A cytidine kinase mutant and use thereof

Abstract

The application belongs to the field of genetic engineering, and particularly relates to a cytidine kinase mutant and application thereof. The application is based on the amino acid of wild-type cytidine kinase (cytidine kinase, EC 2.7.1.213) from Thermus thermophilus, and a cysteine is introduced on the surface of the wild-type cytidine kinase protein to form a disulfide bond in the spatial structure, so as to improve the stability of the three-dimensional structure of the protein, and a cytidine kinase mutant with improved stability is screened. The modified cytidine kinase can maintain an active state for a long time at a catalytic reaction temperature, so as to improve the stability of the catalytic system and improve the efficiency, and meet the needs of industrial production.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a cytidine kinase mutant and its applications. Background Technology

[0002] Cytidine acid itself is used as a food additive, genetic engineering reagent, and pharmaceutical raw material. It promotes gastrointestinal cell development and improves the intestinal and gastrointestinal microecology. It also possesses antiviral, antitumor, lymphocyte immune-enhancing, and nucleic acid metabolism-inhibiting effects, and serves as an intermediate raw material for the production of nucleotide drugs such as citicoline and cytidine triphosphate. Cytidine acid can be synthesized from cytidine using cytidine kinase UDK (EC 2.7.1.213). However, during cytidine acid production, the industrial catalytic environment significantly reduces the activity of the catalytic enzyme. When stored at 35°C for more than 5 hours, the activity of cytidine kinase UDK (EC 2.7.1.213) decreases by more than 50%. To increase the stability of this enzyme under industrial catalytic conditions and improve the conversion rate of cytidine acid, it is necessary to construct a cytidine kinase with enhanced stability for better application in the preparation of cytidine acid. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by introducing cysteine ​​residues on the surface of wild-type cytidine kinase protein to construct a cytidine kinase mutant, so that adjacent amino acid residues in its spatial structure form disulfide bonds, thereby enhancing the stability of the protein's three-dimensional structure.

[0004] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned cytidine kinase mutant in the preparation of cytidine acid.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A cytidine kinase mutant, wherein the amino acid sequence of the cytidine kinase mutant is obtained by mutating lysine at position 19 to cysteine, leucine at position 46 to cysteine, glycine at position 124 to cysteine, proline at position 175 to cysteine, and glutamic acid at position 195 to cysteine.

[0007] Specifically, the mutations are K19C, L46C, L124C, P175C, and E195C.

[0008] The wild-type cytidine kinase is derived from Thermus thermophilus, whose amino acid sequence is shown in SEQ ID NO: 1, and the corresponding nucleotide sequence encoding the wild-type cytidine kinase is shown in SEQ ID NO: 2.

[0009] The amino acid sequence of the cytidine kinase mutant is shown in SEQ ID NO: 3.

[0010] The nucleotide sequence encoding the cytidine kinase mutant is shown in SEQ ID NO: 4.

[0011] A recombinant expression vector comprising the nucleotide sequence encoding the cytidine kinase mutant described above.

[0012] The expression vector can be any plasmid vector conventional in the art, as long as the recombinant expression vector can replicate and be expressed normally in the corresponding expression host. A preferred expression vector is pET28a, which is obtained by extracting the E. coli DHSα / pET28a plasmid (purchased from Nanjing Novizan Biotechnology Co., Ltd.), followed by enzyme digestion, separation, recovery, and purification.

[0013] A recombinant expression transformant containing the nucleotide sequence of the above-described recombinant expression vector or the above-described cytidine kinase mutant. It is prepared by transforming the constructed recombinant expression vector into host cells.

[0014] The host cell can be any conventional host cell in the art, as long as the recombinant expression vector can replicate stably and effectively express the target protein after induction with an inducer. Preferably, the host cell is Escherichia coli BL21(DE3) (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0015] After culturing the recombinant expression transformant under conditions suitable for cytidine kinase mutant expression, the cytidine kinase mutant cells were collected by centrifugation.

[0016] Specifically, the strain was streaked onto LB agar plates containing 50 μg / mL kanamycin resistance and incubated at 37°C for 12 h. Single colonies were picked and transferred to 50 mL centrifuge tubes (containing 10% v / v LB liquid medium with 50 μg / mL kanamycin resistance) and incubated at 37°C and 220 rpm for 12 h. Then, a 10% v / v inoculum was transferred to 1 L shake flasks (containing 25% v / v LB liquid medium with 50 μg / mL kanamycin resistance) and incubated at 37°C and 220 rpm until OD (out of control) was reached. 600When the concentration was 0.8, IPTG was added to a final concentration of 0.2 mM, the temperature was lowered to 30°C, the temperature was increased to 200 rpm, and the cells were cultured for another 10 h to induce expression. The cells were then centrifuged and collected for subsequent catalytic reactions.

[0017] In some embodiments of the present invention, the obtained cytidine kinase mutant bacterial cells were subjected to enzyme activity detection. The detection method is as follows: a 50 mL enzyme activity assay system contained 30 mM cytidine, 100 mM MgCl2·6H2O, 30 mM ATP, 10 g / L xylene, and 20 g / L cytidine kinase mutant. The reaction mixture was stirred at 750 rpm for 1 h at 35 °C, and the pH of the reaction was maintained at 7.5 by adding 5 mol / L alkaline solution. The mixture was stored at 35 °C for 5 h and 10 h, respectively, and the enzyme activity of the cytidine kinase mutant under each environment was detected (with wild-type cytidine kinase as a control). The enzyme activity is defined as the amount of enzyme required to produce 1 μmol CMP (cytidine nucleotide) per minute under the conditions of 30-37 °C and pH = 7-8, preferably at 35 °C and pH 7.5.

[0018] Specifically, the cytidine kinase mutant, when stored in an industrial catalytic environment at 35°C for 5 hours and 10 hours, showed that its enzyme activity was increased by 36.5% and 93.8% respectively compared to that of the wild-type cytidine kinase.

[0019] The application of the above-mentioned cytidine kinase mutant in the catalytic synthesis of cytidine acid is also within the scope of protection of this invention.

[0020] That is, using cytidine as a substrate and ATP as a co-substrate, the synthesis of cytidine nucleotide is catalyzed by a cytidine kinase mutant.

[0021] The catalytic synthesis described herein comprises: 80-150 mM cytidine, 30-80 mM MgCl2·6H2O, 80-150 mM ATP, 5-15 g / L xylene, and 50-150 g / L cytidine kinase mutant; preferably: 100 mM cytidine, 50 mM MgCl2·6H2O, 100 mM ATP, 10 g / L xylene, and 100 g / L cytidine kinase mutant.

[0022] The catalytic synthesis is carried out under the following reaction conditions: 30-37℃, 500-1000rpm, pH=7-8; preferably 35℃, 750rpm, pH=7.5, for 10h or 15h.

[0023] Specifically, the cytidine kinase mutant was catalyzed in an industrial environment at 35°C for 5h, 10h and 15h. The cytidine conversion rate of the cytidine kinase mutant was significantly higher than that of the wild-type cytidine kinase in the later stages. After 10h and 15h of reaction, its cytidine conversion rate was 31.9% and 46.9% higher than that of the wild-type cytidine kinase, respectively.

[0024] Beneficial effects: Compared with the prior art, the present invention constructs a cytidine kinase mutant with enhanced stability. Compared with wild-type cytidine kinase, it has stronger adaptability in the industrial catalytic environment of cytidine synthesis of cytidine acid using cytidine as a raw material. It can maintain its active state for a long time at the catalytic reaction temperature, thereby ensuring the stability of the catalytic system and improving the efficiency of cytidine acid generation. It can better meet the needs of industrial production and thus has broad application prospects. Attached Figure Description

[0025] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0026] Figure 1 This is a diagram showing the plasmid construction of the recombinant expression vector for the cytidine kinase mutant. Detailed Implementation

[0027] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0028] In this invention, the cytidine kinase stability-enhancing mutant M and the cytidine kinase mutant M have the same meaning, both being mutants obtained by site-directed mutation (K19C, L46C, L124C, P175C, E195C) of wild-type cytidine kinase WT.

[0029] Example 1: Construction of wild-type Thermus thermophilus cytidine kinase and construction of its stability-enhancing mutant.

[0030] Wild-type Thermus thermophilus cytidine kinase WT, according to the corresponding sequence described by NCBI, was amplified from the genome using in vitro PCR technology, and its corresponding nucleotide sequence is: (SEQ ID NO:1)

[0031] ATGAGCGCGCCGAAACCGTTTGTGATTGGCATTGCGGGCGGCACCGCGAGCGGCAAAACCACCCTGGCGCAGGCGCTGGCGCGCACCCTGGGCGAACGCGTGGCGCTGCTGCCGATGGATCATTATTATAAAGATCTGGGCCATCTGCCGCTGGAAGAACGCCTGCGCGTGAACTATGATCATCCGGATGCGTTTGATCTGGCGCTGTATCTGGAACATGCGCAGGCGCTGCTGCGCGGCCTGCCGGTGGAAATGCCGGTGTATGATTTTCGCGCGTATACCCGCAGCCCGCGCCGCACCCCGGTGCGCCCGGCGCCGGTGGTGATTCTGGAAGGCATTCTGGTGCTGTATCCGAAAGAACTGCGCGATCTGATGGATCTGAAAGTGTTTGTGGATGCGGATGCGGATGAACGCTTTATTCGCCGCCTGAAACGCGATGTGCTGGAACGCGGCCGCAGCCTGGAAGGCGTGGTGGCGCAGTATCTGGAACAGGTGAAACCGATGCATCTGCATTTTGTGGAACCGACCAAACGCTATGCGGATGTGATTGTGCCGCGCGGCGGCCAGAACCCGGTGGCGCTGGAAATGCTGGCGGCGAAAGCGCTGGCGCGCCTGGCGCGCATGGGCGCGGCG

[0032] The corresponding amino acid sequence is: (SEQ ID NO: 2)

[0033] MSAPKPFVIGIAGGTASGKTTLAQALARTLGERVALLPMDHYYKDLGHLPLEERLRVNYDHPDAFDLALYLEHAQALLRGLPVEMPVYDFRAYTRSPRRTPVRPAPVVILEGILVLYPKELRDLMDLKVFVDADADERFIRRLKRDVLERGRSLEGVVAQYLEQVKPMHLHFVEPTKRYADVIVPRGGQNPVALEMLAAKALARLARMGAA

[0034] The specific operation steps are as follows:

[0035] (1) Thermus thermophilus was inoculated into YP (containing 20 g / L peptone and 30 g / L yeast extract) liquid medium and cultured at 30°C until the logarithmic growth phase. The genome was extracted using a bacterial genome extraction kit (purchased from Solarbio Science & Technology Co., Ltd.) to obtain the Thermus thermophilus genome template.

[0036] Based on the cytidine kinase gene of *Thermus thermophilus* already existing in the NCBI database, primers UDK-F (ACGCGAATTCATGAGCGCGCCGAAACCGTTT) and UDK-R (CGTAAAGCTTCGCCGCGCCCATGCGCGCCAGG) (designed and synthesized by Nanjing GenScript Technology Co., Ltd.) were designed and synthesized for PCR amplification to obtain the PCR reaction solution.

[0037] The PCR reaction system included: 2.5 μL 10×Buffer (Mg 2+ The following reagents were prepared: 2 μL dNTP Mixture (2.5 mM), 3 μL MgCl2 (25 mM), 0.1 μL primer UDK-F (100 μM), 0.1 μL primer UDK-R (100 μM), 1 μL Hermus thermophilus genome template, 0.5 μL PCR amplification high-fidelity enzyme (purchased from Takara), and sterile double-distilled water to a final volume of 25 μL. The PCR reaction parameters were: denaturation at 95°C for 5 minutes, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute, 20 cycles, incubation at 72°C for 15 minutes, followed by incubation at 16°C for 1 hour.

[0038] (2) Dilute 10×TBE buffer 20 times to 0.5×TBE working buffer; prepare a nucleic acid electrophoresis gel containing 1% agarose with Gel Red nucleic acid dye, mix 4μL PCR reaction solution or DL ​​2000 DNA marker (control, purchased from Takara) and 1μL 10×Loading Buffer (purchased from Takara), add the sample to the sample slot of the nucleic acid gel plate with a micropipette, start running the gel at 100V after adding the sample, stop electrophoresis when the blue band in the gel moves to 1.5 cm from the bottom edge, take the gel for observation and use a DNA gel recovery kit (TaKaRa) to cut and recover the gel. The gel-recovered product was ligated into the pMD18T-vector vector (purchased from Takara). The ligation product was transformed into E. coli DHSα competent cells prepared by the calcium chloride method and plated on LB agar plates containing 50 mg / L kanamycin (containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 5 g / L agar). The plates were incubated at 30°C overnight. Single colonies were picked from the LB agar plates and placed in 5 mL of LB liquid medium (containing 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride). The plates were incubated at 30°C and 220 rpm for 12 hours, after which the recombinant plasmid was extracted.

[0039] The extracted recombinant plasmid was verified by double enzyme digestion. The double digestion system included: 8.4 μL recombinant plasmid, 0.3 μL EcoRI, 0.3 μL HindIII, and 1 μL 10× buffer. After enzyme digestion verification, sequencing was performed by Nanjing GenScript Biotech Co., Ltd. Bacterial cells with correct sequencing results were inoculated into 5 ml LB liquid medium and cultured at 30℃ and 220 rpm for 12 hours. The recombinant plasmid was then extracted. The extracted recombinant plasmid was double-digested using the following system: 84 μL recombinant plasmid, 3 μL EcoRI, 3 μL HindIII, and 10 μL 10× buffer. The digestion was incubated overnight at 37℃ for 12 hours. The gene fragment UDK, approximately 600 bp in size, was separated, recovered, and purified by 1% agarose gel electrophoresis.

[0040] (3) E. coli DHSα / pET28a, purchased from Nanjing Novizan Biotechnology Co., Ltd., was inoculated into 5 mL of LB liquid medium and cultured at 37°C and 220 rpm for 12 hours. The plasmid was then extracted. The extracted plasmid was subjected to double enzyme digestion using the same digestion system as above. The digestion was incubated overnight at 37°C for 12 hours. The gene fragment of approximately 5000 bp was separated, recovered, and purified by 1% agarose gel electrophoresis, which was the desired vector fragment pET28a. The purified gene fragment UDK and the vector fragment pET28a were ligated overnight using the following ligation system: 4 μL gene fragment UDK, 1 μL vector fragment pET28a, 5 μL Solution I, ligated overnight at 16°C.

[0041] (4) Escherichia coli BL21(DE3) competent cells (purchased from Nanjing Novizan Biotechnology Co., Ltd.) prepared by converting the ligation product into calcium chloride were plated on LB agar plates containing 50 mg / L kanamycin and incubated overnight at 37°C. Ten single colonies were picked and inoculated into LB liquid medium, double-digested for verification, and sequenced. The correctly verified strains were identified as wild-type Thermus thermophilus cytidine kinase strains.

[0042] Example 2: Construction of Cytidine Kinase Stability Enhancement Mutant M

[0043] Site-directed mutagenesis was performed on the amino acid residue sites of the wild-type cytidine kinase in Example 1. The modification sites and methods are as follows:

[0044] The lysine at position 19 is mutated to cysteine, the leucine at position 46 is mutated to cysteine, the glycine at position 124 is mutated to cysteine, the proline at position 175 is mutated to cysteine, and the glutamic acid at position 195 is mutated to cysteine, namely K19C, L46C, L124C, P175C, E195C.

[0045] The amino acid sequence of the cytidine kinase stability-enhancing mutant M is: (SEQ ID NO:3)

[0046] MSAPKPFVIGIAGGTASGCTTLAQALARTLGERVALLPMDHYYKDCGHLPLEERLRVNYDHPDAFDLALYLEHAQALLRGLPVEMVYDFRAYTRSPRRTPVRPAPVVILEGILVLYPKELRDCMDLKVFVDADADERFIRRLKRDVLERGRSLEGVVAQYLEQVKPMHLHFVECTKRYADVIVPRGGQNPVALCMLAAKALARLARMGAA

[0047] The nucleotide sequence of the corresponding cytidine kinase stability-enhancing mutant M is: (SEQ ID NO:4)

[0048] ATGAGCGCGCCGAAACCGTTTGTGATTGGCATTGCGGGCGGCACCGCGAGCGGCAGCACCACCCTGGCGCAGGCGCTGGCGCGCACCCTGGGCGAACGCGTGGCGCTGCTGCCGATGGATCATTATTATAAAGATAGCGGCCATCTGCCGCTGGAAGA ACGCCTGCGCGTGAACTATGATCATCCGGATGCGTTTGATCTGGCGCTGTATCTGGAACATGCGCAGGCGCTGCTGCGCGGCCTGCCGGTGGAAATGCCGGTGTATGATTTTCGCGCGTATACCCGCAGCCCGCGCCGCACCCCGGTGCGCCCGGCGC CGGTGGTGATTCTGGAAGGCATTCTGGTGCTGTATCCGAAAGAACTGCGCGATAGCATGGATCTGAAAGTGTTTGTGGATGCGGATGCGGATGAACGCTTTATTCGCCGCCTGAAACGCGATGTGCTGGAACGCGGCCGCAGCCTGGAAGGCGTGGTG GCGCAGTATCTGGAACAGGTGAAACCGATGCATCTGCATTTTGTGGAAAGCACCAAACGCTATGCGGATGTGATTGTGCCGCGGCGGCCAGAACCCGGTGGCGCTGAGCATGCTGGCGGCGAAAGCGCTGGCGCGCCTGGCGCGCATGGGCGCGGCG

[0049] The gene sequence of the above-mentioned cytidine kinase stability-enhanced mutant M was obtained using a whole-genome synthesis method (synthesized by Nanjing GenScript Technology Co., Ltd.). It was then constructed through enzyme digestion, ligation, gel recovery, and transformation (same as in Example 1; the plasmid construction diagram of the recombinant expression vector of the cytidine kinase mutant is shown in Figure 1). Figure 1 (As shown) A strain was obtained to obtain the cytidine kinase stability-enhanced mutant M.

[0050] Example 3: Detection of the effect of cytidine kinase stability-enhancing mutant modification

[0051] (1) Culture of wild-type cytidine kinase and its mutant M: The strain was streaked on LB agar plates containing 50 μg / mL kanamycin resistance and incubated at 37°C for 12 h. Single colonies were picked and transferred to 50 mL centrifuge tubes (containing 10% v / v LB liquid medium with 50 μg / mL kanamycin resistance) and incubated at 37°C and 220 rpm for 12 h. Then, 10% v / v inoculation was carried into 1 L shake flasks (containing 25% v / v LB liquid medium with 50 μg / mL kanamycin resistance) and incubated at 37°C and 220 rpm until OD. 600 When the concentration was 0.8, IPTG was added to a final concentration of 0.2 mM, the temperature was lowered to 30°C, the temperature was increased to 200 rpm, and the cells were cultured for another 10 h to induce expression. The cells were then centrifuged and collected for subsequent catalytic reactions.

[0052] (2) Enzyme activity detection of wild-type cytidine kinase and its mutant M: 50 mL enzyme activity assay system contained 30 mM cytidine, 100 mM MgCl2·6H2O, 30 mM ATP, 10 g / L xylene, and 20 g / L wild-type cytidine kinase or mutant M. The reaction mixture was stirred at 750 rpm for 1 h at 35 °C, and the pH of the reaction was maintained at 7.5 by adding 5 mol / L alkaline solution. The mixture was stored at 35 °C for 5 h and 10 h, respectively. The enzyme activity of wild-type cytidine kinase and its mutant M under each environment was detected. The results are shown in Table 1.

[0053] The enzyme activity is defined as the amount of enzyme required to produce 1 μmol of CMP (cytidine monophosphate) per minute under conditions of 35°C and pH 7.5.

[0054] Table 1. Activity differences of wild-type cytidine kinase and its mutant M after storage at industrial catalytic temperatures for different durations.

[0055] Enzyme activity after storage at 35℃ for 0 hours 102±3.1 95±2.5 Enzyme activity after storage at 35℃ for 5 hours 52±3.1 71±4.2 Enzyme activity after storage at 35℃ for 10 hours 32±4.3 62±3.5

[0056] (3) Cytidine conversion reaction of wild-type cytidine kinase and its mutant M: 1 L catalytic system contained 100 mM cytidine, 50 mM MgCl2·6H2O, 100 mM ATP, 10 g / L xylene, and 100 g / L wild-type cytidine kinase or mutant M. The reaction mixture was stirred at 750 rpm for 1 h at 35 °C. The pH of the reaction was maintained at 7.5 by adding 5 mol / L alkaline solution. The reaction was carried out at 35 °C for 5 h, 10 h and 15 h respectively. The conversion rate of cytidine under each environment was detected. The results are shown in Table 2.

[0057] Table 2. Cytidine conversion rates of wild-type cytidine kinase and its mutant M at different reaction times.

[0058] 5h cytidine conversion rate 41％ 38％ 10h cytidine conversion rate 47％ 62％ 15h cytidine conversion rate 49％ 72％

[0059] The results showed that, under initial conditions, the activity of the stability-enhanced mutant M of cytidine kinase was similar to that of the wild-type cytidine kinase. However, long-term industrial catalytic environments weakened enzyme activity. As shown in Table 1, the activity of the wild-type cytidine kinase was significantly inhibited, while the mutant exhibited higher activity than the wild-type, and the degree of activity reduction was significantly less than that of the wild-type. After storage at 35℃ for 5 h and 10 h in an industrial catalytic environment, the enzyme activity of the stability-enhanced mutant M was increased by 36.5% and 93.8% compared to that of the wild-type cytidine kinase, respectively. Table 2 also shows that, with the progression of reaction time, the cytidine conversion rate of the stability-enhanced mutant M was significantly higher than that of the wild-type cytidine kinase in the later stages. After 10 h and 15 h of reaction, its cytidine conversion rate was increased by 31.9% and 46.9% compared to that of the wild-type cytidine kinase, respectively. Therefore, the modified cytidine kinase is more suitable for the industrial catalytic environment for the synthesis of cytidine acid from cytidine, which is beneficial for improving reaction efficiency.

[0060] This invention provides a concept and method for cytidine kinase mutants and their applications. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A cytidine kinase mutant, characterized in that, The amino acid sequence of the cytidine kinase mutant is obtained by mutating lysine at position 19 to cysteine, leucine at position 46 to cysteine, leucine at position 124 to cysteine, proline at position 175 to cysteine, and glutamic acid at position 195 to cysteine ​​from the amino acid sequence of wild-type cytidine kinase. The wild-type cytidine kinase mentioned above is derived from *Thermophilus thermophilus*. Thermus thermophilus Its nucleotide sequence is shown in SEQ ID NO: 1, and the corresponding amino acid sequence is shown in SEQ ID NO:

2.

2. The application of the cytidine kinase mutant according to claim 1 in the catalytic synthesis of cytidine nucleotides; wherein, The catalytic synthesis described herein uses cytidine as a substrate and ATP as an auxiliary substrate, and cytidine is synthesized into cytidine acid by catalysis of cytidine by a cytidine kinase mutant.

3. The application according to claim 2, characterized in that, The catalytic synthesis described herein uses a catalytic system consisting of: 80-150 mM cytidine, 30-80 mM MgCl2·6H2O, 80-150 mM ATP, 5-15 g / L xylene, and 50-150 g / L cytidine kinase mutant.

4. The application according to claim 3, characterized in that, The catalytic synthesis described herein uses a catalytic system consisting of: 100 mM cytidine, 50 mM MgCl2·6H2O, 100 mM ATP, 10 g / L xylene, and 100 g / L cytidine kinase mutant.

5. The application according to claim 2, characterized in that, The catalytic synthesis described herein is performed under the following reaction conditions: 30-37℃, 500-1000 rpm, pH=7-8.

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