CN116064605A - Laccase and gene and application thereof - Google Patents

Abstract

The invention discloses laccase and a gene and application thereof, and belongs to the technical field of genetic engineering. The laccase gene is obtained by codon optimization of the trichuria Cerrena uncolor laccase gene, and the nucleotide sequence is shown as SEQ ID NO. 2. The laccase gene provided by the invention can realize large-scale production of laccase with high enzyme activity in industry by converting Aspergillus niger to carry out recombinant expression, the highest enzyme activity of shake flask culture reaches 39U/ml, and the total enzyme activity of laccase reaches 9234U/ml after 125h of culture in a 7L fermentation tank. Meanwhile, the laccase and the mediator methyl syringate provided by the invention have remarkable damage effect on zearalenone, and are beneficial to grain transportation and storage.

Description

Laccase and gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to laccase as well as a gene and application thereof.

Background

Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase that is widely found in higher plants, fungi, bacteria, insects and lichens. Wherein, laccase from fungi has the characteristics of most wide distribution, highest oxidation-reduction potential, broad substrate spectrum and simple separation, purification and identification. Laccase has considerable application prospect in industrial biocatalysis, and currently, the catalytic substrate is known to be more than 200, and mainly comprises phenols (mainly catechol, hydroquinone and other polyphenols and derivatives thereof), arylamines and derivatives thereof, carboxylic acid and derivatives thereof, steroid hormones, biological pigments, ferrocene compounds and derivatives thereof and other 6 types. Laccase can also further catalyze more non-phenolic substrates such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, azo dyes and organophosphorus pesticides and lignin macromolecular compounds in the presence of redox mediators. At present, laccase has important applications in green chemistry such as environmental remediation, biological monitoring, food processing, fiber modification, dyeing prevention, pharmaceutical and organic synthesis.

At present, laccase is mainly applied to the environmental protection industry in China, in particular to the aspects of sewage treatment, biological bleaching of paper pulp, biological degradation of toxic compounds and the like.

Laccase is known to be produced by a variety of fungi including Aspergillus (Aspergillus), neurospora (Neurospora), podospora (Podospora), botrytis (Botrytis), pleurotus (Pleurotus), phellinus (Fornes), white rot (Phlebia), trametes (Trametes), polyporus (Polyporus), scirpus (Stachybotrys), rhizoctonia (Rhizoctonia), bipolaris (Bipolaris), curvularia (Curvularia), monococcus (amersosporum), lentinus (Lentinus), myceliophthora (myceliophthora), coprinus (Coprinus), rhizopus (Thielavia), myceliophthora (Cerrena), streptomyces (Streptomyces), and melanomyces (melana).

Yao et al, chinese patent No. 110373395B, discloses a method and a method for improving the degradation rate of laccase to mycotoxin by lavender mediator; tian Jian et al, chinese patent No. 105255843B discloses a mutant of E.coli laccase, and encoding gene and application thereof, wherein the 276 th glycine of the wild type E.coli laccase is mutated into the G276R mutant obtained by arginine, and the enzyme activity of the mutant is about 3 times that of the wild type E.coli laccase under the optimal condition, and reaches 12.56U/mg.

The efficient expression of laccase genes in recombinant strains by genetic engineering means is an effective way for large-scale and low-cost production of laccase. The genetic engineering technology is widely applied to the field of biological medicine, and the genetic engineering technology is utilized to obtain recombinant strain with high expression, so as to promote the production and application of laccase in industrial enzymes.

Disclosure of Invention

In order to solve the related problems, the primary aim of the invention is to provide a laccase gene. The gene is obtained by carrying out codon optimization on the trichinella tricolor laccase gene.

It is a further object of the present invention to provide the use of the laccase gene described above.

In order to achieve the above object, the present invention adopts the following technical scheme:

a laccase gene has a nucleotide sequence shown in SEQ ID NO. 2.

Further, the amino acid sequence of the protein encoded by the laccase gene is shown as SEQ ID NO. 1.

An expression cassette comprising a laccase gene as shown in SEQ ID NO. 2.

An expression vector contains laccase gene shown in SEQ ID NO.2 or the expression cassette.

A laccase genetically engineered bacterium is obtained by introducing laccase gene shown in SEQ ID NO.2 into host bacterium.

Further, the host bacterium is selected from Aspergillus niger.

The construction method of the laccase genetically engineered bacterium comprises the following steps:

s1, artificially synthesizing the laccase gene;

s2, carrying out a ligation reaction on the laccase gene subjected to NotI and PmeI double-enzyme digestion and pAN-EXP plasmid to obtain a recombinant plasmid;

s3, introducing the recombinant plasmid into a protoplasm of the Aspergillus niger VT-002, and obtaining laccase genetic engineering bacteria through screening and verification.

The laccase gene, the expression cassette, the expression vector or the laccase genetically engineered bacterium are applied to the production of laccase.

Further, the specific steps of the application are as follows: shake flask culture is carried out on the laccase genetically engineered bacterium, the culture is carried out for 5 days at the temperature of 32 ℃, and the thalli are removed by centrifugation to obtain culture solution supernatant, namely crude enzyme solution; or fermenting and culturing the laccase genetically engineered bacterium, wherein the temperature is 30 ℃, when the pH value in the fermentation process is reduced to 5.0, ammonia is introduced, the pH value is controlled to be 5.0-5.2 by feeding ammonia water, the ventilation rate is 7.9-8.2L/min, the rotating speed is 500-1000 rpm, the feeding DE value is controlled to be 10-30 after 36 hours, the rotating speed is gradually increased in the later period, the culture is carried out for more than 125 hours, and the bacterial body is centrifugally removed to obtain culture supernatant, namely the crude enzyme liquid.

The laccase can be used for degrading mycotoxins such as aflatoxin, zearalenone and the like, and can be combined with any one or more mediators selected from p-hydroxy formic acid, 2, 6-dimethoxy phenol and methyl syringate, so that the effect is remarkable.

Compared with the prior art, the invention has the following advantages and effects:

the laccase gene provided by the invention can realize large-scale production of laccase with high enzyme activity in industry by converting Aspergillus niger to carry out recombinant expression, the highest enzyme activity of shake flask culture reaches 39U/ml, and the total enzyme activity of laccase reaches 9234U/ml after 125h of culture in a 7L fermentation tank. Meanwhile, the laccase and the mediator methyl syringate provided by the invention have remarkable damage effect on zearalenone, and are beneficial to grain transportation and storage.

Drawings

FIG. 1 is a diagram of a recombinant expression vector pAN-EXP-CE-LACCASE of the present invention;

FIG. 2 is a diagram of the recombinant expression vector pAN-EXP-CE-LACCASE-CODE of the present invention;

FIG. 3 is a graph showing the pH profile of the optimum reaction for the recombinant laccase of the invention;

FIG. 4 is a graph showing the optimal reaction temperature for the recombinant laccase of the invention;

FIG. 5 is a graph of fermentation enzyme activity in a fermentor of a recombinant laccase strain of the invention.

Detailed Description

The following examples are given for better illustration of the invention and should not be construed as limiting the invention. The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j. The reagents and biological materials, unless otherwise specified, are commercially available.

Experimental materials and reagents:

1. bacterial species and expression plasmids

Aspergillus niger (Aspergillus niger) VT-002 strain and expression plasmid pAN-EXP are disclosed in the Chinese patent application CN 202011514714.6.

2. Instrument and apparatus

Constant temperature incubator: LHS-150SC in a Shanghai-constant temperature incubator; constant temperature shaking table: hua Li reaches a constant temperature shaking table HZ2410K6; ultra clean bench: suzhou clean bench SW-CJ2FD; shanghai stirring type bioreactor: bairens Biochemical Equipment Co.Ltd.

3. Configuration of culture Medium and reagent

TZ solid medium: beef extract (Guangdong Cryptographic microorganism Co., ltd.) 8g/L, yeast extract 2g/L, peptone 5g/L, naCl 2g/L, starch 10g/L, agar 17g/L, pH 5.8.

CD medium: sucrose 30g/L, naNO ₃ 2g/L，K ₂ HPO ₄ 1g/L，MgSO ₄ 0.5g/L，KCl 0.5g/L，FeSO ₄ 0.01g/L, or 15g/L agar (when preparing solid medium), pH 7.3.

Regeneration medium plates: 8g/L of nutrient juice powder, 2g/L of yeast extract, 5g/L of peptone, 2g/L of NaCl, 10g/L of starch, 17g/L of agar, pH 5.8 and 0.8M KCl or 1M sorbitol.

Shake flask fermentation medium: maltodextrin 80g/L, bean cake powder 20g/L, corn steep liquor 30mL/L, pH5.5, triangular flask liquid volume 100mL/500mL, and 115 ℃ sterilization 20min.

Seed pot medium: maltodextrin 80g/L, bean cake powder 40g/L, corn steep liquor 10mL/L, pH5.5, and sterilizing at 121deg.C for 30min.

Fermentation tank medium: maltodextrin 40g/L, bean cake powder 20g/L, corn steep liquor 20mL/L, (NH 4) ₂ SO ₄ 4g/L, 2g/L of calcium chloride, 2g/L of disodium hydrogen phosphate, 3g/L of potassium dihydrogen phosphate and a proper amount of defoamer, and sterilizing the fermentation tank at the temperature of 121 ℃ for 35min.

Cleavage enzyme solution: 1% lywallase, dissolved with 1M sorbitol.

KCl solution: 0.6M KCl solution

Sorbitol solution: 1M sorbitol

S/C solution: 1M sorbitol, 50mM CaCl ₂

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

Example 1 laccase Gene cloning and codon optimization

1.1 extraction of total genomic DNA from the Achromobacter Cerrena unicolor. And then amplifying by using the genome total DNA as a template and using the upstream primer and the downstream primer.

PCR amplification conditions were 98℃for 2min;98℃for 10s,55℃for 15s,72℃for 1min for 30 cycles; and at 72℃for 5min. And (5) the gel recovery kit recovers PCR amplification products and sends the PCR amplification products to Shanghai workers for sequencing analysis. The result shows that the nucleotide sequence of the amplified product CE-LACCASE is SEQ ID NO. 3, and the encoded amino acid sequence is SEQ ID NO. 1.

1.2 according to the Aspergillus niger codon optimization principle, the LACCASE CE-LACCASE genome codon of the Cerrena unicolor is optimized, and the gene CE-LACCASE-CODE is artificially synthesized. The nucleotide sequence is SEQ ID NO.2, and the coded amino acid sequence is SEQ ID NO. 1.

The DNA band of about 1.5kb was obtained by PCR amplification, and the desired fragment was recovered.

EXAMPLE 2 construction of recombinant vector

2.1 designing primers containing NotI and PmeI enzyme cutting sites by taking the amplified LACCASE original gene fragment and codon optimization as templates, and then respectively expanding an original LACCASE gene CE-LACCASE and a codon optimization gene CE-LACCASE-CODE.

The reaction conditions were the same as in example 1.

After PCR amplification, a DNA band of about 1.5kb was obtained, and the target fragment of laccase gene with cleavage site was recovered.

2.2 restriction enzymes NotI and PmeI are respectively digested with the amplified gene fragment with the restriction sites and the Aspergillus niger expression plasmid pAN-EXP.

Target fragment cleavage reaction System (50. Mu.l): 25 μl of PCR fragment, 5 μl of reaction buffer, 1 μl of NotI, 1 μl of PmeI, and 18 μl of water; cleavage reaction conditions: the reaction was carried out at 37℃for 30 minutes.

Aspergillus niger expression plasmid cleavage system (50 μl): plasmid 15 μl,10 response buffer 5 μl, notI1 μl, pmeI1 μl, water 28 μl; cleavage reaction conditions: the reaction was carried out at 37℃for 60 minutes.

2.3 digestion of the fragment of interest and the Aspergillus niger expression plasmid, followed by electrophoresis to recover the two fragments, respectively, and enzymatic ligation of the fragment of interest and the Aspergillus niger expression plasmid using T4DNA ligase.

Ligation system (50 μl): 2 μl of the target fragment, 1 μl of the plasmid fragment, 1 μl of T4 ligase, 1 μl of 10 reaction buffer, and 5 μl of water; connection reaction conditions: the enzyme was incubated overnight at 16 ℃.

2.4 after completion of the enzymatic ligation reaction, E.coli Top10 competent cell transformation was performed.

Taking 100 mu l of escherichia coli competent cells, adding the competent cells into an Eppendorf tube of a connecting solution under the aseptic condition, uniformly mixing, and placing the mixture into an ice bath for 30min. After ice bath, the cell suspension being transformed was added to a thermostatic water bath at a temperature of 42℃and incubated for 2min, and 1mL of LB medium was rapidly poured and placed on a shaking table at 37℃for 1h. Then, the solution was spread on an LB ampicillin dish and left at room temperature for about 15 minutes, so that the spread solution was dried and prevented from flowing. Then, the cells were placed in an incubator at 37℃overnight. The following day the dishes were removed and single colonies were picked. Colony PCR verifies that the correctly connected transformants are sent to sequencing, and after the sequencing is correct, the correct transformants extract plasmids. Obtaining an Aspergillus niger original gene expression vector pAN-EXP-CE-LACCASE and an Aspergillus niger codon optimized gene expression vector pAN-EXP-CE-LACCASE-CODE for expressing LACCASE.

Example 3 construction of recombinant expressed laccase Strain

3.1 preparation of Aspergillus niger protoplast

Aspergillus niger strain VT-002 was cultured on TZ medium at 32℃for 4d, standard colonies were selected and streaked on CD solid medium, cultured at 32℃for 4d, 4 cc agar blocks were taken from CD plates and placed in a flask of 60ml CD liquid medium, and cultured at 34℃for 4d. Mycelium is collected, washed once by 1M sorbitol, wet weight is weighed, and the mass volume ratio is 1g:25ml of the enzyme solution is added for enzymolysis for 2.5 to 3 hours at the temperature of 30 ℃ and at the speed of 80 r/min. The protoplast solution was filtered, and the filtrate was recovered. Centrifuging at 4000r/min for 10min, and discarding supernatant. Centrifuge with pre-chilled 0.6M KCl solution. The protoplast sediment is resuspended in a proper amount of 0.6M KCl solution, and the concentration of the thallus reaches (1-3) x 10 ⁶ And (5) placing the mixture in an ice bath for standby. The purified protoplast was diluted with an osmotic stabilizer for protoplast regeneration, spread on a plate of a protoplast regeneration medium, and cultured at 32℃for 4 to 5 days, and then observed for regeneration of the protoplast. Meanwhile, sterile water is used as a control after the protoplast is swelled and broken, so that errors caused by colonies formed by non-protoplasts are eliminated.

3.2 transformation of Aspergillus niger protoplasts

About 5. Mu.g of the Aspergillus niger expression vectors pAN-EXP-CE-LACCASE and pAN-EXP-CE-LACCASE-CODE were added to 200. Mu.l of the prepared protoplast suspension, respectively, and gently mixed with a gun head. 50 μl of PEG solution is added, the mixture is gently inverted and mixed uniformly, ice bath is carried out for 20-30 min, 1ml of PEG solution is slowly added, the mixture is placed at room temperature for 20min, 2ml of S/C solution is added, the mixture is gently mixed uniformly, the mixture is coated on a regeneration medium plate of 100 μg/ml hygromycin, and the mixture is cultured for 5-6 days at 34 ℃.

3.3 Aspergillus niger transformant selection and shake flask culture

Observing the growth conditions of pAN-EXP-CE-LACCSE and pAN-EXP-CE-LACCASE-CODE on hygromycin resistance plates after Aspergillus niger conversion, and respectively picking out single colonies with larger 12 colonies to a triangular flask with a shake flask fermentation medium for culture, and carrying out shake culture at 32 ℃ for 5 days. And centrifuging to remove thalli to obtain a culture solution supernatant, namely crude enzyme solution, and carrying out enzyme activity determination and enzymatic property detection.

Example 4 laccase enzyme Activity assay and enzymatic Property detection

4.1 enzyme Activity assay

Enzyme activity determination reagent (ABTS method): (1) 1mmol/L ABTS solution, ABTS is a product of Sigma company in America, 0.0274g of ABTS is accurately weighed, distilled water is used for constant volume to 100mL, and the solution is placed in a brown bottle for standby; (2) Hac-NaAc (pH 4.5) buffer solution, 18g NaAc,9.8ml HAc, was fixed to a volume of 1L with distilled water and pH was adjusted with a pH meter.

Method for measuring enzyme Activity (ABTS method): taking 0.1ml of enzyme solution, adding 2.5ml of Hac-NaAc (pH 4.5) buffer solution, uniformly mixing, adding 0.4ml of ABTS, standing for 30s, and reading the absorbance at 420nm every 15 s. And 6 to 7 numerical values are read in total. Empirically, if the light absorption value increases rapidly, the enzyme solution is diluted by 2-5 times and then measured again until the light absorption value changes less rapidly and the values are all between 0.2 and 0.8.

Calculation of enzyme activity: enzyme activity = slope of absorbance change x 4000 x dilution.

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

4.2 optimal reaction pH

The enzyme activities of laccase are respectively measured in buffer systems with different pH values (2.0-10.0), and relative enzyme activities under different pH values are measured by taking the enzyme activities with the pH value of 5.0 as a reference. The results are shown in table 1 and fig. 3: the pH optimum for the laccase is 4.0.

4.3 optimum reaction temperature

The activity of laccase is measured at 25-80 deg.C with 5 deg.C as interval, and the relative enzyme activity at different temperatures is measured with 25 deg.C as reference. As shown in Table 2 and FIG. 4, the laccase of the invention can always keep more than 97% of enzyme activity at 25-80 ℃, wherein the most suitable temperature is 50 ℃ and the relative enzyme activity can reach 192%. Therefore, the laccase provided by the invention has a wide action temperature range, and can still keep 97% of enzyme activity at a high temperature of 80 ℃, so that the laccase is beneficial to industrial application.

TABLE 1 relative enzyme activities at different pH values

TABLE 2 relative enzyme activities at different temperatures

Reaction temperature (DEG 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

Example 5 fermentation of laccase Strain

In the embodiment, a 7L stirring type bioreactor (Shanghai Bairen Biochemical equipment Co., ltd.) is adopted, the initial liquid loading amount is 4L, the inoculation amount is 500mL, and the recombinant engineering bacteria are subjected to fermentation process optimization. Seed transfer conditions: the concentration of the thalli is increased, the thalli is deeply dyed, the visual field is clear, the bacteria are free from impurities, the temperature is 30 ℃, when the pH value in the fermentation process is reduced to 5.0, ammonia is introduced, the pH value is controlled to be 5.0-5.2 by feeding ammonia water, the ventilation rate is 7.9-8.2L/min, the rotating speed is 500-1000 rpm, and the feeding DE value of the tank is controlled to be 10-30 after 36 hours. The rotation speed is gradually increased in the later period, and the culture is carried out for 125 hours, and the enzyme activity is measured by sampling at regular time. And centrifuging to remove thalli to obtain a fermentation supernatant which is crude enzyme liquid, and carrying out enzyme activity determination and enzymatic property detection. The results are shown in Table 3 and FIG. 5. The result shows that the fermentation culture medium is utilized to carry out 7L fermentation tank fermentation culture on recombinant engineering bacteria, wherein the laccase total enzyme activity of the recombinant strain reaches 9234U/mL.

TABLE 3 fermentation enzyme activity data in fermentors

Example 6 laccase degradation test for mycotoxins

6.1 reagents, raw materials

Standard substance: the aflatoxin and zearalenone standard substance are dissolved by pure methanol to prepare a standard substance solution with the concentration of 10 mug/ml (10 ppm).

Succinic acid-sodium hydroxide buffer at ph 4.5: 4.5g succinic acid is dissolved in water, the pH value is regulated to 4.50+/-0.01 by sodium hydroxide, and the volume is fixed to 1L.

Mediator: a total of 3 are respectively: 2, 6-dimethoxyphenol, p-hydroxy carboxylic acid, methyl syringate: liquid samples were prepared at a concentration of 0.1g/ml with 95% ethanol.

6.2 Experimental methods

Enzyme extract: the enzyme samples were extracted with pH4.5 buffer and diluted to about 100U/ml enzyme solution.

Enzyme adding group: adding an enzyme sample (laccase concentration is 1U/ml in a reaction system), adding 10ml of succinic acid-sodium hydroxide buffer solution with pH of 4.5, adding 10 mu l of mycotoxin standard substance and 10 mu l of mediator solution, shaking and mixing uniformly, and carrying out enzymolysis for 24 hours at 30 ℃ and 250r/min respectively. And after the enzymolysis is finished, determining the content of mycotoxin in the sample liquid.

Control group: adding an inactivated enzyme sample (the laccase concentration in the reaction system is 1U/ml, the laccase is inactivated at 95 ℃ for 5 min), adding 10ml of succinic acid-sodium hydroxide buffer solution with pH of 4.5, adding 10 μl of mycotoxin standard substance and 10 μl of mediator solution, shaking and mixing uniformly, and carrying out enzymolysis at 30 ℃ and 250r/min for 24 hours respectively. And after the enzymolysis is finished, determining the content of mycotoxin in the sample liquid.

TABLE 4 decomposition efficiency of aflatoxin AF B1

TABLE 5 decomposition efficiency of aflatoxin AF B1

The results are shown in tables 4 and 5. The laccase and the mediator methyl syringate have remarkable damage effect on the zearalenone, and the degradation rate reaches 96.4%. The degradation rate of the laccase and the mediator p-hydroxy formic acid on the zearalenone reaches 46.2%, which indicates that the laccase has very strong degradation rate on the zearalenone degradation efficiency and very remarkable effect on toxin degradation.

The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.

Claims (10)

1. A laccase gene characterized in that: the nucleotide sequence is shown as SEQ ID NO. 2.

2. The laccase gene according to claim 1, characterized in that: the amino acid sequence of the protein coded by the laccase gene is shown as SEQ ID NO. 1.

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

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

5. A laccase genetically engineered bacterium is characterized in that: and (3) introducing laccase gene shown in SEQ ID NO.2 into host bacteria.

6. The laccase genetically engineered bacterium of claim 5, wherein:

the host bacteria are selected from Aspergillus niger.

7. The method for constructing laccase genetically engineered bacteria in claim 6, which is characterized in that: the method comprises the following steps:

s1, artificially synthesizing the laccase gene;

s2, carrying out a ligation reaction on the laccase gene subjected to NotI and PmeI double-enzyme digestion and pAN-EXP plasmid to obtain a recombinant plasmid;

s3, introducing the recombinant plasmid into a protoplasm of the Aspergillus niger VT-002, and obtaining laccase genetic engineering bacteria through screening and verification.

8. Use of the laccase gene of claim 1 or the expression cassette of claim 3 or the expression vector of claim 4 or the laccase engineering bacterium of any one of claims 5 to 6 for producing laccase.

9. The use according to claim 8, characterized in that:

the specific steps of the application are as follows: shake flask culture is carried out on the laccase genetically engineered bacterium, the culture is carried out for 5 days at the temperature of 32 ℃, and the supernatant of the culture solution is obtained by removing thalli through centrifugation, thus obtaining crude enzyme solution.

10. The use according to claim 8, characterized in that:

the specific steps of the application are as follows: fermenting and culturing the laccase genetically engineered bacterium, wherein the temperature is 30 ℃, ammonia is introduced when the pH value in the fermentation process is reduced to 5.0, the pH value is controlled to be 5.0-5.2 by adding ammonia water, the ventilation rate is 7.9-8.2L/min, the rotating speed is 500-1000 rpm, the feeding DE value is controlled to be 10-30 after 36 hours, the rotating speed is gradually increased in the later period, the culture is carried out for more than 125 hours, and the bacterial body is centrifugally removed to obtain culture supernatant, namely crude enzyme liquid.

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