CN110551781A

CN110551781A - Method for preparing 5' -guanylic acid by enzyme method

Info

Publication number: CN110551781A
Application number: CN201910801969.1A
Authority: CN
Inventors: 张玲; 夏海洋
Original assignee: Taizhou University
Current assignee: Taizhou University
Priority date: 2019-08-28
Filing date: 2019-08-28
Publication date: 2019-12-10

Abstract

The invention discloses a method for preparing 5' -guanylic acid by an enzymatic method, belonging to the technical field of food processing. The preparation method comprises the following steps: adding pure acid phosphotransferase mutant enzyme with an amino acid sequence shown as SEQ ID NO.1 as a catalyst into a reaction solution containing guanosine and sodium pyrophosphate and having a pH value of 3.6-9.0, carrying out catalytic reaction at 25-60 ℃, obtaining a mixed solution containing 5 '-guanylic acid after the reaction is finished, and separating and purifying to obtain the 5' -guanylic acid. The acid phosphotransferase mutant selected by the invention has the advantages of high phosphotransferase activity, low hydrolase activity and high substrate specificity; the method has the advantages of environment-friendly process, mild reaction conditions, low cost, high yield of the obtained target product, simple preparation method, suitability for industrial production of 5' -guanylic acid and wide application prospect.

Description

Method for preparing 5' -guanylic acid by enzyme method

Technical Field

The invention relates to the technical field of food processing, in particular to a method for preparing 5' -guanylic acid by an enzymatic method.

Background

5 ' -guanylic acid (Guanosine-5 ' -monophosphate, i.e., 5 ' -GMP) is a nucleotide seasoning widely used in foods, and it is used in combination with sodium glutamate in the process of food processing to constitute a basic seasoning for foods, and has a remarkable flavor-enhancing effect, and therefore, it is also called as a flavor-providing nucleotide. The prior production methods of the flavor-developing nucleotide mainly comprise four methods: chemical synthesis, microbial fermentation, enzymolysis, and biocatalysis.

The chemical synthesis method mainly uses nucleoside as raw material to make phosphorylation reaction. The commonly used phosphorylating agents are mainly phosphoric acid or reactive derivatives of pyrophosphoric acid, such as acid chloride pyrophosphate, bis-p-nitrophenyl pyrophosphate, monochloro and dichloro derivatives of phosphoric acid, etc., while phosphorus oxychloride is widely used in industry. In order to obtain 5 '-nucleotides by introducing a phosphate group into the 5' -hydroxyl group of a nucleoside, the 2 '-and 3' -hydroxyl groups of the ribose of the nucleoside are protected with protecting groups before phosphorylation reaction, and the protecting groups are removed after phosphorylation is completed. The overall reaction yield of the method is reduced due to the protection and deprotection steps. The reagents used in the chemical synthesis method are expensive and toxic, so that the reagents are generally used for producing some 5' -nucleotide and derivatives thereof with special purposes, and the reagents are difficult to be used for industrial production.

Microbial fermentation processes are divided into one-step and two-step processes. One-step method, namely direct fermentation method: directly producing 5 '-inosinic acid and 5' -xanthylic acid by microbial fermentation. The two-step method is a method combining a fermentation method and a chemical method: firstly, the nucleoside is produced by microbial fermentation, and then is phosphorylated by a chemical method, and finally, the 5 '-inosinic acid and the 5' -guanylic acid are obtained. The microbial fermentation method has few byproducts and low cost, but the application of the method is greatly limited by the characteristics of microorganisms.

the enzymolysis method has become a classical method for preparing various natural nucleotides, has the longest history and the most mature technology, and most of the nucleotides are produced by the enzymolysis method at present. The mixture of 4 nucleotides can be obtained by preparing the nucleotides by an enzymolysis method once, the utilization rate of the enzyme is high, but the difficulty of obtaining 4 high-purity products by extraction in the later period is high, so that the production period is long, the separation and purification process is complicated, and the product purity is not high.

A biocatalytic method: the biocatalytic nucleoside phosphorylation method mainly utilizes biocatalytic catalysis. Currently, enzymes available for catalyzing the synthesis of 5' -guanylic acid are mainly kinases, acid phosphotransferase (AP/PTAse), and the like.

Kinases are a class of phosphotransferases that transfer phosphate groups from ATP to other phosphate acceptors, and can be used to synthesize a variety of 5' -nucleotides, according to the following equation: ATP + nucleoside ADP + 5' -nucleotide. The disadvantage of this method is that ATP is always consumed in the catalytic process, so that it is necessary to culture ATP-consuming microorganisms capable of regenerating reaction or to add a large amount of ATP with high price to complete the reaction, which is expensive, and thus the method is limited.

The method for synthesizing 5 '-guanylic acid by catalyzing guanosine and phosphate group donor with acid phosphotransferase has the characteristics of simplicity, high efficiency, short period, low cost, environmental protection requirement conformity and the like, is suitable for industrial production, and has great development potential compared with the disclosed process for preparing 5' -guanylic acid. At present, the technology for producing guanosine by a fermentation method in China is mature and industrialization is realized, and the key for further developing and producing 5' -guanylic acid on the basis of the technology is how to realize the specific phosphorylation of enzyme catalytic guanosine. Therefore, a production process which is simple and safe to operate, low in cost, high in product purity and environment-friendly is urgently needed.

Disclosure of Invention

The invention aims to provide an acid phosphotransferase capable of specifically catalyzing guanosine phosphorylation, which is used for preparing 5' -guanylic acid by an enzyme method, and provides a production process which is simple and safe to operate, mild in reaction conditions, low in production cost, high in yield and purity and suitable for industrial production.

In order to achieve the purpose, the invention adopts the following technical scheme:

The application of the acid phosphotransferase mutant with the amino acid sequence shown in SEQ ID NO.1 in catalyzing guanosine to prepare 5' -guanylic acid.

The acid phosphotransferase mutant is obtained by modifying acid phosphotransferase (AP/PTase) from Morganella morganii, and the specific modification method comprises the following steps: the first 20 amino acid sequences were deleted and site-directed mutated at 2 amino acid positions (G72D, I151T) using the acid phosphotransferase primary amino acid sequence published by Genebank (Genebank accession number P28581).

The research of the invention shows that the modified acid phosphotransferase mutant shows higher substrate specificity for catalyzing guanosine, has the characteristics of high phosphotransferase activity and low hydrolase activity, and can effectively convert guanosine to synthesize 5' -guanylic acid. The reaction formula is as follows:

In the reaction formula, Acid Phosphotransferase is Acid Phosphotransferase; acid Phosphotase is an Acid phosphohydrolase. The acid phosphotransferase mutant has a bidirectional effect.

The invention also provides a coding gene of the acid phosphotransferase mutant, codon optimization is carried out according to the codon frequency of escherichia coli, and the optimized nucleotide sequence is shown as SEQ ID NO. 2.

The invention also provides a method for preparing 5' -guanylic acid by an enzymatic method, which comprises the following steps: adding pure acid phosphotransferase mutant enzyme with an amino acid sequence shown as SEQ ID NO.1 as a catalyst into a reaction solution containing guanosine and sodium pyrophosphate and having a pH value of 3.6-9.0, carrying out catalytic reaction at 25-60 ℃, obtaining a mixed solution containing 5 '-guanylic acid after the reaction is finished, and separating and purifying to obtain the 5' -guanylic acid.

Preferably, in the reaction system, the concentration of guanosine is 1 to 20mg/mL, the concentration of sodium pyrophosphate is 150 to 300mg/mL, and the concentration of pure acid phosphotransferase mutant enzyme is 20 to 120. mu.g/mL. More preferably, the molar conversion of guanosine in the reaction system under the conditions that the concentration of guanosine was 1mg/mL, the concentration of sodium pyrophosphate was 150mg/mL, and the concentration of pure acid phosphotransferase mutant enzyme was 20. mu.g/mL, was 97%.

Preferably, sodium acetate buffer solution with the pH value of 3.6-5.8 is adopted as a reaction medium in the reaction solution.

more preferably, the reaction medium is a sodium acetate buffer at pH 4.0. The research shows that the acidic phosphotransferase mutant catalyzes 5' -guanylic acid to synthesize at the fastest pH condition of 4.0, and the pH value is the optimal reaction pH value of the enzyme.

Preferably, the temperature of the catalytic reaction is 25-40 ℃. More preferably, the temperature of the catalytic reaction is 35 ℃. The research shows that the temperature condition that the acid phosphotransferase mutant catalyzes 5' -guanylic acid to synthesize at the fastest speed is 35 ℃.

Preferably, the time of the catalytic reaction is 6-8 h. Research shows that in the initial stage of the catalytic reaction, the concentration of the 5 '-guanylic acid of the reaction product is approximately linearly increased along with the extension of the reaction time, and when the reaction time reaches 6-8 hours, the concentration of the 5' -guanylic acid reaches the highest.

Preferably, the preparation method of the pure enzyme of the acid phosphotransferase mutant comprises the steps of culturing recombinant escherichia coli containing coding genes of the acid phosphotransferase mutant until OD ₆₀₀ is 0.6-0.8, adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mM, inducing at 25 ℃ for 10-12 h, collecting thalli, performing ultrasonic disruption to obtain cell lysate, centrifuging to obtain supernatant, and further separating and purifying to obtain the pure enzyme of the acid phosphotransferase mutant.

The method of the invention is not only suitable for preparing 5 '-guanylic acid, but also can be universally used for preparing 5' -monophosphate nucleotide ester compounds.

The invention has the beneficial effects that:

(1) The invention adopts a new enzyme method process to prepare 5' -guanylic acid, successfully solves the technical problems existing in the prior art, and the selected acid phosphotransferase mutant has the advantages of high phosphotransferase activity, low hydrolase activity and high substrate specificity; the preparation method is simple, greatly simplifies the industrial production steps of 5' -guanylic acid, and successfully solves the technical problems of raw material toxicity, complex process and the like in the existing chemical synthesis process.

(2) The raw material source is wide, and the requirement on equipment is low.

(3) The method has the advantages of environment-friendly process, mild reaction conditions, low cost, high yield of the obtained target product, suitability for industrial production of 5' -guanylic acid and wide application prospect.

Drawings

FIG. 1 shows the diagram of the purification electrophoresis of acid phosphotransferase, wherein M is protein Marker, lane 1 is whole cell, lane 2 is supernatant after cell disruption, lane 3 is pellet after cell disruption, and lane 4 is purified protein.

FIG. 2 is a graph showing the effect of pH on the catalytic performance of acid phosphotransferases.

FIG. 3 is a graph showing the effect of temperature on the catalytic performance of acid phosphotransferases.

FIG. 4 shows the phosphotransferase activity studies at different substrate concentrations.

FIG. 5 is a graph showing a study of phosphotransferase activity amplification at guanosine concentrations of 10mg/mL and 20 mg/mL.

Detailed Description

The technical solution and the effects of the present invention are further described by the following specific examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Simple modifications of the invention applying the inventive concept are within the scope of the invention as claimed.

In the following examples, all starting materials are commercially available unless otherwise indicated.

guanosine was purchased from bio-engineering (shanghai) gmbh;

Expression vectors pET28a and e.coli BL21(DE3) were purchased from invitrogen.

EXAMPLE 1 acquisition of acid phosphotransferase mutants

Construction of recombinant E.coli expressing acid phosphotransferase:

Removing the first 20 amino acid sequences from an original amino acid sequence (AP/PTAse, Genebank accession number P28581) of acid phosphotransferase disclosed by Genebank, designing mutation on 2 amino acid sites (G72D, I151T) in the original amino acid sequence, carrying out codon optimization according to the codon frequency of escherichia coli, obtaining a target fragment by a whole-gene synthesis mode through the optimized nucleotide sequence, and inserting the target fragment into an expression vector pET28a between an NdeI site and a BamHI site to construct a recombinant expression plasmid; the recombinant expression plasmid is transferred into an expression host of Escherichia coli E.coli BL21(DE3) to test the expression of the target protein.

Obtaining of thallus:

Inoculating Escherichia coli BL21(DE3) containing the above acid phosphotransferase recombinant expression plasmid into LB liquid medium containing corresponding antibiotic, culturing at 37 deg.C and 200rpm overnight, inoculating into new LB medium containing corresponding antibiotic according to 2% inoculum size, culturing at 37 deg.C and 200rpm until OD is 0.6-0.8, adding IPTG with final concentration of 1mM, and inducing at 25 deg.C overnight; centrifuging at 10000rpm for 15min, collecting thallus, and freezing and preserving at-20 deg.C for use.

Obtaining pure enzyme:

And taking the thalli obtained by centrifugal collection, placing the thalli in a phosphate buffer solution with the pH value of 8.0 and the concentration of 50mM for resuspension, and carrying out ultrasonic disruption (carrying out ultrasonic treatment in an ice water bath for 15-30min, wherein the ultrasonic time is 2s each time and the interval is 4s) to obtain a cell lysate. 12000rpm, 4 ℃, centrifuging for 10min, taking supernatant as crude enzyme liquid, purifying by Ni-NTA affinity chromatography one step, dialyzing eluted target protein to remove high-concentration salt, wherein an acid phosphotransferase purification electrophoretogram is shown in figure 1, and an amino acid sequence of the acid phosphotransferase mutant is shown in SEQ ID No. 1. Freezing and preserving at-80 ℃ for later use.

Example 2 phosphotransferase mutant substrate specificity Studies

After 20. mu.g/mL of the acid phosphotransferase pure enzyme prepared in example 1 was added to reaction systems containing 2.5mg/mL of guanosine, inosine, cytidine, adenosine, uridine, and 150mg/mL of sodium pyrophosphate, respectively, the reaction was terminated with 2MHCl at 30 ℃ and pH4.0, and the supernatant was diluted 10-fold by centrifugation, and the relative enzyme activity was measured by HPLC, and the results are shown in Table 1. The phosphatase has high substrate specificity for guanosine and inosine.

TABLE 1 study of the specificity of acid phosphotransferases to substrates

Example 3 phosphotransferase mutant reaction kinetics assay

The K _m and V _max values of guanosine, 5 '-guanylic acid, inosine and 5' -inosinic acid by the acid phosphotransferase obtained in example 1 were measured by the double reciprocal mapping method, and the results are shown in Table 2, which indicates that the acid phosphatase has a high phosphotransferase activity and a low hydrolase activity and is advantageous for the synthesis of guanylic acid.

TABLE 2 acid phosphotransferase kinetics study

Example 4 determination of the optimum reaction pH for phosphotransferase mutants

To a reaction system containing 2.5mg/mL of guanosine and 150mg/mL of sodium pyrophosphate, 20. mu.g/mL of the acid phosphotransferase pure enzyme prepared in example 1 was added, and after reaction at 30 ℃ and different pH (3.6 to 9.0) for 10min, the reaction was terminated with 2M HCl, the supernatant was centrifuged and diluted 10-fold, and the concentration of 5 '-guanylic acid was measured by HPLC to examine the effect of different pH on the catalytic synthesis of 5' -guanylic acid by the pure enzyme. As a result, as shown in FIG. 2, the condition that the synthesis rate of 5' -guanylic acid was the fastest was pH4.0, which was the optimum reaction pH of the enzyme, and it was revealed that the enzyme was the acid phosphatase.

Example 5 determination of optimal reaction temperature for phosphotransferase mutants

To a reaction system containing 2.5mg/mL of guanosine and 150mg/mL of sodium pyrophosphate, 20. mu.g/mL of the acid phosphotransferase purified enzyme prepared in example 1 was added, and after reaction at pH4.0 and various temperatures (25 to 60 ℃) for 10min, the reaction was terminated with 2M HCl, the supernatant was centrifuged and diluted 10-fold, and the concentration of 5 '-guanylic acid was measured by HPLC to examine the influence of various temperatures on the catalytic synthesis of 5' -guanylic acid by the purified enzyme. As shown in FIG. 3, the temperature condition at which the synthesis rate of 5' -guanylic acid is the fastest was 35 ℃ and this temperature was the optimum temperature for the enzymatic reaction.

EXAMPLE 6 phosphotransferase Activity Studies at different substrate concentrations

To reaction systems containing 1mg/mL, 2.5mg/mL, 4mg/mL, 5.5mg/mL guanosine and 150mg/mL sodium pyrophosphate, respectively, 20. mu.g/mL of the acid phosphotransferase pure enzyme prepared in example 1 was added, and then a catalytic reaction was carried out at an optimum pH of 4.0 and an optimum temperature of 35 ℃, samples were taken for 0.5h, 1h, 2h, 4h, 6h and 24h, the reaction was terminated with 2M HCl, the supernatant was centrifuged and diluted 10-fold, and the concentration of 5 '-guanylic acid was measured by HPLC, as shown in FIG. 4, the concentration of 5' -guanylic acid increased approximately linearly with the increase of the reaction time, the concentration of 5 '-guanylic acid reached 6h and after the concentration of 5' -guanylic acid reached 6h at the maximum, the concentration of 5 '-guanylic acid started to decrease, and the concentration of 5' -guanylic acid remained at a constant level after the concentration of 24h, the mutant strain selected by the invention has higher phosphotransferase activity and lower hydrolase activity, and can be used for preparing 5' -guanylic acid. And the molar conversion rate of guanylic acid reaches 97% when the concentration of guanosine is 1 mg/mL.

Example 710 amplification of phosphotransferase Activity at guanosine concentration at mg/mL

60. mu.g/mL of the acid phosphotransferase pure enzyme prepared in example 1 was added to a reaction system containing 10mg/mL of guanosine and 200mg/mL of sodium pyrophosphate, and then a catalytic reaction was carried out at an optimum pH of 4.0 and an optimum temperature of 35 ℃, samples of 1h, 2h, 3h, 4h, 6h, 8h and 24h were taken, 2M HCl was used to terminate the reaction, the supernatant was centrifuged and diluted 10-fold, and the concentration of 5 ' -guanylic acid was measured by HPLC, as shown in FIG. 5, the concentration of 5 ' -guanylic acid increased approximately linearly with the increase of the reaction time within 1h to 8h, the concentration of 5 ' -guanylic acid reached 8.5mg/mL at the maximum, after 8h, the concentration of 5 ' -guanylic acid started to decrease, and the concentration of 5 ' -guanylic acid reached 24h was 6.7mg/mL, indicating that the mutant strain selected by the present invention had a higher hydrolase activity of phosphotransferase, can be used for preparing 5' -guanylic acid.

example 820 mg/mL guanosine concentration phosphotransferase Activity amplification study

Adding 120 μ g/mL of the acid phosphotransferase pure enzyme prepared in example 1 to a reaction system containing 20mg/mL of guanosine and 250mg/mL of sodium pyrophosphate, then carrying out catalytic reaction at an optimal pH of 4.0 and an optimal temperature of 35 ℃, sampling 1h, 2h, 3h, 4h, 6h, 8h and 24h, terminating the reaction by 2M HCl, centrifuging to take the supernatant and diluting the supernatant by 10 times, and measuring the concentration of 5 ' -guanylic acid by HPLC (high performance liquid chromatography), wherein the detection result is shown in FIG. 5, the concentration of 5 ' -guanylic acid is approximately linearly increased along with the extension of the reaction time within 1h-8h, the concentration of 5 ' -guanylic acid reaches 8h, the concentration of 5 ' -guanylic acid starts to be reduced after 8h, and the concentration of 5 ' -guanylic acid reaches 10.9mg/mL when the reaction time reaches 24h, which indicates that the mutant strain selected by the invention has higher hydrolase activity with lower phosphotransferase activity, can be used for preparing 5' -guanylic acid.

sequence listing

<110> Taizhou college

<120> method for preparing 5' -guanylic acid by enzyme method

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 229

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Ile Pro Ala Gly Asn Asp Ala Thr Thr Lys Pro Asp Leu Tyr Tyr

1 5 10 15

Leu Lys Asn Glu Gln Ala Ile Asp Ser Leu Lys Leu Leu Pro Pro Pro

20 25 30

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

35 40 45

Lys Gly Arg Met Leu Arg Asn Thr Glu Arg Gly Lys Gln Ala Gln Ala

50 55 60

Asp Ala Asp Leu Ala Ala Gly Asp Val Ala Thr Ala Phe Ser Gly Ala

65 70 75 80

Phe Gly Tyr Pro Ile Thr Glu Lys Asp Ser Pro Glu Leu Tyr Lys Leu

85 90 95

Leu Thr Asn Met Ile Glu Asp Ala Gly Asp Leu Ala Thr Arg Ser Ala

100 105 110

Lys Glu His Tyr Met Arg Ile Arg Pro Phe Ala Phe Tyr Gly Thr Glu

115 120 125

Thr Cys Asn Thr Lys Asp Gln Lys Lys Leu Ser Thr Asn Gly Ser Tyr

130 135 140

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

145 150 155 160

Glu Val Asn Pro Ala Asn Gln Asp Ala Ile Leu Glu Arg Gly Tyr Gln

165 170 175

Leu Gly Gln Ser Arg Val Ile Cys Gly Tyr His Trp Gln Ser Asp Val

180 185 190

Asp Ala Ala Arg Ile Val Gly Ser Ala Ala Val Ala Thr Leu His Ser

195 200 205

Asp Pro Ala Phe Gln Ala Gln Leu Ala Lys Ala Lys Gln Glu Phe Ala

210 215 220

Gln Lys Ser Gln Lys

225

<210> 2

<211> 690

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcaattccgg caggtaacga tgcaaccact aaaccggatc tgtactacct gaaaaacgaa 60

caggcgattg attctctgaa actgctgccg ccgccgccgg aagttggttc tatccagttc 120

ctgaacgatc aagcgatgta cgaaaaaggc cgtatgctgc gtaacactga acgtggtaaa 180

caggctcaag ctgatgctga tctggctgct ggtgatgtgg caaccgcctt ctccggtgcg 240

tttggctatc cgatcaccga aaaagattct ccggaactgt ataaactgct gactaatatg 300

atcgaagatg cgggtgatct ggcgacccgt agcgcgaaag aacattacat gcgtatccgt 360

ccgttcgcgt tctacggcac cgaaacctgc aacaccaaag atcagaaaaa actgagcacc 420

aacggcagct acccgagcgg ccacaccagc accggctggg cgaccgcgct ggttctggcg 480

gaagttaacc cggcgaacca ggatgcgatc ctggaacgtg gctaccagct gggccagagc 540

cgtgttatct gcggctacca ctggcagagc gatgttgatg cggcgcgtat cgttggtagc 600

gcggcggttg cgaccctgca cagcgatccg gcgttccagg cgcagctggc gaaagcgaaa 660

caggaattcg cgcagaaaag ccaaaaataa 690

Claims

1. The application of the acid phosphotransferase mutant with the amino acid sequence shown in SEQ ID NO.1 in catalyzing guanosine to prepare 5' -guanylic acid.

2. A method for preparing 5' -guanylic acid by an enzymatic method, which is characterized by comprising the following steps: adding pure acid phosphotransferase mutant enzyme with an amino acid sequence shown as SEQ ID NO.1 as a catalyst into a reaction solution containing guanosine and sodium pyrophosphate and having a pH value of 3.6-9.0, carrying out catalytic reaction at 25-60 ℃, obtaining a mixed solution containing 5 '-guanylic acid after the reaction is finished, and separating and purifying to obtain the 5' -guanylic acid.

3. The method according to claim 2, wherein the concentration of guanosine is 1 to 20mg/mL, the concentration of sodium pyrophosphate is 150 to 300mg/mL, and the concentration of pure acid phosphotransferase mutant enzyme is 20 to 120. mu.g/mL in the reaction system.

4. The enzymatic method for preparing 5' -guanylic acid according to claim 2, wherein a sodium acetate buffer solution having a pH of 3.6 to 5.8 is used as a reaction medium in the reaction solution.

5. The enzymatic process of claim 4 wherein the reaction medium is sodium acetate buffer at pH 4.0.

6. The enzymatic method of producing 5' -guanylic acid according to claim 2, wherein the temperature of the catalytic reaction is 25 to 40 ℃.

7. The enzymatic method of preparing 5' -guanylic acid according to claim 6, wherein the temperature of the catalytic reaction is 35 ℃.

8. The enzymatic method for preparing 5' -guanylic acid according to claim 2, wherein the time for catalytic reaction is 6-8 hours.

9. The enzymatic method for preparing 5' -guanylic acid according to claim 2, wherein the method for preparing the pure enzyme of the acid phosphotransferase mutant comprises culturing recombinant Escherichia coli containing the gene encoding the acid phosphotransferase mutant to OD ₆₀₀ of 0.6-0.8, adding IPTG with a final concentration of 1mM, inducing at 25 ℃ for 10-12 h, collecting the bacterial cells, performing ultrasonic disruption to obtain cell lysate, centrifuging to obtain supernatant, and further separating and purifying to obtain the pure enzyme of the acid phosphotransferase mutant.