WO2022052191A1 - Histidinol-phosphate aminotransferase mutant, engineering bacteria and use - Google Patents

Abstract

A histidinol-phosphate aminotransferase mutant, engineering bacteria and the use of the histidinol-phosphate aminotransferase mutant in the production of indole-3-pyruvic acid. A histidinol-phosphate aminotransferase gene is derived from a Streptomyces luteoverticillatus strain HY61, and the nucleotide sequence of the gene is as shown in SEQ ID NO: 1, and the amino acid sequence of the gene is as shown in SEQ ID NO: 2. The mutant is obtained by means of single mutation or multi-site mutations at position 66, position 138, position 197, position 226 and position 311 of the amino acid sequence as shown in SEQ ID NO: 2.

Description

A kind of histidine alcohol phosphate aminotransferase mutant, engineering bacteria and application technical field

The invention relates to histidine alcohol phosphate aminotransferase mutants, engineering bacteria and applications, and belongs to the field of biotechnology.

Background technique

3-Indolepyruvate (IPA) is an important intermediate compound and the key to the manufacture of 3-indoleacetic acid (plant growth hormone), 3-indoleethanol, DL-tryptophan and D-tryptophan. It is a precursor, and itself is a therapeutic drug for the nervous system. It has a wide range of uses in the fields of agriculture, food and medicine, and has broad application prospects. The traditional chemical synthesis of IPA requires a large amount of base and pyridine aldehyde, which is expensive, and has harsh reaction conditions, complicated product purification, low yield, and the risk of environmental pollution by by-products. Oxidative deamination using L-amino acid oxidase is also an important method for synthesizing IPA, but the by-product hydrogen peroxide will lead to the degradation of IPA, and the yield and purity are difficult to guarantee. Patent CN200880104143.X utilizes Escherichia coli engineering to express the amino acid oxidase derived from Providencia rettgeri AJ2770 strain, transforms L-tryptophan to produce IPA, and in the 200mmol/L substrate tryptophan reaction system, The highest concentration of IPA was only 129 mM, and the product yield was low.

Transaminase (Transaminase), also known as aminotransferase (Aminotransferase), catalyzes the aminotransferase between the amino group and the ketone group, and can use the cheap raw material L-tryptophan to produce IPA through transamination. It produces by-products (such as hydrogen peroxide) that destabilize ketoacids, which has great application prospects.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a histidine alcohol phosphate aminotransferase-producing mutant, recombinant bacteria and whole cells thereof as catalysts for the synthesis of 3-indolepyruvic acid.

The present invention is achieved through the following technical solutions:

The invention provides a histidine phosphate aminotransferase mutant, the histidine phosphate aminotransferase gene is derived from Streptomyces luteoverticillatus strain HY61 (Streptomyces luteoverticillatus) CGMCC15060, and its nucleotide sequence is such as SEQ ID NO: 1, the amino acid sequence is shown in SEQ ID NO: 2. The mutant is obtained by single mutation or multi-site mutation at the 66th position, 138th position, 197th position, 226th position and 311th position of the amino acid sequence shown in SEQ ID NO: 2.

Further, the mutant is one of the following, the 66th valine is mutated to isoleucine (hisC-V66I), the 138th asparagine is mutated to glutamine (hisC-N138Q), the 197th serine Histidine phosphate aminotransferase mutated to histidine (hisC-S197H) or proline 226 to phenylalanine and arginine 311 to tyrosine (hisC-P226F-R311Y) The mutant, more preferably the mutant is hisC-P226F-R311Y.

The present invention also relates to a gene encoding the histidine alcohol phosphate aminotransferase mutant.

The present invention provides engineered bacteria containing the encoded gene.

The present invention also provides an application of the histidine alcohol phosphate aminotransferase mutant in catalyzing the synthesis of 3-indolepyruvate from L-tryptophan.

Further, the described application method is to use the bacterial cell harvested by fermentation and centrifugation of the engineered bacteria containing the histidine alcohol phosphate aminotransferase mutant gene as a whole-cell catalyst, use L-tryptophan as a substrate, and use L-chromogen as a substrate. Pyruvate with an amino acid molar concentration of 1.2 times is an amino acceptor, adding 25mg/L coenzyme pyridoxal phosphate, 1g/L stabilizing protective agent to the conversion system, ammonia water is adjusted to pH 7.5, nitrogen is used to drive oxygen, and the temperature is 30 The reaction produces 3-indolepyruvic acid under the condition of ~40°C.

Further, in the transformation system, the amount of catalyst used is 10-40 g/L in terms of wet cell weight, the substrate concentration is 50-200 mM, and the pyruvate concentration is 60-240 mM.

Further, the engineered bacteria containing the histidine alcohol phosphate aminotransferase mutant gene are prepared as follows: the recombinant Escherichia coli containing the histidine alcohol phosphate aminotransferase mutant gene is inserted into the LB slant medium and cultured at 37°C for 12 to 18 hours; Connect 1 ring of slanted inoculum to LB liquid seed medium, shake at 37°C and 180r/min for 4-10h; put 2.0L of medium into a 3L fermenter, and insert the seed liquid with a volume ratio of 2-12% of the inoculum. Fermentation medium, the initial rotation speed is 220r/min, and the initial ventilation flow rate is 1.0L/min. With the increase of bacterial concentration, the rotation speed and ventilation flow rate are adjusted to maintain the dissolved oxygen value at 20-30% air saturation. The mass-volume ratio is 25% ammonia water was used to adjust the pH value to be stable at 6.7. After culturing at 37°C for 4-12h, lactose was added to the final concentration of 1-5g/L and cooled to 20-30°C to induce expression; during the fermentation process, 500g/L glycerol solution was added to maintain glycerol. After the fermentation at a concentration of 5-10 g/L, the cells were collected by centrifugation at 10,000 r/min and 4°C for 10 min, and washed twice with sterile saline to obtain histidine alcohol phosphate aminotransferase mutant whole-cell catalysts.

Further, the composition and final concentration of the LB slant medium: yeast extract powder 5g/L, peptone 10g/L, NaCl 5g/L, ampicillin 100mg/L, agar 20g/L, pH7.0～7.2, 121 ℃ high pressure steam sterilization for 20min;

Further, the composition and final concentration of the LB liquid seed medium: yeast extract powder 5g/L, peptone 10g/L, NaCl 5g/L, ampicillin 100mg/L, pH7.0～7.2, 121 ℃ of high pressure steam sterilization bacteria 20min;

Further, the composition and final concentration of the fermentation medium: glycerol 20g/L, peptone 20g/L, yeast extract 5g/L, MgSO ₄ 2g/L, KH ₂ PO ₄ 12.5g/L, (NH ₄ ) ₂ SO ₄ 2g/L, citric acid 1g/L, corn steep liquor powder 3g/L, CaCl ₂ 1g/L, D-biotin 20μg/L, pH 6.7～7.0, 121 ℃ high pressure steam sterilization for 20min.

Further, the stabilizing protective agent is one or a combination of sodium sulfite and phytic acid.

The beneficial effects of the present invention compared with the prior art are:

The invention adopts the error-prone PCR technology to successfully mutate the histidine alcohol phosphate aminotransferase derived from Streptomyces garciniae and constructs an engineering strain. The screened mutant enzyme activity is up to 120% higher than the original, and is used for 3-indole For the enzymatic synthesis of pyruvate, the stability of 3-indolepyruvic acid is effectively improved through the combined use of nitrogen-filled protection and stabilizer. At the same time, the raw materials used in the production method of the present invention are cheap and easy to obtain, the production process is simple and feasible, and the production cost is low. The established whole-cell transformation system solves the problems of harsh reaction conditions for chemical synthesis of IPA, complicated product purification, low yield, environmental pollution risks of by-products, and the influence of L-amino acid oxidase by-product hydrogen peroxide on the product. Destroy the impact and achieve high-yield green production of IPA.

detailed description

The technical solutions of the present invention are further explained below through the examples, but the protection scope of the present invention is not limited in any form by the examples.

Example 1: Construction of expression vector and engineered bacteria

According to the strain HY61 of Streptomyces garcinata, the biological deposit number is CGMCC 15060 (this strain has been biologically deposited and disclosed in the patent 2018100327172 before the application date) gene hisC sequence (SEQ ID No. 1) is designed to contain Nco I digestion The upstream primer of the site hisC-NcoI-F: 5′-aaccatggcaatgacgcgattacacgagctcc-3′, and the downstream primer hisC-XhoI-R containing the Xho I restriction site:

5′-aactcgagtcaggcggccgccggtccggcg-3′, the black italic is the restriction site. Bacterial genomic DNA extraction kit was used to extract the genomic DNA of Streptomyces garcinata, and the genomic DNA was used as a template for PCR amplification. The PCR reaction system included 2×GC Buffer I 25 μL, dNTP Mix 8 μL, TaKaRa LA Taq 0.5 μL, template 0.5 μL, upstream and downstream primers 1.0 μL each, supplemented to 50 μL with dd H ₂ O. PCR reaction parameters were: 94°C for 1 min, 1 cycle; 94°C for 30 s, 60°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min, 1 cycle. PCR amplification products were detected by 1% agarose gel, and target DNA fragments were purified by AxyPrep DNA gel recovery kit. The vector pET32a and its fragment were digested with Nco I and Xho I, respectively, and then the hisC fragment was ligated with the vector with T4 DNA ligase and transformed into E. coli DH5α. After the positive clones were verified by colony PCR and enzyme digestion, the pET32a-hisC recombinant vector was obtained.

The recombinant vector pET32a-hisC was used as a template for amplification by error-prone PCR. The reaction system of error-prone PCR is: PCR Grade Water 39μL, 10×TITANIUM Taq Buffer 5μL, MnSO4 (8mM) 1μL, dGTP (2mM) 1μL, 50×Diversify dNTP Mix 1μL, Primer mix 1μL, Template 1μL, TITANIUM Taq Polym 1μL , a total of 50 μL of the system (wherein the Primer mix in the reaction system is a mixed solution of 0.5 μL of each of the upstream and downstream primers described in Example 1). The error-prone PCR reaction conditions were: 94°C for 30s, 1 cycle; 94°C for 30s, 68°C for 1min30s, 25 cycles; 68°C for 1min, 1 cycle. Error-prone PCR amplification products were detected on a 1% agarose gel and purified using the AxyPrep DNA Gel Recovery Kit. The vector pET32a and the amplified product were digested with Nco I and Xho I respectively, and then the amplified product was connected with the vector with T4 DNA ligase, and the connected recombinant vector was thermally transferred into E.coli BL21 (DE3) competent cells, The cells were incubated at 37°C at 200 r/min for 1 h for activation, and the activated recombinant cells were spread on LB plates containing 0.1 mg/mL ampicillin resistance, and incubated overnight at 37°C upside down to obtain histidine alcohol amino phosphate. Transferase mutant expression library.

From the obtained histidine phosphate aminotransferase mutant expression library colonies, high-throughput screening methods were used to screen high-activity mutant strains from these single colonies. The specific screening method is as follows: randomly pick 600 single colonies from the plate, insert it into a 96-deep-well plate, the medium is LB liquid medium, cultivate at 37°C for 9 hours at 180 r/min, collect the bacteria by centrifugation, and remove the supernatant. Fermentation medium was added after the medium, cultured at 37°C, 220r/min for 6h, lactose was added to the final concentration of 2g/L, and the temperature was lowered to 20°C to induce expression; the cells were collected by centrifugation, and the enzyme activity was determined. Standard enzyme activity detection system: 25g/L wet bacterial cells, 50mM substrate L-tryptophan, 25mg/L coenzyme pyridoxal phosphate, 60mM pyruvate, the reaction medium is phosphate buffer at pH 7.5, and the total system is 1mL . Definition of unit enzyme activity: Under standard reaction conditions, the amount of enzyme required to generate 1 μmol of 3-indolepyruvate per minute is one unit of enzyme activity U. Four highly active histidine alcohol phosphate aminotransferase mutant strains were obtained by screening, and after sequencing, the 66th valine in the amino acid sequence shown in SEQ ID NO: 2 was mutated to isoleucine (hisC-V66I), the Asparagine 138 was mutated to glutamine (hisC-N138Q), serine 197 was mutated to histidine (hisC-S197H), proline 266 was mutated to phenylalanine, and arginine 311 was mutated Histidine alcohol phosphate aminotransferase mutants mutated to tyrosine (hisC-P226F-R311Y), the corresponding engineering strains are E.coli BL21(DE3)/pET32a-hisC-V66I, E.coli BL21(DE3) /pET32a-hisC-N138Q, E.coli BL21(DE3)/pET32a-hisC-S197H, E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y. Among them, the engineering strain E. coli BL21(DE3)/pET32a-hisC-P226F-R311Y had the highest activity (Table 1).

Table 1 Histidine alcohol phosphate aminotransferase mutant enzyme activity

Example 2: Production of histidine alcohol phosphate aminotransferase mutants (hisC-N138Q, hisC-P226F-R311Y) by recombinant bacteria fermentation and enzymatic transformation of L-tryptophan to produce IPA

(1) E.coli BL21(DE3)/pET32a-hisC-N138Q and E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y were respectively inserted into LB slant medium and cultured at 37℃ for 18h; Planted in LB liquid seed medium, 37°C, 180r/min shaking culture for 9h; 2.0L medium was placed in a 3L fermenter, the seed liquid was inserted into the fermentation medium at a volume ratio of 4% inoculum, and the initial speed was 220r/min , the initial ventilation flow is 1.0L/min, and the speed and ventilation flow are adjusted with the increase of bacterial concentration to maintain the dissolved oxygen value at 20-30% air saturation, and the pH value is stabilized at 6.7 with a mass-volume ratio of 25% ammonia water. , after culturing at 37°C for 6h, add lactose to a final concentration of 2g/L and cool down to 20°C to induce expression; during the fermentation process, 500g/L glycerol solution was added to maintain the glycerol concentration at 5-10g/L after fermentation, 10000r/min, 4 The cells were collected by centrifugation at ℃ for 10 min, and the cells were washed twice with sterile normal saline to obtain the complete and complete samples of E. Cell Catalyst.

(2) The whole cell catalyzed production of 3-indolepyruvate using histidine alcohol phosphate aminotransferase mutant, the transformation conditions are: prepare 2L of 50mM L-tryptophan solution, add pyruvate with a tryptophan molar concentration of 1.2 times and 25mg/L of pyridoxal phosphate, 1g/L of phytic acid, ammonia water to adjust pH to 7.5, add 10g of wet cells, flow nitrogen to drive oxygen at a flow rate of 0.5L/min for 15min, maintain the temperature at 35°C, and ferment at 3L. Transformation on the tank for 3h, the IPA concentration of E.coli BL21(DE3)/pET32a-hisC-N138Q as catalyst was 45.7mM, and the molar conversion rate was 91.4%; E.coli BL21(DE3)/pET32a-hisC-P226F- The IPA concentration of R311Y as the catalyst was 47.6 mM, and the molar conversion was 95.2%.

Example 3: Production of histidine alcohol phosphate aminotransferase mutant (hisC-P226F-R311Y) by recombinant bacteria fermentation and enzymatic transformation of L-tryptophan to produce IPA

(1) The genetically engineered bacteria E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y was inserted into LB slant medium for 15 hours at 37°C; then a ring of slant strains was placed in LB liquid seed medium at 37°C, 180r /min shaking culture for 10h; 2.0L medium was placed in the 3L fermenter, the seed liquid was inserted into the fermentation medium at a volume ratio of 5% inoculum, the initial speed was 220r/min, and the initial ventilation flow was 1.0L/min. The concentration of bacteria was increased to adjust the rotational speed and ventilation flow to maintain the dissolved oxygen value at 20-30% air saturation. The pH value was adjusted to 6.7 with a mass-volume ratio of 25% ammonia water. After culturing at 37 °C for 4 hours, lactose was added to the final concentration of 1 g/L and cooled to 22 °C to induce expression; during the fermentation process, 500 g/L glycerol solution was added to maintain the glycerol concentration at 5-10 g/L. After the fermentation, the cells were collected by centrifugation at 10000 r/min and 4 °C for 10 min, and sterile saline was used. The cells were washed twice to obtain the whole cell catalyst.

(2) The whole cell catalyzed production of 3-indolepyruvic acid using histidine alcohol phosphate aminotransferase mutant (hisC-P226F-R311Y), the transformation conditions are: prepare 2L of 100mM L-tryptophan solution, add tryptophan Pyruvate with a molar concentration of 1.2 times, pyridoxal phosphate 25mg/L, sodium sulfite 1g/L, ammonia water to adjust the pH to 7.5, add 20g of wet bacteria, flow nitrogen to drive oxygen at a flow rate of 0.5L/min for 15min, and maintain the temperature At 35°C, the transformation was carried out on a 3L fermentor for 5h, the IPA concentration was 87.4mM, and the molar conversion rate was 87.4%.

Example 4: Production of histidine alcohol phosphate aminotransferase mutant (hisC-P226F-R311Y) by recombinant bacteria fermentation and enzymatic transformation of L-tryptophan to produce IPA

(1) The genetically engineered bacteria E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y was inserted into LB slant medium for 15 hours at 37°C; then a ring of slant strains was placed in LB liquid seed medium at 37°C, 180r /min shaking culture for 10h; 2.0L medium was placed in the 3L fermenter, the seed liquid was inserted into the fermentation medium at a volume ratio of 5% inoculum, the initial speed was 220r/min, and the initial ventilation flow was 1.0L/min. The concentration of bacteria was increased to adjust the rotational speed and ventilation flow to maintain the dissolved oxygen value at 20-30% air saturation. The pH value was adjusted to 6.7 with a mass-volume ratio of 25% ammonia water. After culturing at 37 °C for 4 hours, lactose was added to the final concentration of 1 g/L and cooled to 22 °C to induce expression; during the fermentation process, 500 g/L glycerol solution was added to maintain the glycerol concentration at 5-10 g/L. After the fermentation, the cells were collected by centrifugation at 10000 r/min and 4 °C for 10 min, and sterile saline was used. The cells were washed twice to obtain the whole cell catalyst.

(2) The whole cell catalyzed production of 3-indolepyruvic acid using histidine alcohol phosphate aminotransferase mutant (hisC-P226F-R311Y), the transformation conditions are: prepare 2L of 100mM L-tryptophan solution, add tryptophan 1.2 times the molar concentration of pyruvate, 25mg/L of pyridoxal phosphate, 1g/L of sodium sulfite, 1g/L of phytic acid, ammonia water to adjust the pH to 7.5, add 20g of wet cells, and pass through at a flow rate of 0.5L/min. Nitrogen was used to drive oxygen for 15 minutes, the temperature was maintained at 35°C, and the reaction was carried out in a 3L fermenter for 5 hours. The concentration of IPA was 92.2 mM, and the molar conversion rate was 92.2%.

Example 5: Production of histidine alcohol phosphate aminotransferase mutant (hisC-P226F-R311Y) by recombinant bacteria fermentation and enzymatic transformation of L-tryptophan to produce IPA

(1) The genetically engineered bacteria E.coli BL21(DE3)/pET32a-hisC-P226F-R311Y was inserted into LB slant medium for 14 hours at 37°C; then a ring of slant strains was placed in LB liquid seed medium at 37°C, 180r /min shaking culture for 8h; 2.0L medium was placed in the 3L fermenter, the seed liquid was inserted into the fermentation medium at a volume ratio of 5% inoculum, the initial rotation speed was 220r/min, and the initial ventilation flow was 1.0L/min. The concentration of bacteria was increased to adjust the rotational speed and ventilation flow to maintain the dissolved oxygen value at 20-30% air saturation. The pH value was adjusted to 6.7 with a mass-volume ratio of 25% ammonia water. After culturing at 37 °C for 4 hours, lactose was added to the final concentration of 4g/L and cooled to 20°C to induce expression; during the fermentation process, 500g/L glycerol solution was added to maintain the glycerol concentration at 5-10g/L. After the fermentation, the cells were collected by centrifugation at 10,000r/min and 4°C for 10min, and sterile saline The cells were washed twice to obtain the whole cell catalyst.

(2) The whole cell catalyzed production of 3-indolepyruvic acid using histidine alcohol phosphate aminotransferase mutant (hisC-P226F-R311Y), the transformation conditions are: prepare 2L of 200mM L-tryptophan solution, add tryptophan 1.2 times molar concentration of pyruvate, 25mg/L pyridoxal phosphate, 1g/L sodium sulfite, 1g/L phytic acid, ammonia water to adjust the pH to 7.5, add 30g of wet cells, and pass through at a flow rate of 0.5L/min. Nitrogen was used to drive oxygen for 15 min, the temperature was maintained at 37 °C, and the reaction was carried out in a 3 L fermentor for 12 h. The concentration of IPA was 179.8 mM, and the molar conversion rate was 89.9%.

Claims (10)

1. A kind of histidine alcohol phosphate aminotransferase mutant, it is characterized in that histidine alcohol phosphate aminotransferase gene is derived from Streptomyces garcinias strain HY61, its nucleotide sequence is as shown in SEQ ID NO: 1, the amino acid sequence As shown in SEQ ID NO: 2; the mutant is a single mutation or multiple sites at the 66th, 138th, 197th, 226th and 311th positions of the amino acid sequence shown in SEQ ID NO: 2 Obtained by mutation, the specific mutation is the mutation of valine at position 66 to isoleucine, the mutation of asparagine at position 138 to glutamine, the mutation of serine at position 197 to histidine, or the mutation of proline at position 226 to Phenylalanine and arginine at position 311 were mutated to tyrosine.
2. A gene encoding the histidine alcohol phosphate aminotransferase mutant of claim 1.
3. The engineering bacteria containing the coding gene of claim 2.
4. The application of the histidine alcohol phosphate aminotransferase mutant of claim 1 in catalyzing the synthesis of 3-indolepyruvate from L-tryptophan.
5. The application according to claim 4, characterized in that the application method is to use the somatic cells harvested by fermentation and centrifugation of the engineered bacteria containing the histidine alcohol phosphate aminotransferase mutant gene as a whole-cell catalyst, and use L-color Amino acid as substrate, pyruvate with 1.2 times the molar concentration of L-tryptophan as amino acceptor, adding 25mg/L coenzyme pyridoxal phosphate, 1g/L stabilizing protective agent to the conversion system, ammonia water to adjust pH It is 7.5, and nitrogen is used to drive out oxygen, and 3-indolepyruvic acid is produced by reaction under the condition of temperature of 30-40 °C.
6. The application according to claim 5, characterized in that in the transformation system, the amount of the catalyst is 10-40 g/L by wet cell weight, the substrate concentration is 50-200 mM, and the pyruvate concentration is 60-240 mM.
7. The preparation method of the engineered bacteria containing histidine phosphate aminotransferase mutant gene according to claim 3, it is characterized in that the method is specifically as follows: the recombinant Escherichia coli containing histidine phosphate aminotransferase mutant gene is connected Into LB slant medium at 37°C for 12-18h; connect 1 ring of slant strains to LB liquid seed medium, shake at 37°C, 180r/min for 4-10h; put 2.0L of medium in a 3L fermenter, and use the volume The seed liquid is connected to the fermentation medium at a ratio of 2 to 12% of the inoculum, the initial speed is 220r/min, and the initial ventilation flow is 1.0L/min. With the increase of bacterial concentration, the speed and ventilation flow are adjusted to maintain the dissolved oxygen value at 20 ～30% air saturation, adjust the pH value to be stable at 6.7 with 25% ammonia water in mass volume ratio, add lactose to the final concentration of 1～5g/L after culturing at 37℃ for 4～12h, and cool down to 20～30℃ to induce expression; During the fermentation process, 500g/L glycerol solution was added to maintain the glycerol concentration at 5-10g/L. After the fermentation was completed, the cells were collected by centrifugation at 10,000r/min and 4°C for 10min. The cells were washed twice with sterile saline to obtain histidine alcohol phosphate amino groups. Transferase mutant whole-cell catalysts.
8. method according to claim 7, is characterized in that the composition and final concentration of described LB slant medium: yeast extract powder 5g/L, peptone 10g/L, NaCl 5g/L, ampicillin 100mg/L, agar 20g /L, pH7.0～7.2, 121℃ high pressure steam sterilization for 20min;
9. method according to claim 7, is characterized in that the composition and final concentration of described LB liquid seed medium: yeast extract powder 5g/L, peptone 10g/L, NaCl 5g/L, ampicillin 100mg/L, pH7 .0～7.2, 121℃ high pressure steam sterilization for 20min;
10. The method according to claim 7, characterized in that the composition and final concentration of the fermentation medium: glycerol 20g/L, peptone 20g/L, yeast extract 5g/L, MgSO ₄ 2g/L, KH ₂ PO ₄ 12.5g/L, (NH ₄ ) ₂ SO ₄ 2g/L, citric acid 1g/L, corn steep liquor 3g/L, CaCl ₂ 1g/L, D-biotin 20μg/L, pH6.7～7.0, Sterilize with high pressure steam at 121° C. for 20 min; the stabilized protective agent is one or a combination of sodium sulfite and phytic acid.