[0103] Table 3 Results of pure enzyme reactions

[0104]

[0105] The detection spectra of reactions 2392-1, 2452-1, and 2453-1 are as follows: Figure 2-4 As shown.

[0106] The results above indicate that the purity of 2-αGG in the products of the two mutant strains was significantly improved compared with that of the wild-type strain.

[0107] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A sucrose phosphorylase mutant, which has an amino acid sequence of: SEQ ID NO: 3, which is a mutant of SEQ ID NO: 1 with P at position 134 replaced by Q; or SEQ ID NO: 5, which is a mutant of SEQ ID NO: 1 with L at position 341 replaced by W. 2.A gene encoding the sucrose phosphorylase mutant of claim 1. The nucleotide sequence of the gene is shown in SEQ ID NO. 4 or SEQ ID NO.

6. 4.An expression vector comprising the gene of claim 2 or 3.

3. The gene of claim 2, wherein The vector is selected from a pET-28a (+) plasmid, a pET-28b (+) plasmid or a pET-20b (+) plasmid. 6.A host cell comprising the sucrose phosphorylase mutant of claim 1.

5. The expression vector of claim 4, wherein, The host cell is selected from Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica and Bacillus subtilis. 8.Use of the sucrose phosphorylase mutant of claim 1 or the host cell of claim 6 or 7 in the enzymatic synthesis of glycerol glucoside, particularly 2-α-glycerol glucoside.

7. The host cell of claim 6, wherein The method comprises the step of adding the sucrose phosphorylase mutant of claim 1 or an expression system containing the enzyme to a reaction system containing substrates sucrose and glycerol. The glycerol concentration in the reaction system is 1.5-3.5 mol / L; the sucrose concentration is 0.8-1.6 mol / L; the pH is 5.5-7.5; and the reaction temperature is 25-37 ℃.

9. A method for preparing a glycerol glucoside, characterized by, ​ 10. The production method according to claim 9, wherein ​

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