CN109423485B

CN109423485B - Sucrose phosphorylase mutant and application thereof

Info

Publication number: CN109423485B
Application number: CN201710740257.4A
Authority: CN
Inventors: 胡美荣; 王雷; 陈紫薇; 彭颖; 陶勇
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2017-08-25
Filing date: 2017-08-25
Publication date: 2021-12-24
Anticipated expiration: 2037-08-25
Also published as: CN109423485A

Abstract

The invention discloses a sucrose phosphorylase mutant and application thereof. The protein provided by the invention is obtained by replacing the 341 th amino acid residue of sucrose phosphorylase with other amino acid residue from leucine. The protein provided by the invention can be BaSP/L341D protein obtained by replacing the 341 th amino acid residue of sucrose phosphorylase with leucine into aspartic acid, and also can be BaSP/L341R protein obtained by replacing the 341 th amino acid residue of sucrose phosphorylase with leucine into arginine. The specificity of producing 2-glycerol glucoside by taking sucrose and glycerol as raw materials is remarkably improved and the yield of the byproduct 1-glycerol glucoside is reduced by point mutation of the 341 th amino acid residue primer of wild sucrose phosphorylase from bifidobacterium adolescentis. Has great application value in the field of 2-glycerol glucoside production.

Description

Sucrose phosphorylase mutant and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a sucrose phosphorylase mutant and application thereof.

Background

Sucrose phosphorylase (EC 2.4.1.7) belongs to family 13 glycosyltransferases (GH13) which catalyze the reversible phosphorylation of sucrose molecules in vivo (phosphate group as the glucose acceptor, products α -D-glucose-1 phosphate and D-fructose). Because of the stepwise reaction mechanism of the enzyme and the special structure of the active site, the glucosyl group acceptor in the reaction can be other molecules with biological activity such as ascorbic acid, hydroquinone, hesperidin, glycerol and the like besides the phosphoryl group. Glycosylation of these bioactive molecules can improve their stability, increase their solubility, enhance or even confer new physiological activities. The corresponding glycosylation product can be used as a functional additive of food or cosmetics, thereby having great application value and market prospect.

The alpha-glucosyl glycerol is also called 2-glycerol glucoside, and the structural formula is shown as a formula (I). The structural formula of the 1-glycerol glucoside is shown as a formula (II). The 2-glyceroglucoside can up-regulate the expression of aquaporin (aquaporin), has the physiological effect of moistening skin, can be used as a functional additive of cosmetics, and has larger market demand. The moisturizing effect of the 2-glycerol glucoside is better than that of the 1-glycerol glucoside. The 2-glycerol glucoside can be produced by taking sucrose and glycerol as raw materials under the action of sucrose phosphorylase. However, the prior art has a problem that 2-glyceroglucoside is produced and a large amount of 1-glyceroglucoside is produced as a by-product.

Disclosure of Invention

The invention aims to provide a sucrose phosphorylase mutant and application thereof.

The protein provided by the invention is obtained by replacing the 341 th amino acid residue of sucrose phosphorylase with other amino acid residue from leucine (L).

The protein provided by the invention can be BaSP/L341D protein. The BaSP/L341D protein is a mutant protein obtained by replacing the 341 th amino acid residue of sucrose phosphorylase from leucine (L) to aspartic acid (D).

The protein provided by the invention can be BaSP/L341R protein. The BaSP/L341R protein is a mutant protein obtained by replacing the 341 th amino acid residue of sucrose phosphorylase with leucine (L) to arginine (R).

The sucrose phosphorylase may be specifically (a1) or (a2) or (a3) as follows:

(a1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(a2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 1, has the function of sucrose phosphorylase and is derived from the sequence 1;

(a3) a protein derived from bifidobacterium adolescentis and having more than 90% identity with the protein shown in the sequence 1.

The gene coding the BaSP/L341D protein also belongs to the protection scope of the invention. The gene encoding the BaSP/L341D protein may specifically be (b1) or (b2) or (b5) or (b6) as follows.

The gene coding the BaSP/L341R protein also belongs to the protection scope of the invention. The gene encoding the BaSP/L341R protein may specifically be (b3) or (b4) or (b5) or (b6) as follows.

(b1) The coding region is a DNA molecule which mutates the 1021-1023 bit nucleotide of the double-stranded DNA molecule shown in the sequence 2 of the sequence table from "ctc" to "GAC".

(b2) The double-stranded DNA molecule shown in the sequence 2 of the sequence table has the mutation of the 1021-1023 bit nucleotide from "ctc" to "GAC".

(b3) The coding region is a DNA molecule which mutates the 1021-1023 bit nucleotide of the double-stranded DNA molecule shown in the sequence 2 of the sequence table from 'ctc' to 'CGC'.

(b4) The double-stranded DNA molecule shown in the sequence 2 of the sequence table has the mutation of the 1021-1023 bit nucleotide from "ctc" to "CGC".

(b5) A DNA molecule which hybridizes with the DNA sequence defined in (b1) or (b2) or (b3) or (b4) under stringent conditions and encodes the protein.

(b6) A DNA molecule which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or more homology with the DNA sequence defined in (b1) or (b2) or (b3) or (b4) and encodes the protein.

The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.

Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the gene encoding the BaSP/L341D protein belong to the scope of protection of the present invention. The recombinant bacterium containing the gene encoding the BaSP/L341D protein was designated as recombinant bacterium BaSP/L341D. The recombinant bacterium, BaSP/L341D, may be specifically a recombinant bacterium obtained by introducing a recombinant plasmid pBAD-BaSP/L341D into Escherichia coli BW 25113. The recombinant plasmid pBAD-BaSP/L341D is a recombinant plasmid obtained by inserting a gene encoding a BaSP/L341D protein into the multiple cloning site (for example, between XhoI and PstI cleavage sites) of the vector pBAD/HisB.

Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the gene encoding the BaSP/L341R protein belong to the scope of protection of the present invention. The recombinant bacterium containing the gene encoding the BaSP/L341R protein was designated as recombinant bacterium BaSP/L341R. The recombinant bacterium, BaSP/L341R, may be specifically a recombinant bacterium obtained by introducing a recombinant plasmid pBAD-BaSP/L341R into Escherichia coli BW 25113. The recombinant plasmid pBAD-BaSP/L341R is a recombinant plasmid obtained by inserting a gene encoding a BaSP/L341R protein into the multiple cloning site (for example, between XhoI and PstI cleavage sites) of the vector pBAD/HisB.

The invention also protects the application of the BaSP/L341D protein in the production of 2-glycerol glucoside.

The invention also protects the application of the recombinant bacterium BaSP/L341D in the production of 2-glycerol glucoside.

The invention also protects the application of the BaSP/L341D protein in the production of 2-glycerol glucoside by using sucrose and glycerol as raw materials.

The invention also protects the application of the recombinant bacterium BaSP/L341D in the production of 2-glycerol glucoside by taking sucrose and glycerol as raw materials.

The invention also protects the application of the BaSP/L341R protein in the production of 2-glycerol glucoside.

The invention also protects the application of the recombinant bacterium BaSP/L341R in the production of 2-glycerol glucoside.

The invention also protects the application of the BaSP/L341R protein in the production of 2-glycerol glucoside by using sucrose and glycerol as raw materials.

The invention also protects the application of the recombinant bacterium BaSP/L341R in the production of 2-glycerol glucoside by taking sucrose and glycerol as raw materials.

The invention also provides a method for producing 2-glycerol glucoside, which comprises the following steps: sucrose and glycerol are used as raw materials, and under the action of BaSP/L341D protein, 2-glycerol glucoside is obtained.

The invention also provides a method for producing 2-glycerol glucoside, which comprises the following steps: taking sucrose and glycerol as raw materials, and obtaining the 2-glycerol glucoside under the action of a recombinant bacterium BaSP/L341D.

The method comprises the following steps: culturing a recombinant bacterium BaSP/L341D, performing L-arabinose induction in the culture process, and then centrifuging to collect the bacterium; and adding the thalli, glycerol and sucrose into a buffer system, and reacting to obtain the 2-glycerol glucoside. The mass ratio of the thalli, the glycerol and the sucrose is as follows: 10: 200: 200. in the reaction system, the initial concentrations of the components were as follows: 10g/L of thallus, 200g/L of glycerol and 200g/L of sucrose. The mass of the cells is wet weight. The buffer system is phosphate buffer solution with pH 6.0. The reaction conditions are specifically as follows: shaking at 30 deg.C and 80rpm for 60 h.

The method specifically comprises the following steps: culturing recombinant bacteria BaSP/L341D to OD in liquid LB culture medium_600nm0.6-0.8 (specifically 0.7), adding L-arabinose to the culture system to make the concentration of the L-arabinose be 0.2g/100mL, culturing at 30 ℃ for 12 hours under the condition of 200rpm oscillation, and centrifuging to collect the thallus; and adding the thalli, glycerol and sucrose into a buffer system, and reacting to obtain the 2-glycerol glucoside. The mass ratio of the thalli, the glycerol and the sucrose is as follows: 10: 200: 200. in the reaction system, the initial concentrations of the components were as follows: 10g/L of thallus, 200g/L of glycerol and 200g/L of sucrose. The mass of the cells is wet weight. The buffer system is phosphate buffer solution with pH 6.0. The reaction conditions are specifically as follows: shaking at 30 deg.C and 80rpm for 60 h.

The invention also provides a method for producing 2-glycerol glucoside, which comprises the following steps: sucrose and glycerol are used as raw materials, and under the action of BaSP/L341R protein, 2-glycerol glucoside is obtained.

The invention also provides a method for producing 2-glycerol glucoside, which comprises the following steps: taking sucrose and glycerol as raw materials, and obtaining the 2-glycerol glucoside under the action of a recombinant bacterium BaSP/L341R.

The method comprises the following steps: culturing a recombinant bacterium BaSP/L341R, performing L-arabinose induction in the culture process, and then centrifuging to collect the bacterium; and adding the thalli, glycerol and sucrose into a buffer system, and reacting to obtain the 2-glycerol glucoside. The mass ratio of the thalli, the glycerol and the sucrose is as follows: 10: 200: 200. in the reaction system, the initial concentrations of the components were as follows: 10g/L of thallus, 200g/L of glycerol and 200g/L of sucrose. The mass of the cells is wet weight. The buffer system is phosphate buffer solution with pH 6.0. The reaction conditions are specifically as follows: shaking at 30 deg.C and 80rpm for 60 h.

The method specifically comprises the following steps: culturing recombinant bacteria BaSP/L341R to OD in liquid LB culture medium_600nm0.6-0.8 (specifically 0.7), adding L-arabinose to the culture system to make the concentration of the L-arabinose be 0.2g/100mL, culturing at 30 ℃ for 12 hours under the condition of 200rpm oscillation, and centrifuging to collect the thallus; and adding the thalli, glycerol and sucrose into a buffer system, and reacting to obtain the 2-glycerol glucoside. The mass ratio of the thalli, the glycerol and the sucrose is as follows: 10: 200: 200. in the reaction system, the initial concentrations of the components were as follows: 10g/L of thallus, 200g/L of glycerol and 200g/L of sucrose. The mass of the cells is wet weight. The buffer system is phosphate buffer solution with pH 6.0. The reaction conditions are specifically as follows: shaking at 30 deg.C and 80rpm for 60 h.

The invention obviously improves the specificity of producing 2-glycerol glucoside by taking sucrose and glycerol as raw materials and reduces the yield of a byproduct 1-glycerol glucoside by introducing point mutation into the 341 th amino acid residue of wild sucrose phosphorylase from bifidobacterium adolescentis. The invention has great application value in the field of production of 2-glycerol glucoside.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Unless otherwise specified, all phosphate buffers in the examples were 50mM PBS buffer. For chromatograms of different reactions under the same condition parameters, the retention time of a target peak has a certain error range, and generally, the difference can be regarded as an error within 0.1 min. 2-glycerol glucoside standard: carbofuran, cat # J60-H943140. In the examples, the cell weights were wet weights.

Bifidobacterium adolescentis (Bifidobacterium adolescentis): CGMCC 1.2190. Vector pBAD/HisB: invitrogen, catalog number V430-01. Coli BW 25113: biovector NTCC INC, cat # 355297.

Example 1 construction of recombinant bacterium

Construction of recombinant bacterium BaSP

1. Extracting the genome DNA of the bifidobacterium adolescentis.

2. And (3) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and adopting a primer pair consisting of F1 and R1, and recovering a PCR amplification product.

F1：5’-GCCTGGTGCCGCGCGGCAGCCTCGAGatgaaaaacaaggtgcag-3’；

R1：5’-CAGCTGCAGACCGAGCTCACCCTGCAGtcaggcgacgacaggcggattg-3’。

3. And (3) taking the PCR amplification product obtained in the step (2), carrying out double enzyme digestion by using restriction enzymes XhoI and PstI, and recovering the enzyme digestion product.

4. The vector pBAD/HisB was digested with restriction enzymes XhoI and PstI, and the vector backbone of about 4000bp was recovered.

5. And (4) connecting the enzyme digestion product in the step (3) with the vector framework in the step (4) to obtain the recombinant plasmid pBAD-BaSP.

According to the sequencing result, the structure of the recombinant plasmid pBAD-BaSP is described as follows: the small fragment between the XhoI and PstI cleavage sites of the vector pBAD/HisB was substituted for the double-stranded DNA molecule shown in sequence 2 of the sequence listing. The DNA molecule shown in the sequence 2 of the sequence table encodes BaSP protein shown in the sequence 1 of the sequence table.

6. The recombinant plasmid pBAD-BaSP is introduced into escherichia coli BW25113 to obtain a recombinant bacterium BaSP.

Secondly, constructing a recombinant bacterium BaSP/L341D

1. The recombinant plasmid pBAD-BaSP is used as a template, and a primer pair consisting of F2 and R2 is adopted to introduce single-point mutation, so that the recombinant plasmid pBAD-BaSP/L341D is obtained.

F2：5’-TCCAATGACGACCTCTACCAGGTCAACAG-3’；

R2：5’-GAGGTCGTCATTGGATGCGGCGGCGCCAG-3’。

According to the sequencing results, the recombinant plasmid pBAD-BaSP/L341D was structurally described as follows: the double-stranded DNA molecule 1021-1023 nucleotide shown in the sequence 2 of the sequence table in the recombinant plasmid pBAD-BaSP is mutated from "ctc" to "GAC". The mutated DNA molecule encodes the BaSP/L341D protein. The difference between the BaSP/L341D protein and the BaSP protein is only that the 341 th amino acid residue of the BaSP protein is mutated from leucine (L) to aspartic acid (D).

2. The recombinant plasmid pBAD-BaSP/L341D was introduced into Escherichia coli BW25113 to obtain recombinant strain BaSP/L341D.

Thirdly, constructing a recombinant bacterium BaSP/L341R

1. The recombinant plasmid pBAD-BaSP is used as a template, and a primer pair consisting of F3 and R3 is adopted to introduce single-point mutation, so that the recombinant plasmid pBAD-BaSP/L341R is obtained.

F3：5’-TCCAATCGCGACCTCTACCAGGTCAACAG-3’；

R3：5’-GAGGTCGCGATTGGATGCGGCGGCGCCAG-3’。

According to the sequencing results, the recombinant plasmid pBAD-BaSP/L341R was structurally described as follows: the double-stranded DNA molecule 1021-1023 nucleotide shown in the sequence 2 of the sequence table in the recombinant plasmid pBAD-BaSP is mutated from "ctc" to "CGC". The mutated DNA molecule encodes the BaSP/L341R protein. The difference between the BaSP/L341R protein and the BaSP protein is only that the 341 th amino acid residue of the BaSP protein is mutated from leucine (L) to arginine (R).

2. The recombinant plasmid pBAD-BaSP/L341R was introduced into Escherichia coli BW25113 to obtain recombinant strain BaSP/L341R.

Fourthly, constructing recombinant bacteria pBAD

The vector pBAD/HisB was introduced into E.coli BW25113 to obtain recombinant strain pBAD.

Example 2 preparation of 2-Glycerol glucoside Using recombinant bacteria

Respectively carrying out the following steps on the recombinant bacterium BaSP, the recombinant bacterium BaSP/L341D, the recombinant bacterium BaSP/L341R or the recombinant bacterium pBAD:

1. taking a monoclonal of the recombinant bacteria, inoculating the monoclonal to a liquid LB culture medium, and carrying out shaking culture at 37 ℃ and 220rpm until the recombinant bacteria reach OD_600nm0.7 (OD in practical use)_600nm0.6-0.8).

2. After completion of step 1, L-arabinose was added to the culture system so that the concentration thereof in the culture system became 0.2g/100mL, and the culture was carried out at 30 ℃ for 12 hours with shaking at 200 rpm.

3. After the step 2 is completed, the whole culture system is taken, centrifuged at 6000rpm for 15min at 4 ℃, and thalli precipitates are collected.

4. Preparing a reaction system.

The reaction system consists of the thallus precipitate obtained in the step 3, glycerol, sucrose and phosphate buffer solution with pH of 6.0. In the reaction system, the initial concentrations of the components were as follows: 10g/L of thallus, 200g/L of glycerol and 200g/L of sucrose.

Reaction conditions are as follows: shaking at 30 deg.C and 80rpm for 60 h.

5. After the step 4 is completed, taking the reaction system, and detecting the content of the 2-glycerol glucoside in the reaction system, wherein the specific steps are as follows:

(1) the reaction system was centrifuged at 12000rpm for 2min, and the supernatant was collected.

(2) Diluting the supernatant obtained in the step (1) with distilled water to obtain a supernatant.

(3) And (3) taking the sample liquid obtained in the step (2), and detecting the content of the 2-glycerol glucoside by adopting a high performance liquid chromatography. HPLC system: agilent 1260; a chromatographic column: waters Amide columns (34.6X 150mm,3.5 μm); the mobile phase consists of 800 parts by volume of acetonitrile and 200 parts by volume of 1 per mill of ammonia water;

the sample volume is 10 mu L; the column temperature is 30 ℃; the flow rate is 1 mL/min; and detecting by an RID detector.

Under the chromatographic conditions, the peak position of the 2-glycerol glucoside standard substance is 8.141min, and the peak position of the 1-glycerol glucoside standard substance is 9.108 min.

The standard curve equation of the content of the 2-glycerol glucoside and the peak area is established by using the 2-glycerol glucoside standard substance as follows: 29060x (R)²0.9999); wherein x is the peak area in the HPLC chromatogram, and y is the concentration of 2-glycerol glucoside, and the unit is g/L.

The standard curve equation of the content of the 1-glycerol glucoside and the peak area is established by using the 1-glycerol glucoside standard substance as follows: Y29060X (R)²0.9999); wherein X is the peak area in an HPLC chromatogram, and Y is the concentration of 1-glycerol glucoside, and the unit is g/L.

In the reaction system of the step 4 for completing the recombinant bacterium BaSP, the concentration of 2-glycerol glucoside is 45g/L (average value of 10 repeated tests), the concentration of 1-glycerol glucoside is 30g/L (average value of 10 repeated tests), and the yield ratio of 2-glycerol glucoside to 1-glycerol glucoside is 1.5: 1.

In the reaction system of the recombinant bacterium BaSP/L341D in the step 4, the concentration of 2-glyceroglucoside is 60g/L (average value of 10 repeated tests), the concentration of 1-glyceroglucoside is 21g/L (average value of 10 repeated tests), and the yield ratio of 2-glyceroglucoside to 1-glyceroglucoside is 2.86: 1.

In the reaction system of the recombinant bacterium BaSP/L341R in the step 4, the concentration of 2-glyceroglucoside is 55g/L (average value of 10 repeated tests), the concentration of 1-glyceroglucoside is 17g/L (average value of 10 repeated tests), and the yield ratio of 2-glyceroglucoside to 1-glyceroglucoside is 3.24: 1.

The recombinant pBAD has a concentration of 2-glycerol glucoside (average value of 10 repeated tests) and a concentration of 1-glycerol glucoside (average value of 10 repeated tests) in the reaction system for completing the step 4.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> sucrose phosphorylase mutant and application thereof

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Met Lys Asn Lys Val Gln Leu Ile Thr Tyr Ala Asp Arg Leu Gly Asp

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Gly Thr Ile Lys Ser Met Thr Asp Ile Leu Arg Thr Arg Phe Asp Gly

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Val Tyr Asp Gly Val His Ile Leu Pro Phe Phe Thr Pro Phe Asp Gly

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Ala Asp Ala Gly Phe Asp Pro Ile Asp His Thr Lys Val Asp Glu Arg

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Leu Gly Ser Trp Asp Asp Val Ala Glu Leu Ser Lys Thr His Asn Ile

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Met Val Asp Ala Ile Val Asn His Met Ser Trp Glu Ser Lys Gln Phe

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Gln Asp Val Leu Ala Lys Gly Glu Glu Ser Glu Tyr Tyr Pro Met Phe

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Leu Thr Met Ser Ser Val Phe Pro Asn Gly Ala Thr Glu Glu Asp Leu

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Ala Gly Ile Tyr Arg Pro Arg Pro Gly Leu Pro Phe Thr His Tyr Lys

130 135 140

Phe Ala Gly Lys Thr Arg Leu Val Trp Val Ser Phe Thr Pro Gln Gln

145 150 155 160

Val Asp Ile Asp Thr Asp Ser Asp Lys Gly Trp Glu Tyr Leu Met Ser

165 170 175

Ile Phe Asp Gln Met Ala Ala Ser His Val Ser Tyr Ile Arg Leu Asp

180 185 190

Ala Val Gly Tyr Gly Ala Lys Glu Ala Gly Thr Ser Cys Phe Met Thr

195 200 205

Pro Lys Thr Phe Lys Leu Ile Ser Arg Leu Arg Glu Glu Gly Val Lys

210 215 220

Arg Gly Leu Glu Ile Leu Ile Glu Val His Ser Tyr Tyr Lys Lys Gln

225 230 235 240

Val Glu Ile Ala Ser Lys Val Asp Arg Val Tyr Asp Phe Ala Leu Pro

245 250 255

Pro Leu Leu Leu His Ala Leu Ser Thr Gly His Val Glu Pro Val Ala

260 265 270

His Trp Thr Asp Ile Arg Pro Asn Asn Ala Val Thr Val Leu Asp Thr

275 280 285

His Asp Gly Ile Gly Val Ile Asp Ile Gly Ser Asp Gln Leu Asp Arg

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Ser Leu Lys Gly Leu Val Pro Asp Glu Asp Val Asp Asn Leu Val Asn

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Ala Ala Ser Asn Leu Asp Leu Tyr Gln Val Asn Ser Thr Tyr Tyr Ser

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Ala Leu Gly Cys Asn Asp Gln His Tyr Ile Ala Ala Arg Ala Val Gln

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Phe Phe Leu Pro Gly Val Pro Gln Val Tyr Tyr Val Gly Ala Leu Ala

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Gly Lys Asn Asp Met Glu Leu Leu Asn Lys Thr Asn Asn Gly Arg Asp

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Ile Asn Arg His Tyr Tyr Ser Thr Ala Glu Ile Asp Glu Asn Leu Lys

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Arg Pro Val Val Lys Ala Leu Asn Ala Leu Ala Lys Phe Arg Asn Glu

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Ser Ile Ser Phe Thr Trp Arg Gly Glu Thr Ser Glu Ala Thr Leu Thr

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Met Leu Glu Trp His Asp Ser Ala Gly Asp His Arg Ser Asp Asp Leu

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Ile Ala Asn Pro Pro Val Val Ala

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atgaaaaaca aggtgcagct catcacttac gccgaccgcc ttggcgacgg caccatcaag 60

tcgatgaccg acattctgcg cacccgcttc gacggcgtgt acgacggcgt tcacatcctg 120

ccgttcttca ccccgttcga cggcgccgac gcaggcttcg acccgatcga ccacaccaag 180

gtcgacgaac gtctcggcag ctgggacgac gtcgccgaac tctccaagac ccacaacatc 240

atggtcgacg ccatcgtcaa ccacatgagt tgggaatcca agcagttcca ggacgtgctg 300

gccaagggcg aggagtccga atactatccg atgttcctca ccatgagctc cgtgttcccg 360

aacggcgcca ccgaagagga cctggccggc atctaccgtc cgcgtccggg cctgccgttc 420

acccactaca agttcgccgg caagacccgc ctcgtgtggg tcagcttcac cccgcagcag 480

gtggacatcg acaccgattc cgacaagggt tgggaatacc tcatgtcgat tttcgaccag 540

atggccgcct ctcacgtcag ctacatccgc ctcgacgccg tcggctatgg cgccaaggaa 600

gccggcacca gctgcttcat gaccccgaag accttcaagc tgatctcccg tctgcgtgag 660

gaaggcgtca agcgcggtct ggaaatcctc atcgaagtgc actcctacta caagaagcag 720

gtcgaaatcg catccaaggt ggaccgcgtc tacgacttcg ccctgcctcc gctgctgctg 780

cacgcgctga gcaccggcca cgtcgagccc gtcgcccact ggaccgacat acgcccgaac 840

aacgccgtca ccgtgctcga tacgcacgac ggcatcggcg tgatcgacat cggctccgac 900

cagctcgacc gctcgctcaa gggtctcgtg ccggatgagg acgtggacaa cctcgtcaac 960

accatccacg ccaacaccca cggcgaatcc gaagcagcca ctggcgccgc cgcatccaat 1020

ctcgacctct accaggtcaa cagcacctac tattcggcgc tcgggtgcaa cgaccagcac 1080

tacatcgccg cccgcgcggt gcagttcttc ctgccgggcg tgccgcaagt ctactacgtc 1140

ggcgcgctcg ccggcaagaa cgacatggag ctgctgaaca agacgaataa cggccgcgac 1200

atcaatcgcc attactactc caccgcggaa atcgacgaga acctcaagcg tccggtcgtc 1260

aaggccctga acgcgctcgc caagttccgc aacgagctcg acgcgttcga cggcacgttc 1320

tcgtacacca ccccgaccga cacgtccatc agcttcacct ggcgcggcga aaccagcgaa 1380

gccacgctga cgttcgagcc gaagcgcggt ctcggtgtgg acaacactac gccggtcgcc 1440

atgttggaat ggcatgattc cgcgggagac caccgttcgg atgatctgat cgccaatccg 1500

cctgtcgtcg cctga 1515

Claims

1. A protein, which is obtained by replacing the 341 th amino acid residue of sucrose phosphorylase with another amino acid residue from leucine; the other amino acid residue is aspartic acid or arginine; the sucrose phosphorylase is a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein: the gene is a DNA molecule of (b1) or (b2) as follows:

(b1) the coding region is a DNA molecule which mutates the 1021-1023 bit nucleotide of the double-stranded DNA molecule shown in the sequence 2 of the sequence table from 'ctc' to 'GAC';

(b2) the coding region is a DNA molecule which mutates the 1021-1023 bit nucleotide of the double-stranded DNA molecule shown in the sequence 2 of the sequence table from 'ctc' to 'CGC'.

4. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the gene of claim 2 or 3.

5. Use of the protein of claim 1 or the recombinant bacterium of claim 4 for producing 2-glyceroglucoside.

6. Use of the protein of claim 1 or the recombinant bacterium of claim 4 for producing 2-glyceroglucoside from sucrose and glycerol.

7. A method for producing 2-glyceroglucoside, comprising the steps of: 2-glycerol glucoside is obtained by using sucrose and glycerol as raw materials under the action of the protein of claim 1.

8. A method for producing 2-glyceroglucoside, comprising the steps of: taking sucrose and glycerol as raw materials, and obtaining the 2-glycerol glucoside under the action of the recombinant bacterium disclosed in claim 4.