Laccase, Its Genes and Applications

Abstract

The present invention discloses a laccase, its gene and application, belonging to the technical field of genetic engineering. The laccase gene of the present invention is obtained by codon optimization of the laccase gene of Cerrena unicolor, and the nucleotide sequence is as shown in SEQ ID NO.2. The laccase gene provided by the present invention is recombinantly expressed by transforming Aspergillus niger, and high-activity laccase can be mass-produced industrially. The highest enzyme activity in shake flask culture reaches 39 U / ml, and after culturing in a 7L fermenter for 125 h, the total laccase activity reaches 9234 U / ml. At the same time, the laccase provided by the present invention and the mediator methyl syringate have a significant destructive effect on zearalenone, which is beneficial to grain transportation and storage.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a laccase, its gene and applications. Background Art

[0002] Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase that widely exists in higher plants, fungi, bacteria, insects and lichens. Among them, laccase from fungi is the most widely distributed, and has the characteristics of the highest redox potential, broad substrate spectrum, simple separation, purification and identification. Laccase has considerable application prospects in industrial biocatalysis. Currently, more than 200 catalytic substrates are known, mainly including six categories such as phenols (mainly polyphenols and their derivatives such as catechol and hydroquinone), aromatic amines and their derivatives, carboxylic acids and their derivatives, steroid hormones, biological pigments, ferrocene compounds and their derivatives. In the presence of redox mediators, laccase can further catalyze more non-phenolic substrates, such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, azo dyes, organophosphorus pesticides and lignin-like macromolecular compounds. Currently, laccase has important applications in green chemistry such as environmental remediation, biological monitoring, food processing, fiber modification, anti-staining, pharmaceutical and organic synthesis.

[0003] Currently, in China, laccase is mainly applied in the environmental protection industry, especially in aspects such as sewage treatment, biological bleaching of pulp and biodegradation of toxic compounds.

[0004] It is known that laccase is produced by a variety of fungi, including species of Aspergillus, Neurospora, Podospora, Botrytis, Pleurotus, Fornes, Phlebia, Trametes, Polyporus, Stachybotrys, Rhizoctonia, Bipolaris, Curvularia, Amerosporium, Lentinus, Myceliophtora, Coprinus, Thielavia, Cerrena, Streptomyces and Melanocarpus.

[0005] The Chinese invention patent CN110373395B of Yao Bin et al. discloses the basis and method for the mediator of lavender to improve the degradation rate of laccase on mycotoxins; the Chinese invention patent CN105255843B of Tian Jian et al. discloses an Escherichia coli laccase mutant, its encoding gene and application. Among them, the mutant G276R obtained by mutating glycine at the 276th position of wild-type Escherichia coli laccase to arginine has an enzyme activity about 3 times that of wild-type Escherichia coli laccase under optimal conditions, reaching 12.56 U / mg.

[0006] Making the laccase gene highly expressed in recombinant strains by genetic engineering means is an effective way to produce laccase on a large scale at low cost. Genetic modification technology has been widely used in the biomedical field. Using genetic engineering technology to obtain recombinant strains with high expression can promote the production and application of laccase in industrial enzymes. Summary of the Invention

[0007] To solve related problems, the primary object of the present invention is to provide a laccase gene. The gene is obtained by codon optimization of the laccase gene of Cerrena unicolor.

[0008] Another object of the present invention is to provide the application of the above laccase gene.

[0009] To achieve the above invention objects, the present invention adopts the following technical solutions:

[0010] A laccase gene, whose nucleotide sequence is shown in SEQ ID NO.2.

[0011] Furthermore, the amino acid sequence of the protein encoded by the laccase gene is shown in SEQ ID NO.1.

[0012] An expression cassette containing the laccase gene shown in SEQ ID NO.2.

[0013] An expression vector containing the laccase gene shown in SEQ ID NO.2 or the above expression cassette.

[0014] A laccase gene engineering bacterium obtained by introducing the laccase gene shown in SEQ ID NO.2 into a host bacterium.

[0015] Furthermore, the host bacterium is selected from Aspergillus niger.

[0016] The construction method of the above laccase gene engineering bacterium includes the following steps:

[0017] S1. Artificially synthesize the laccase gene;

[0018] S2. Ligate the laccase gene digested with NotI and PmeI and the pAN-EXP plasmid to obtain a recombinant plasmid;

[0019] S3. Introduce the recombinant plasmid into the protoplasts of Aspergillus niger VT-002, and through screening and verification, obtain the laccase gene engineering bacteria.

[0020] The application of the above laccase gene, expression cassette, expression vector or laccase gene engineering bacteria in the production of laccase.

[0021] Furthermore, the specific steps of the above application are: culture the obtained laccase gene engineering bacteria in a shake flask at 32 °C for 5 days, centrifuge to remove the bacteria to obtain the supernatant of the culture solution, which is the crude enzyme solution; or ferment and culture the laccase gene engineering bacteria at a temperature of 30 °C. When the pH drops to 5.0 during the fermentation process, start to introduce ammonia and control the pH at 5.0 - 5.2 by adding ammonia water dropwise. The ventilation volume is 7.9 - 8.2 L / min, the rotation speed is 500 - 1000 rpm, harvest the fermenter after 36 hours, control the fed-batch DE value at 10 - 30, gradually increase the rotation speed in the later stage, culture for more than 125 h, centrifuge to remove the bacteria to obtain the supernatant of the culture solution, which is the crude enzyme solution.

[0022] The laccase produced can be used for the degradation of mycotoxins such as aflatoxin and zearalenone, and can be used in combination with any one or more mediators among p-hydroxybenzoic acid, 2,6-dimethoxyphenol, and methyl syringate, with significant effects.

[0023] The present invention has the following advantages and effects compared with the prior art:

[0024] The laccase gene provided by the present invention is recombinantly expressed by transforming Aspergillus niger, and can achieve large-scale industrial production of laccase with high enzyme activity. The highest enzyme activity in shake flask culture reaches 39 U / ml, and after culturing in a 7 L fermenter for 125 h, the total laccase enzyme activity reaches 9234 U / ml. At the same time, the laccase provided by the present invention and the mediator methyl syringate have a significant destructive effect on zearalenone, which is beneficial to grain transportation and storage. Brief Description of the Drawings

[0025] Figure 1 It is the map of the recombinant expression vector pAN-EXP-CE-LACCASE of the present invention;

[0026] Figure 2 It is the map of the recombinant expression vector pAN-EXP-CE-LACCASE-CODE of the present invention;

[0027] Figure 3 It is the optimal reaction pH curve graph of the recombinant laccase of the present invention;

[0028] Figure 4 It is the optimal reaction temperature curve graph of the recombinant laccase of the present invention;

[0029] Figure 5 This is the fermentation enzyme activity curve graph of the recombinant laccase strain of the present invention in a fermenter. Detailed implementation manners

[0030] The following implementation methods are for better explaining the present invention and should not be construed as limiting the purpose of the present invention. For the molecular biology experimental methods not specifically described in the following examples, they are all carried out according to the specific methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the kit and product instructions. The reagents and biological materials, unless otherwise specified, can be obtained from commercial channels.

[0031] Experimental materials and reagents:

[0032] 1. Bacterial strains and expression plasmids

[0033] The Aspergillus niger VT-002 strain and the expression plasmid pAN-EXP have both been disclosed in Chinese Patent Application CN202011514714.6.

[0034] 2. Instruments and equipment

[0035] Constant temperature incubator: Shanghai Yiheng Constant Temperature Incubator LHS-150SC; Constant temperature shaker: Hualida Constant Temperature Shaker HZ2410K6; Ultra-clean bench: Suzhou Jinghua Ultra-clean Bench SW-CJ2FD; Shanghai Stirred Bioreactor: Bailun Biochemical Equipment Co., Ltd.

[0036] 3. Preparation of culture media and reagents

[0037] TZ solid medium: Beef extract (Guangdong Huankai Microbial Technology Co., Ltd.) 8 g / L, yeast extract 2 g / L, peptone 5 g / L, NaCl 2 g / L, starch 10 g / L, agar 17 g / L, pH 5.8.

[0038] CD medium: Sucrose 30 g / L, NaNO3 2 g / L, K2HPO4 1 g / L, MgSO4 0.5 g / L, KCl 0.5 g / L, FeSO4 0.01 g / L, or add 15 g / L agar (when preparing solid medium), pH 7.3.

[0039] Regeneration medium plate: Nutrient juice powder 8 g / L, yeast extract 2 g / L, peptone 5 g / L, NaCl 2 g / L, starch 10 g / L, agar 17 g / L, pH 5.8, plus 0.8 M KCl or sorbitol 1 M.

[0040] Flask fermentation medium: maltodextrin 80 g / L, soybean cake powder 20 g / L, corn steep liquor 30 mL / L, pH 5.5, liquid volume in 500 mL Erlenmeyer flask is 100 mL, sterilized at 115 °C for 20 min.

[0041] Seed tank medium: maltodextrin 80 g / L, soybean cake powder 40 g / L, corn steep liquor 10 mL / L, pH 5.5, sterilized at 121 °C for 30 min.

[0042] Fermentation tank medium: maltodextrin 40 g / L, soybean cake powder 20 g / L, corn steep liquor 20 mL / L, (NH4)2SO4 4 g / L, calcium chloride 2 g / L, disodium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 3 g / L, appropriate amount of antifoaming agent, control the pH of the fermentation tank at 5.2 - 5.5, sterilized at 121 °C for 35 min.

[0043] Lysozyme solution: 1% lywallzyme, dissolved in 1 M sorbitol.

[0044] KCl solution: 0.6 M KCl solution

[0045] Sorbitol solution: 1 M sorbitol

[0046] S / C solution: 1 M sorbitol, 50 mM CaCl2

[0047] PEG solution: 25% (g / 100 mL) PEG8000, 50 mM CaCl2, 10 mM Tris-Hcl, pH 7.5.

[0048] Example 1: Cloning and codon optimization of laccase gene

[0049] 1.1 Extract the total genomic DNA of Cerrena unicolor. Then, using the total genomic DNA as a template, amplify it with upstream and downstream primers.

[0050] PCR amplification conditions are as follows: 98 °C for 2 min; 98 °C for 10 s, 55 °C for 15 s, 72 °C for 1 min 30 cycles; 72 °C for 5 min. Recover the PCR amplification product with a gel recovery kit and send it to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis. The results show that the nucleotide sequence of the amplification product CE-LACCASE is SEQ ID NO:3, and the amino acid sequence it encodes is SEQ ID NO:1.

[0051] 1.2 According to the codon optimization principle of Aspergillus niger, optimize the codons of the laccase CE-LACCASE genomic of Cerrena unicolor, and artificially synthesize the gene CE-LACCASE-CODE. Its nucleotide sequence is SEQ ID NO:2, and the amino acid sequence it encodes is SEQ ID NO:1.

[0052] A DNA band of approximately 1.5 kb was obtained by PCR amplification, and the target fragment was recovered.

[0053] Example 2. Construction of Recombinant Vector

[0054] 2.1 Using the amplified original laccase gene fragment and codon optimization as templates above, primers containing NotI and PmeI restriction sites were designed, and then the original laccase gene CE-LACCASE and the codon-optimized gene CE-LACCASE-CODE were amplified separately.

[0055] The reaction conditions were the same as in Example 1.

[0056] After PCR amplification, a DNA band of approximately 1.5 kb was obtained, and the target fragment of the laccase gene with restriction sites was recovered.

[0057] 2.2 The above amplified gene fragments with restriction sites and the Aspergillus niger expression plasmid pAN-EXP were separately digested with the restriction enzymes NotI and PmeI.

[0058] Target fragment digestion reaction system (50 μl): 25 μl of PCR fragment, 5 μl of reaction buffer, 1 μl of NotI, 1 μl of PmeI, 18 μl of water; digestion reaction conditions: 37 °C, reaction for 30 minutes.

[0059] Aspergillus niger expression plasmid digestion system (50 μl): 15 μl of plasmid, 5 μl of 10× reaction buffer, 1 μl of NotI, 1 μl of PmeI, 28 μl of water; digestion reaction conditions: 37 °C, reaction for 60 minutes.

[0060] 2.3 After digesting the target fragment and the Aspergillus niger expression plasmid, these two fragments were recovered by electrophoresis respectively, and the target fragment and the Aspergillus niger expression plasmid were ligated with T4 DNA ligase.

[0061] Ligation system (50 μl): 2 μl of digested target fragment, 1 μl of digested plasmid fragment, 1 μl of T4 ligase, 1 μl of 10× reaction buffer, 5 μl of water; ligation reaction conditions: ligate overnight at 16 °C.

[0062] 2.4 After the ligation reaction was completed, Escherichia coli Top10 competent cells were transformed.

[0063] Take 100 μl of Escherichia coli competent cells. Under sterile conditions, add them to the Eppendorf tube containing the ligation solution, mix well, and place them in an ice bath for 30 min. After the ice bath, add the cell suspension undergoing the transformation reaction to a thermostatic water bath adjusted to 42 °C, incubate for 2 min, quickly pour in 1 mL of LB culture medium, and place it on a shaker at 37 °C for 1 h. Then, spread it on an LB ampicillin culture dish, place it at room temperature for about 15 min to allow the spread bacterial solution to dry and not flow. Next, invert it and place it in an incubator at 37 °C to culture overnight. Take out the culture dish the next day and pick single colonies. Verify the correctly ligated transformants by colony PCR and send them for sequencing. After correct sequencing, extract the plasmids from the correctly transformed strains. Obtain the original Aspergillus niger gene expression vector pAN-EXP-CE-LACCASE and the Aspergillus niger codon-optimized gene expression vector pAN-EXP-CE-LACCASE-CODE that express laccase.

[0064] Example 3. Construction of Recombinant Laccase-Expressing Strains

[0065] 3.1 Preparation of Aspergillus niger Protoplasts

[0066] The Aspergillus niger strain VT-002 was cultured on TZ medium at 32 °C for 4 d. Select standard colonies and streak them on a CD solid medium, and culture at 32 °C for 4 d. Take 4 cubic centimeters of agar blocks from the CD plate and put them into a triangular flask containing 60 ml of CD liquid medium, and culture at 34 °C for 4 d. Collect the mycelia, wash them once with 1 M sorbitol, weigh the wet weight, add the lytic enzyme solution at a mass-to-volume ratio of 1 g:25 ml, and enzymatically digest at 30 °C and 80 r / min for 2.5 - 3 h. Filter the protoplast solution and recover the filtrate. Centrifuge at 4000 r / min for 10 min and discard the supernatant. Centrifuge with pre-cooled 0.6 M KCl solution. Resuspend the protoplast precipitate in an appropriate amount of 0.6 M KCl solution until the cell concentration reaches (1 - 3)×10 6 cells / ml, and place them in an ice bath for standby. For protoplast regeneration, dilute the purified protoplasts with an osmotic stabilizer and spread them on a protoplast regeneration medium plate, and culture at 32 °C. Observe the protoplast regeneration after 4 - 5 d. At the same time, use sterile water to lyse the protoplasts as a control to eliminate the error caused by colonies formed by non-protoplasts.

[0067] 3.2 Transformation of Aspergillus niger Protoplasts

[0068] Take the prepared 200 μl protoplast suspension, and add about 5 μg of Aspergillus niger expression vectors pAN-EXP-CE-LACCASE and pAN-EXP-CE-LACCASE-CODE respectively. Gently mix them with a pipette tip. Add 50 μl of PEG solution, gently invert and mix well, incubate in an ice bath for 20 - 30 min, slowly add 1 ml of PEG solution, let it stand at room temperature for 20 min, then add 2 ml of S / C solution, gently mix well, spread it on a regeneration medium plate containing 100 μg / ml hygromycin, and culture at 34 °C for 5 - 6 days.

[0069] 3.3 Screening of Aspergillus niger transformants and shake flask culture

[0070] Observe the growth of Aspergillus niger transformed with pAN-EXP-CE-LACCSE and pAN-EXP-CE-LACCASE-CODE on the hygromycin resistance plate. Pick 12 single colonies with larger colonies respectively and transfer them to Erlenmeyer flasks containing shake flask fermentation medium for culture. Culture on a shaker at 32 °C for 5 days. Centrifuge to remove the cells and obtain the supernatant of the culture solution, which is the crude enzyme solution, and perform enzyme activity determination and enzyme property detection.

[0071] Example 4: Laccase enzyme activity determination and enzyme property detection

[0072] 4.1 Enzyme activity determination

[0073] Reagents for enzyme activity determination (ABTS method): (1) 1 mmol / L ABTS solution. ABTS is a product of Sigma Company, USA. Accurately weigh 0.0274 g of ABTS, dissolve it in distilled water and make up to 100 mL, and place it in a brown bottle for later use; (2) Hac-NaAc (pH 4.5) buffer solution. Dissolve 18 g of NaAc and 9.8 ml of HAc in distilled water and make up to 1 L, and calibrate its pH value with a pH meter.

[0074] Method for enzyme activity determination (ABTS method): Take 0.1 ml of the enzyme solution, add 2.5 ml of Hac-NaAc (pH 4.5) buffer solution, mix well, add 0.4 ml of ABTS, let it stand for 30 s, and read the absorbance value at 420 nm every 15 s. Read a total of 6 - 7 values. Judging from experience, if the absorbance value increases rapidly, dilute the enzyme solution by 2 - 5 times and measure again until the change in the absorbance value is not too fast and the values are all between 0.2 and 0.8.

[0075] Calculation of enzyme activity: Enzyme activity = slope of absorbance change × 4000 × dilution factor.

[0076] The results show that the highest enzyme activity of the codon-optimized strain CE-LACCSE-CODE reaches 39 U / ml, which is significantly higher than that of the original strain (7.68 U / ml).

[0077] 4.2 Optimal reaction pH

[0078] The enzyme activity of laccase was measured in buffer systems with different pH values (2.0 - 10.0). Taking the enzyme activity at pH 5.0 as the control, the relative enzyme activities at different pH values were measured. The results are shown in Table 1 and Figure 3 as follows: The optimal pH value for the action of this laccase is 4.0.

[0079] 4.3 Optimal reaction temperature

[0080] At 25°C - 80°C, at intervals of 5°C, the activity of laccase was measured respectively. Taking the enzyme activity at 25°C as the control, the relative enzyme activities at different temperatures were measured. The results are shown in Table 2 and Figure 4 as follows: It was found that for the laccase of the present invention at 25°C - 80°C, the laccase could always maintain an enzyme activity of more than 97%. Among them, the most suitable temperature is 50°C, and the relative enzyme activity can reach 192%. Thus, it can be seen that the laccase of the present invention has a wide range of action temperatures, and still can maintain an enzyme activity of 97% under the condition of high temperature 80°C, which is beneficial to industrial applications.

[0081] Table 1. Relative enzyme activities at different pH values

[0082]

[0083]

[0084] Table 2. Relative enzyme activities at different temperatures

[0085] Reaction temperature (°C) Relative enzyme activity (%) 25 100 30 114 35 133 40 158 45 181 50 192 55 183 60 180 65 155 70 144 75 129 80 97

[0086] Example 5. Fermentation of laccase strain

[0087] In this example, a 7L stirred bioreactor (Shanghai Bailun Biochemical Equipment Co., Ltd.) was used, with an initial liquid loading of 4L and an inoculation amount of 500mL. The fermentation process of the recombinant engineering bacteria was optimized. Transfer conditions: The cell concentration increased, the cells were stained deeply under microscopic examination, the field of view was clear without contaminants, the temperature was 30°C. When the pH dropped to 5.0 during the fermentation process, ammonia was introduced, and the pH was controlled at 5.0 - 5.2 by adding ammonia water dropwise. The ventilation rate was 7.9 - 8.2L / min, the rotation speed was 500 - 1000rpm, and the fermentation was terminated after 36 hours. The DE value of the fed-batch was controlled at 10 - 30. The rotation speed was gradually increased later, and the culture was carried out for 125h. Samples were taken regularly to measure the enzyme activity. The cells were removed by centrifugation to obtain the fermentation supernatant, which was the crude enzyme solution, and the enzyme activity was measured and the enzymatic properties were detected. The results are shown in Table 3 and Figure 5 as follows. The results showed that when the recombinant engineering bacteria were fermented and cultured in a 7L fermenter using the fermentation medium, the total enzyme activity of laccase of the recombinant strain reached 9234U / mL.

[0088] Table 3. Enzyme activity data of fermentation in fermenter

[0089]

[0090]

[0091] Example 6: Laccase degradation of mycotoxins

[0092] 6.1 Reagents and raw materials

[0093] Standards: Aflatoxin and zearalenone standards were dissolved in pure methanol to prepare a standard solution with a concentration of 10 μg / ml (10 ppm).

[0094] pH 4.5 Succinic acid-sodium hydroxide buffer: Dissolve 4.5 g of succinic acid in water, adjust the pH to 4.50 ± 0.01 with sodium hydroxide, and make up to 1 L.

[0095] Mediators: 3 in total, namely: 2,6-dimethoxyphenol, p-hydroxyformic acid, methyl syringate: Prepare liquid samples with a concentration of 0.1 g / ml using 95% ethanol.

[0096] 6.2 Experimental methods

[0097] Enzyme extract: The enzyme sample was extracted with pH 4.5 buffer and diluted to approximately 100 U / ml enzyme solution.

[0098] Enzyme group: Add enzyme sample (laccase concentration in the reaction system is 1 U / ml), add 10 ml of pH 4.5 succinic acid-sodium hydroxide buffer, add 10 μl of mycotoxin standard, and 10 μl of mediator solution. After vortexing and mixing, perform enzymatic hydrolysis at 30°C and 250 rpm for 24 hours. After completion of enzymatic hydrolysis, measure the mycotoxin content in the sample solution.

[0099] Control: Add inactivated enzyme sample (laccase concentration in the reaction system is 1 U / ml, inactivated at 95°C for 5 minutes), add 10 ml of pH 4.5 succinic acid-sodium hydroxide buffer, add 10 μl of mycotoxin standard and 10 μl of mediator solution, shake and mix, and then enzymatically digest at 30°C, 250 rpm for 24 hours. After completion of enzymatic digestion, measure the mycotoxin content in the sample solution.

[0100] Table 4. Decomposition efficiency of aflatoxin AF B1

[0101]

[0102] Table 5. Decomposition efficiency of aflatoxin AF B1

[0103]

[0104]

[0105] The results are shown in Tables 4 and 5. This laccase and the mediator methyl syringate have a significant destructive effect on zearalenone, and the degradation rate reaches 96.4%. The degradation rate of this laccase and the mediator p-hydroxybenzoic acid on zearalenone reaches 46.2%, indicating that this laccase has a very strong degradation rate on the decomposition efficiency of zearalenone and has a very significant effect on toxin degradation.

[0106] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A laccase gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

2.

2. An expression cassette, characterized in that: It contains the laccase gene shown in SEQ ID NO.

2.

3. An expression vector, characterized in that: It contains the laccase gene shown in SEQ ID NO.2 or the expression cassette described in claim 2.

4. A laccase genetically engineered bacterium, characterized in that: It is obtained by introducing the laccase gene shown in SEQ ID NO.2 into a host bacterium.

5. The laccase genetic engineering bacterium according to claim 4, characterized in that: The host bacterium is selected from Aspergillus niger.

6. Use of the laccase gene described in claim 1 or the expression cassette described in claim 2 or the expression vector described in claim 3 in the production of laccase.

7. Use of the laccase genetic engineering bacterium described in any one of claims 4 - 5 in the production of laccase.

8. The use according to claim 7, characterized in that: The specific steps of the use are: culturing the obtained laccase genetic engineering bacterium in a shake flask for 5 days at 32°C, centrifuging to remove the bacterial cells to obtain the supernatant of the culture solution, which is the crude enzyme solution.

9. The use according to claim 7, characterized in that: The specific steps of the use are: fermenting and culturing the obtained laccase genetic engineering bacterium at a temperature of 30°C. When the pH drops to 5.0 during the fermentation process, ammonia is introduced. The pH is controlled at 5.0 - 5.2 by adding ammonia water dropwise. The ventilation rate is 7.9 - 8.2 L / min, the rotation speed is 500 - 1000 rpm. The fermentation tank is emptied and fed after 36 hours, and the DE value is controlled at 10 - 30. The rotation speed is gradually increased in the later stage, and the culture is carried out for more than 125 h. Centrifuging to remove the bacterial cells to obtain the supernatant of the culture solution, which is the crude enzyme solution.

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