Enzyme mutant and application thereof in catalytic production of feruloyl tyramine

Abstract

The invention relates to the technical field of bioengineering, in particular to an enzyme mutant and application thereof in catalytic production of feruloyl tyramine, and the amino acid sequence of the enzyme is shown as SEQ ID NO: 1. According to the mutant, T at the 421st site of enzyme is mutated into hydrophobic amino acid. The enzyme can adopt ferulic acid as a substrate to catalytically produce feruloyl tyramine, so that the production cost can be greatly reduced. By mutating peripheral amino acid residues of the enzyme, a mutant with higher catalytic conversion activity is finally obtained, and the production cost can be further reduced.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to an enzyme and an application thereof in the preparation of feruloyltyramide. Background Art

[0002] The active ingredient of traditional Chinese medicine, feruloyl tyramide, has free radical scavenging and antioxidant effects. It also has antibacterial, melanin synthesis inhibition, insulin secretion stimulation and other activities. Studies have shown that ferulic acid amide derivatives represented by ferulic acid tyramide are significantly superior to ferulic acid and ferulic acid ester derivatives in terms of antioxidant activity and thermal stability. Their heat resistance temperature is above 200°C, especially in the frying oil industry as a high-stability natural antioxidant, showing great application prospects (Jin Wenhua, Zhang Jinfang, Gao Liang, Wang Xingguo, Wang Xiaosan. Evaluation of the antioxidant properties of different addition amounts of ferulic acid amide substances in soybean oil frying system [J]. China Oils and Fats, 2021, 46(05): 89-92+116.). At present, feruloyl tyramide is mainly extracted from traditional Chinese medicines such as citronella and Lycium bark. Due to the limited natural resources of traditional Chinese medicine and the low content in plants, relying solely on direct extraction cannot meet people's long-term needs.

[0003] Existing preparation methods of feruloyl tyramide include chemical synthesis and biological preparation. Chemical synthesis: At present, the chemical synthesis method of feruloyl tyramide is mainly the acyl chloride method. The reaction method has poor reaction atom economy, large energy loss, and is prone to waste of resources; the added coupling agent and organic solvent are prone to environmental pollution. Biological preparation: The enzymes used to catalyze the synthesis reaction of feruloyl tyramide are mostly lipases and individual acyltransferases. The catalysis of lipase needs to be carried out in an organic phase system. If these solvents cannot be recovered or recycled, their waste treatment will cause pollution to the environment; and acyltransferases need to use coenzyme A (CoA) as an acyl donor, and CoA and its derivatives (such as feruloyl coenzyme A) are usually expensive, which will increase production costs. For example, Chinese patent CN109082433 A provides a method for preparing feruloyl tyramide, which uses feruloyl coenzyme A as an acyl donor to prepare feruloyl tyramide, and its preparation cost is relatively high.

[0004] As mentioned above, so far, since the production process of feruloyl tyramine is still mainly based on traditional chemical synthesis, it is usually necessary to protect and deprotect active groups such as hydroxyl groups, and use toxic and harmful coupling agents such as DCC or DMAP or expensive chemical catalysts to activate the carboxyl group in situ, which directly leads to the high production cost of the product, thereby restricting its further promotion and application. In contrast, the use of bioenzymatic synthesis has shown great advantages in the synthesis of feruloyl tyramine due to its mild reaction conditions, ingredients, raw materials and green reaction process. However, the problem of high production cost of bioenzymatic method still needs to be further solved. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of an enzyme and a mutant thereof in catalytic production of feruloyltyramine.

[0006] The technical solution of the present invention is as follows:

[0007] The invention discloses an application of an enzyme and / or a mutant thereof in catalyzing the production of feruloyltyramine. The amino acid sequence of the enzyme is shown in SEQ ID NO: 1.

[0008] The enzyme of the invention is an amide bond synthase Azo (Uniprot: D3NZ98) derived from Azospirillum lipoferum B.510. The enzyme is used as a template for transformation, and the peripheral amino acid residues are mutated to finally obtain a mutant with high catalytic activity for producing feruloyltyramine.

[0009] The mutant is a mutant in which the T at position 421 of the enzyme is mutated into a hydrophobic amino acid.

[0010] In the present invention, the complete protein sequence of Azospirillum lipogenicum (Uniprot: D3NZ98) is referred to, and its amino acid sequence is shown in SEQ ID NO: 1, and its gene sequence is shown in SEQ ID NO: 5. A 6×His tag and a thrombin protease cleavage site are introduced upstream of the gene encoding the protein sequence, and then on the basis of the parent sequence with the amino acid sequence of SEQ ID NO: 1, the 421st position in the parent sequence is mutated to isoleucine (I) and valine (V), respectively, and its amino acid sequences are SEQ ID NO: 2 and SEQ ID NO: 3, and the nucleotide sequences are SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Finally, the efficient synthesis of feruloyltyramide is achieved, which can be used for industrial catalytic preparation.

[0011] In some specific embodiments, the mutation sites in the mutant are: T421I (SEQ ID NO: 2); T421V (SEQ ID NO: 3).

[0012] The present invention also provides a nucleic acid sequence encoding the enzyme of the present invention.

[0013] Furthermore, the present invention also provides an expression vector, which comprises the nucleic acid encoding the present invention.

[0014] In some embodiments, the backbone vector of the expression vector is selected from the pET series vectors. In some specific embodiments, the backbone vector of the expression vector is pET28a.

[0015] Furthermore, the present invention also provides a host cell, the genome of which is integrated with the aforementioned nucleic acid, or which is transformed or transfected with the aforementioned expression vector.

[0016] In some embodiments, the host cell is Escherichia coli. In some specific embodiments, the host cell is genetically engineered Escherichia coli BL21 (DE3)

[0017] In some specific embodiments, the host cell is constructed by a method comprising: transforming the constructed expression vector into the host cell by heat shock transformation.

[0018] Furthermore, the present invention also provides a method for preparing the enzyme, comprising: culturing the host cell as described above, and inducing the expression of the enzyme.

[0019] Furthermore, the present invention also provides the use of the enzyme, nucleic acid, expression vector, host cell or the product obtained by the preparation method as described above in the preparation of feruloyltyramide.

[0020] The present invention provides the enzyme, nucleic acid, expression vector, host cell or the product prepared by the preparation method as described above, and uses them in promoting the synthesis reaction of ferulic acid and tyramine, thereby realizing the efficient synthesis of feruloyltyramine.

[0021] The reaction of the present invention uses ferulic acid and tyramine as substrates. Furthermore, the reaction system also includes ATP and / or a substance capable of generating ATP, and Mg 2+ and Tris buffer.

[0022] Furthermore, the reaction system also includes polyphosphate kinase (PPK) and polyphosphate (PolyP). In the process of amide bond synthetase catalyzing the reaction between ferulic acid and tyramine, ATP needs to be continuously consumed to generate AMP. PPK enzyme can provide phosphate groups through polyphosphate and continuously convert the generated AMP into ATP, thereby realizing the reuse of ATP and greatly reducing the demand for ATP.

[0023] Furthermore, the present invention also provides a method for preparing feruloyltyramine. The preparation method of the present invention can provide suitable enzymatic reaction conditions in vitro to obtain the target product.

[0024] In a specific embodiment of the present invention, the preparation method of feruloyltyramine comprises: reacting ferulic acid and tyramine in the system as described above to obtain feruloyltyramine.

[0025] More specifically, the reaction system comprises:

[0026] Ferulic acid 0.5~3mM Tyramine 10~100mM ATP 2~14mM AMP 2~14mM <![CDATA[MgCl 2 ]]> 2~16mM Tris-HCl pH 9.0 50~200mM PolyP 4–8 mg / mL Azo Pure Enzyme 5~55μM CHU Pure Enzyme 20~40μM

[0027] In one embodiment, the reaction system includes: the reaction system includes the enzyme or host cell or the product obtained by the preparation method and:

[0028]

[0029]

[0030] In another embodiment, the reaction system comprises: the reaction system comprises the enzyme or host cell or the product obtained by the preparation method and:

[0031] Ferulic acid 0.5~3mM Tyramine 10~100mM AMP 2~14mM <![CDATA[MgCl 2 ]]> 2~16mM Tris-HCl pH 9.0 50~200mM PolyP 4–8 mg / mL Azo Pure Enzyme 5~55μM CHU Pure Enzyme 20~40μM

[0032] The reaction conditions of the present invention include: 20-40° C., pH=7.0-9.0, and stirring in a water bath.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) The enzyme of the present invention can use ferulic acid as a substrate to catalyze the production of feruloyltyramine, which can greatly reduce the production cost.

[0035] (2) The present invention utilizes the protein "Azo" derived from Azospirillum lipoferum B.510

[0036] (Uniprot: D3NZ98) was used as a template for modification, and by mutating its peripheral amino acid residues, a mutant with higher catalytic conversion activity was finally obtained, which is conducive to further reducing production costs.

[0037] (3) The method of the present invention is superior to the chemical synthesis preparation process and shows outstanding advantages in production cost, energy consumption, product quality and green index. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the SDS-PAGE electrophoresis of Azo purification; M: Protein Marker; Lane 1: crude enzyme; Lane 2: sample flow-through Lane 3: 200 mM imidazole elution.

[0039] Figure 2 This is the electrophoresis diagram of the expression and purification of the Azo mutant; M: Protein Marker Lane 1: T421I supernatant; Lane 2: T421V supernatant; Lane 3: T421I; Lane 4: T421V.

[0040] Figure 3 This is a graph showing the HPLC detection results of the product obtained from the reaction of the Azo mutant (Azo-T421I) in Example 3.

[0041] Figure 4 This is the mass spectrum of the product obtained from the reaction of the Azo mutant (Azo-T421I) in Example 3. DETAILED DESCRIPTION

[0042] The present invention provides the application of enzyme and its mutant in the preparation of feruloyltyramine, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0043] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0044] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0045] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0046] The term "and / or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

[0047] Unless otherwise defined, all scientific and technical terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terms in this area, professionals can refer specifically to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0048] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for direct expression of the enzyme of the invention in vitro and / or in vivo, e.g., in a host or patient.

[0049] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0050] Enzyme related information:

[0051] The amide bond synthetase template protein of the present invention is derived from Azospirillumlipoferum

[0052] B.510) amide bond synthetase Azo (Uniprot: D3NZ98) was used as a template for modification. The following is the specific mutation site information (Azo-T421I, Azo-T421V).

[0053] Polyphosphate kinase (PPK), derived from Cytophaga harveyi (Cytophaga hutchinsonii) CHU (Uniprot ID: A0A6N4SMB5) The present invention is further described below in conjunction with the embodiments:

[0054]

[0055]

[0056] The DNA sequences of Azo, Azo-T421I, Azo-T421V and CHU enzymes are SEQ ID NOs: 5-8, respectively, see the sequence listing for details.

[0057] The present invention will be further described below in conjunction with embodiments:

[0058] Example 1: Construction of wild-type Azo and mutant recombinant expression vectors and expression strains

[0059] The following steps are involved:

[0060] 1.1 The synthesized Azo gene was seamlessly cloned and connected with the purified gene fragment and plasmid pET28a (+) using primers, transformed into E. coli-Top10 competent cells, and spread on LB (containing 100 μg / mL kanamycin) plates. Positive clones were picked for colony PCR and gene sequencing. The pET28a-Azo recombinant plasmid of wild-type Azo was extracted using a plasmid extraction kit.

[0061] 1.2 Design mutant primers as shown in the following table

[0062]

[0063] Note: The underlined sequence is the mutation site sequence

[0064] 1.3 Using the plasmid pET28a(+)-Azo containing Azo as a template, PCR amplification was performed using the primers in Table 1 to obtain a plasmid containing a mutant gene. The PCR amplification system is shown in the following table, and the PCR amplification program is shown in the small table. The mutant of the present invention was constructed using the overlapping PCR method.

[0065] PCR amplification system

[0066]

[0067]

[0068] PCR amplification procedure

[0069]

[0070] After the PCR amplification product was confirmed by 1% agarose gel electrophoresis, the PCR product was digested and purified using restriction endonuclease Dpn I to digest the template and remove the original template contained in the system. The digestion system is shown in the following table.

[0071] Digestion reaction system

[0072]

[0073] The above system was kept at a constant temperature of 37°C for 30-60 min.

[0074] 1.4 The PCR products were purified from the enzyme digestion products and the purified products were confirmed by nucleic acid electrophoresis.

[0075] 1.5 The purified product was mixed with competent E. coli cells TOP10, placed on ice for 30 min, heat-shocked at 42°C for 90 s, and quickly placed on ice for 2 min.

[0076] 1.6 Add the competent cells obtained in the previous step to 1ml LB medium and activate at 37℃, 200rpm for 45min. After activation, centrifuge the competent cells at 8000rpm for 2min, remove most of the medium, resuspend the cells and evenly spread the resulting transformation solution on an LB plate containing 100μg / mL kanamycin. After static culture in a 37℃ incubator overnight, pick a single colony on the plate and inoculate it in 5ml LB liquid medium (containing 100μg / mL kanamycin), culture overnight at 37℃, 200rpm, and extract the plasmid for use after confirming the amplification results by gene sequencing.

[0077] Example 2: Fermentation of wild-type Azo and its mutant recombinant expression strains and purification of recombinant proteins

[0078] 2.1 Expression of mutants

[0079] The plasmid of the Azo mutant obtained in Example 1 was transformed into E. coli competent cells BL21 (DE3), and the transformation solution was spread on an LB plate containing 100 μg / mL kanamycin, and cultured at 37° C. overnight. The single colony grown was the expression strain.

[0080] A single colony of the Azo mutant was picked and inoculated into 5 ml of LB medium and cultured overnight at 37°C and 220 rpm until OD 600 After the inoculum was about 0.8-0.9, it was inoculated into a 500 mL conical flask containing 100 mL of kanamycin-resistant LB liquid medium at a 2% (v / v) inoculum and cultured in a constant temperature shaker at 37°C and 220 rpm until the OD 600 After the concentration is about 0.8-0.9, it is amplified to 500 ml of autoinduction medium (1% (w / v) peptone, 0.5% (w / v) yeast extract, 0.05% (w / v) glucose, 0.2% (w / v) lactose, 0.5% (w / v) glycerol, 25 mM Na 2 HPO 4 , 25mM KH 2 PO 4 , 50 mM NH 4 Cl, 5 mM Na 2 SO 4 , 2 mM MgSO 4 ) for expansion culture.600 When the culture temperature reaches about 1.0, the culture temperature is lowered from 37°C to 16°C, and the speed is adjusted to 200 rpm for induction. The induction time is 20 hours. After the induction is completed, the bacteria are harvested. The fermentation broth is centrifuged at 4°C and 8000 rpm for 20 minutes, and then the culture medium is discarded to collect the bacteria. The bacteria obtained after centrifugation are washed twice with pre-cooled Tris-HCl buffer (pH 9.0) to remove the residual culture medium components in the bacteria.

[0081] 2.2 Purification of mutants

[0082] The obtained bacteria were resuspended in Tris-HCl buffer (pH 9.0) and the cells were disrupted by ultrasound. The cell disruption solution was centrifuged (10000r / min, 30min) and the supernatant was obtained as the prepared crude enzyme solution of amide bond synthetase Azo or mutant.

[0083] The bacteria were resuspended in Tris-HCl buffer (pH 9.0) at a ratio of 1:10 (m / v) and ultrasonically disrupted on ice for 20 minutes. When the bacterial solution was clear and uniform, it was considered that the disruption was sufficient. The disrupted solution was centrifuged at 4°C and 11,000 rpm for 30 minutes, and the supernatant crude enzyme solution was collected and filtered with a 0.45 μm water-based microporous filter membrane to remove insoluble impurities and tiny bacterial fragments in the crude enzyme solution. The amide bond synthetase was purified using the AKTA Purifier protein purification system, and the chromatography column used was the affinity chromatography column His Prep TM FF16 / 10 and salt exchange column HiPrep TM 26 / 10, all solutions entering the AKTA protein purifier are filtered through a 0.45μm water-based microporous filter membrane. The specific operation steps are as follows: first, use deionized water to drain the alcohol protection liquid in the protein purifier, and then use the loading buffer to balance the affinity chromatography column HisPrep at a flow rate of 5mL / min. TM FF16 / 10, to OD 280 After the conductivity value reaches equilibrium, the crude enzyme solution is pumped into the protein purifier at a flow rate of 3 mL / min. After the binding sites on the Ni column are fully occupied by the target protein, the affinity chromatography column is equilibrated with Tris-HCl buffer (pH 9.0) containing 50 mM imidazole at a flow rate of 5 mL / min to remove unbound impurities. 280 The target protein was then eluted with Tris-HCl buffer (pH 9.0) containing 200 mM imidazole at 3 mL / min and collected in a centrifuge tube. TM26 / 10 salt change, first use salt change buffer to balance the system at a flow rate of 5mL / min, then pump the collected target protein into the protein purifier at an appropriate volume, and then use salt change buffer to change the salt until the end. The final target protease solution is concentrated to a suitable concentration using a 10kDa ultrafiltration tube, packaged, pre-frozen in liquid nitrogen, and stored at -80℃. The target protein band is attached Figure 1 , 2 .

[0084] 2.2 Detection of mutant protein concentration

[0085] The concentration of mutant protein was determined using the Brandford protein assay kit. A series of concentrations of bovine serum albumin were prepared as protein concentration standard solutions to prepare a standard curve. 10 μl of standard protein of different concentrations were added to a 96-well plate, and then 200 μl of Brandford reagent was added. After reacting at room temperature for 5 minutes, the absorbance was measured at 595 nm. The protein concentration standard curve was obtained based on the known protein concentration and the corresponding absorbance. The absorbance of the mutant protein at 595 nm was measured under the same reaction conditions, and the parallel test was performed more than three times. The mutant protein concentration was obtained after comparing the protein concentration standard curve.

[0086] Example 3: Preparation of feruloyltyramide by enzyme-catalyzed reaction using wild-type Azo and mutants (Azo-T421I, Azo-T421V).

[0087] The reaction system for catalytic synthesis of feruloyltyramide is shown in the table below. Each solution in the table is added to a 2mL centrifuge tube, and then placed in a constant temperature metal bath, and reacted for 36 hours at 30°C and 500rpm. After the reaction is completed, an equal volume of methanol is added to the reaction system as a cosolvent, and vortex extraction is performed for 3min. Subsequently, the enzyme protein is removed by centrifugation at 10,000rpm for 5min, and the upper organic phase is filtered through a needle filter and then transferred to a chromatographic bottle for subsequent HPLC analysis and detection. The blank group uses the corresponding buffer solution instead of the enzyme solution, and the other conditions are the same. Three parallel groups are set for each group of experiments.

[0088] Reaction system for the synthesis of feruloyltyramide catalyzed by Azo mutant

[0089]

[0090] The HPLC test results showed that the conversion rate of Azo wild-type catalyzed synthesis of feruloyltyramide was 76%, while the corresponding conversion rates of Azo-T421I and Azo-T421V were 84% and 82%, respectively. Figure 3 ; The product was confirmed to be feruloyl tyramide by mass spectrometry. The mass spectrometry results are shown in Figure 4 .

[0091] Example 4: Preparation of feruloyltyramide using Azo wild type and mutants (Azo-T421I, Azo-T421V) and ATP regeneration system

[0092]

[0093] The reaction system for catalytic synthesis of feruloyltyramide is shown in the table below. Each solution in the table is added to a 2mL centrifuge tube, and then placed in a constant temperature metal bath, and reacted for 36 hours at 30°C and 500rpm. After the reaction is completed, an equal volume of methanol is added to the reaction system as a cosolvent, and vortex extraction is performed for 3min. Subsequently, the enzyme protein is removed by centrifugation at 10,000rpm for 5min, and the upper organic phase is filtered through a needle filter and then transferred to a chromatographic bottle for subsequent HPLC analysis and detection. The blank group uses the corresponding buffer solution instead of the enzyme solution, and the other conditions are the same. Three parallel groups are set for each group of experiments.

[0094] Reaction system for synthesis of feruloyltyramide catalyzed by Azo mutant and ATP regeneration system

[0095]

[0096] HPLC detection results showed that the production rate of feruloyltyramide catalyzed by wild-type Azo was 65%, while the corresponding production rates of Azo-T421I and Azo-T421V were 75% and 71%, respectively.

[0097] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an enzyme and / or its mutant in catalyzing the production of feruloyltyramide, wherein the amino acid sequence of the enzyme is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: The mutant is a mutant in which the T at position 421 of the enzyme is mutated into a hydrophobic amino acid.

3. The use according to claim 2, characterized in that: The mutant is an enzyme in which the T at position 421 is mutated to I or V.

4. The use according to claim 1, 2 or 3, characterized in that: The reaction uses ferulic acid and tyramine as substrates.

5. The use according to claim 4, characterized in that: The reaction system also includes ATP and / or substances that can generate ATP, as well as Mg 2+ and Tris buffer.

6. The use according to claim 5, characterized in that: The reaction system also includes polyphosphate kinase and polyphosphate.

7. The use according to claim 6, characterized in that: The reaction system includes: 0.5-3 mM ferulic acid; 10-100 mM tyramine; 2-14 mM AMP and / or ATP; 2-16 mM MgCl2; 50-200 mM Tris-HCl; 4-8 mg / ml polyphosphoric acid; 5-55 μM enzyme and / or its mutant; and 20-40 μM polyphosphate kinase.

8. The use according to claim 1, 2 or 3, characterized in that: The reaction conditions include: 20-40° C., pH=7.0-9.0, and stirring in a water bath.

9. The mutant according to claim 2 or 3.

10. A nucleic acid sequence encoding the mutant according to claim 9, characterized in that: The nucleic acid sequence is SEQ ID NO:6 or SEQ ID NO:7.

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