Method for improving heterologous soluble expression efficiency of cytochrome P450

Abstract

The invention discloses a method for improving the heterologous soluble expression efficiency of cytochrome P450, which is characterized in that plasmids containing coding genes of fungus-derived cytochrome P450 truncated protein and plasmids containing molecular chaperones are jointly transferred into host bacteria to obtain recombinant genetically engineered bacteria for improving the heterologous soluble expression efficiency of cytochrome P450. Through host screening, molecular chaperonin and cytochrome P450 gene modification, the soluble expression efficiency of cytochrome P450 in Escherichia coli is improved, and the problems of poor protein solubility and low expression efficiency when CYP450 is expressed in Escherichia coli at present are solved.

Description

(1) Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a method for improving the heterologous soluble expression efficiency of cytochrome P450. (2) Background Art

[0002] Cytochrome P450 (CYP450) is a superfamily of heme-containing monooxygenases, which are widely present in bacteria, fungi, and higher organisms. This enzyme can catalyze reactions such as hydroxylation, peroxidation, epoxidation, dehalogenation, and deamination, showing rich catalytic diversity and having broad application prospects in organic chemical industry. The high-level expression of CYP450 is a prerequisite for its industrial application. However, when eukaryotic-source P450 enzymes are heterologously expressed in Escherichia coli, they often form inclusion bodies due to misfolding, resulting in extremely low levels of soluble proteins and non-functional expressed proteins. In addition, when eukaryotic-source CYP450 is heterologously expressed in Escherichia coli, it faces problems such as insufficient cofactor supply, lack of redox proteins, and differences in domain composition.

[0003] Therefore, exploring the soluble and high-level expression of fungal-source P450 enzymes in Escherichia coli can help their industrial application. (3) Summary of the Invention

[0004] The object of the present invention is to provide a method for improving the heterologous soluble expression efficiency of cytochrome P450. The method improves the soluble expression efficiency of cytochrome P450 in Escherichia coli by screening host bacteria, molecular chaperone proteins, and modifying cytochrome P450 genes, and solves the problems of poor protein solubility and low expression efficiency when existing CYP450 is expressed in Escherichia coli.

[0005] The technical solution adopted by the present invention is as follows:

[0006] The present invention provides a method for improving the heterologous soluble expression efficiency of cytochrome P450. The method is to co-transfer a plasmid containing the coding gene of a truncated protein of cytochrome P450 from a fungus and a plasmid containing a molecular chaperone into a host bacterium to obtain a recombinant genetically engineered bacterium with improved heterologous soluble expression efficiency of cytochrome P450. The host bacterium includes one of Rosetta(DE3), BL21-Codon plus(DE3)-RIPL, Super Arctic Express(DE3)RP, and BL21(DE3). The plasmid containing the molecular chaperone includes one of pTf16, pKJE7, pGro7, pG-Tf2, and pG-KJE8. The coding gene of the truncated protein of cytochrome P450 is the coding gene after removing 5-38 amino acids from the N-terminus of cytochrome P450.

[0007] Furthermore, the nucleotide sequence of the cytochrome P450 gene is as shown in SEQ ID NO.1, and the amino acid sequence is as shown in SEQ ID NO.2.

[0008] Furthermore, the host bacterium is Escherichia coli BL21-Codon plus(DE3)-RIPL, and the plasmid containing the molecular chaperone is pGro7.

[0009] Furthermore, 5, 10, 15, 20, 25, 30, 35 or 38 amino acids are removed from the N-terminus of cytochrome P450, preferably 35 amino acids are removed.

[0010] Furthermore, the recombinant genetically engineered bacterium is constructed according to the following steps: 35 amino acids are removed from the N-terminus of cytochrome P450 and then inserted into the BamHI site of the pET-28a expression vector to construct the recombinant plasmid pET-28a-CYP5150AP3-35; the recombinant plasmid pET-28a-CYP5150AP3-35 and the plasmid pGro7 are co-transformed into the competent Escherichia coli BL21-Codon plus(DE3)-RIPL, spread on an LB plate containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured overnight at 37 °C in an inverted manner, and positive clones are selected to obtain the recombinant genetically engineered bacterium.

[0011] Furthermore, the recombinant genetically engineered bacterium is induced to express according to the following method: The recombinant genetically engineered bacterium is inoculated into an LB medium containing 50 μg / mL kanamycin or an LB medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37 °C and 180 - 220 rpm for 8 h; the bacterial liquid is taken and inoculated into a TB medium containing 50 μg / mL kanamycin or an LB medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol at a volume concentration of 1 - 3%, and cultured at 37 °C and 180 - 220 rpm until the cell concentration OD 600 = 0.6 - 0.8, add IPTG with a final concentration of 0.025 - 0.2 mM (0.1 mM), 0.5 mM 5-ALA (5-aminolevulinic acid hydrochloride), 0.5 mM FeSO4, and induce and culture at 18 - 25 °C and 180 rpm for 12 - 20 h (preferably 16 h); centrifuge at 8000 rpm for 10 min at 4 °C to collect the wet bacterial cells; the wet bacterial cells are prepared into a bacterial suspension with a cell concentration of 5 - 15 g / L (preferably 10 g / L) using a phosphate buffer solution of 100 mM and pH 6.0, and broken in an ultrasonic crusher under the conditions of 300 W, broken for 2 s, paused for 1 s for 20 min; the obtained crude enzyme solution is centrifuged at 12000 rpm for 10 min to obtain the supernatant containing the soluble protein of cytochrome P450.

[0012] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0013] The present invention provides a method for heterologously expressing fungal-derived cytochrome P450 in Escherichia coli and improving its soluble expression efficiency. By optimizing the host strain and molecular chaperone system, the correct folding of cytochrome P450 is improved, and the formation of inclusion bodies is reduced. In addition, by removing the disordered Loop structure at the N-terminus of the P450 enzyme to reduce membrane localization signal interference and optimizing the protein folding pathway, the generation of inclusion bodies is further reduced. The method of the present invention significantly enhances the soluble expression of fungal-derived cytochrome P450 in Escherichia coli, up to 96.22%, provides an effective method for the high-efficiency expression of CYP450 derived from fungi in heterologous hosts, and contributes to its functional research and industrial applications. (IV) BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 SDS-PAGE electrophoresis patterns of CYP5150AP3 expressed in different host strains in Example 1; 1: Whole cells of Rosetta(DE3); 2: Precipitation of Rosetta(DE3); 3: Supernatant of Rosetta(DE3); 4: Whole cells of BL21-Codon plus(DE3)-RIPL; 5: Precipitation of BL21-Codon plus(DE3)-RIPL; 6: Supernatant of BL21-Codon plus(DE3)-RIPL; 7: Whole cells of Super Arctic Express(DE3)RP; 8: Precipitation of Super Arctic Express(DE3)RP; 9: Supernatant of Super Arctic Express(DE3)RP; 10: Whole cells of BL21(DE3); 11: Precipitation of BL21(DE3); 12: Supernatant of BL21(DE3).

[0015] Figure 2, SDS-PAGE electrophoresis pattern after the N-terminal optimization expression of CYP5150AP3 in Example 2; 1: -5AA whole cell; 2: -5AA precipitate; 3: -5AA supernatant; 4: -10AA whole cell; 5: -10AA precipitate; 6: -10AA supernatant; 7: -15AA whole cell; 8: -15AA precipitate; 9: -15AA supernatant; 10: -20AA whole cell; 11: -20AA precipitate; 12: -20AA supernatant; 13: -25AA whole cell; 14: -25AA precipitate; 15: -25AA supernatant; 16: -30AA whole cell; 17: -30AA precipitate; 18: -30AA supernatant; 19: -35AA whole cell; 20: -35AA precipitate; 21: -35AA supernatant; 22: -38AA whole cell; 23: -38AA precipitate; 24: -38AA supernatant.

[0016] Figure 3 , SDS-PAGE electrophoresis pattern after the N-terminal optimization of CYP5150AP3 and co-expression with the molecular chaperone system in Example 3; 1: control whole cell; 2: control precipitate; 3: control supernatant; 4: pG-KJE8 whole cell; 5: pG-KJE8 precipitate; 6: pG-KJE8 supernatant; 7: pKJE7 whole cell; 8: pKJE7 precipitate; 9: pKJE7 supernatant; 10: pG-Tf2 whole cell; 11: pG-Tf2 precipitate; 12: pG-Tf2 supernatant; 13: pTf16 whole cell; 14: pTf16 precipitate; 15: pTf16 supernatant; 16: pGro7 whole cell; 17: pGro7 precipitate; 18: pGro7 supernatant.

[0017] Figure 4 , Column chart of the percentage of soluble protein expression of CYP5150AP3 before and after optimization in the host bacterium; CYP5150AP3-RIPL represents the expression of the CYP5150AP3 target gene in BL21-Codon plus(DE3)-RIPL in Example 1; CYP5150AP3-35AA represents the expression of CYP5150AP3 after deleting 35 amino acids at the N-terminus in BL21-Codonplus(DE3)-RIPL in Example 2; CYP5150AP3-pGro7 represents the co-expression of CYP5150AP3 after deleting 35 amino acids at the N-terminus and pGro7 in BL21-Codon plus(DE3)-RIPL in Example 3; CYP5150AP3-BL21 represents the expression of the CYP5150AP3 target gene in BL21(DE3) in Example 1. (V) Specific implementation manners

[0018] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0019] In the embodiments of the present invention, the process equipment or devices not specifically specified are all conventional equipment or devices in the art. The plasmids of the molecular chaperones used in the embodiments of the present invention are all purchased from Baori Biotechnology Co., Ltd., and the host bacteria are purchased from Beijing Huayuanyang Co., Ltd.

[0020] Example 1: Screening of Host Bacteria Expressing CYP5150AP3

[0021] 1. Synthesis of Target Gene Fragment

[0022] The gene fragment (MG721490.1) annotated as "cytochrome P450 family protein" derived from Thanatephorus cucumeris in NCBI was optimized according to the codon preference of Escherichia coli to improve the expression efficiency and stability in Escherichia coli, and the gene fragment was artificially synthesized, denoted as CYP5150AP3 gene. The nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0023] SEQ ID NO.2

[0024] MDPLLKYFLVAREHPFEIGVGLLTAFGAHLALKAFRRANAFRQLDGPTPSS

[0025] SLWGDEALLYDIKTSLTIHDELLSQYGSVCKIKGPLGEDRVWIADPLALSDIVV

[0026] KGFDDFHEVEGFVAWFALTQGPTIITTSGHKHKMQRKILNPVFTAAHMRNLTPT

[0027] FNSIAHHLLEIVTSEVRTSGGNTAVVDMHHWMSNVSLEMIGQAGMGHSFGVM

[0028] TGKEPEYIEASRQLFPLISEMWYIRPFLPALMKIGSAGFRRFVVSYIPFGPVQRL

[0029] RNITETMDKTAAGIYNQKKRALEDGTLESEITAGNDIMSMMLKQNKNVPPED

[0030] QMNEAEIQAQVNGLLFAGHDTTSAALDRTLHLLAHNLEVQDRLRQEVREAHG

[0031] LHGKNLDYDQLNSLQYLDAVCRESLRLWAPGQLVERTAAKDWNLPLRYPIKS

[0032] KDGKTMISNLHVRKGTHLYLSLGSVNRDKQTWGDDASHFKPTRWLTPLPGSV

[0033] AESKIPGVYSNMMTFLGGPRSCIGFKFSQLEMKVILSTLISSFRFETGPEEHFWVASGVLKPHTRRDDGTIDSISSLRLKITLVDY。

[0034] 2. Construction of recombinant plasmid

[0035] The CYP5150AP3 gene was inserted into the BamHI site of the pET-28a expression vector to construct the recombinant plasmid pET-28a-CYP5150AP3.

[0036] 3. Expression of the target gene in different host bacteria

[0037] Using the heat shock method, the recombinant plasmid pET-28a-CYP5150AP3 was transformed into the competent cells of Escherichia coli BL21(DE3), Rosetta(DE3), BL21-Codon plus(DE3)-RIPL, and Super ArcticExpress(DE3)RP, respectively.

[0038] The engineered bacteria BL21(DE3) and Super Arctic Express(DE3)RP containing the CYP5150AP3 gene were spread on an LB plate containing 50 μg / mL kanamycin antibiotic and cultured overnight at 37°C in an inverted position to select positive clones.

[0039] The engineered bacteria Rosetta(DE3) and BL21-Codon plus(DE3)-RIPL containing the CYP5150AP3 gene were spread on an LB plate containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol and cultured overnight at 37°C in an inverted position to select positive clones.

[0040] 4. Induced expression of engineered bacteria

[0041] The engineered bacteria BL21(DE3) and Super Arctic Express(DE3)RP positive clones containing the CYP5150AP3 gene screened in step 3 were inoculated into LB liquid medium containing 50 μg / mL kanamycin antibiotic and cultured at 37 °C and 220 rpm for 8 h; 4 mL of the bacterial solution was taken and added to 200 mL of TB medium containing 50 μg / mL kanamycin, and cultured at 37 °C and 220 rpm until the cell concentration OD 600 = 0.6 - 0.8, then IPTG with a final concentration of 0.1 mM, 5-ALA with a concentration of 0.5 mM, and FeSO4 with a concentration of 0.5 mM were added, and induced and cultured at 25 °C and 180 rpm for 14 h. Finally, centrifuged at 8000 rpm for 10 min at 4 °C to collect the wet bacteria. The wet bacteria were prepared into a bacterial suspension with a cell concentration of 10 g / L using a phosphate buffer solution of 100 mM and pH 6.0, and broken in an ultrasonic crusher at 300 W for 20 min (broken for 2 s, paused for 1 s). The obtained crude enzyme solution was centrifuged at 12000 rpm for 10 min, the supernatant was aspirated, and the precipitate was mixed with the same volume of phosphate buffer solution of 100 mM and pH 6.0. Detect CYP5150AP3 in the bacterial suspension (i.e., whole cells), supernatant, and precipitate by SDS-PAGE electrophoresis.

[0042] The engineered bacteria Rosetta(DE3) and BL21-Codon plus(DE3)-RIPL containing the CYP5150AP3 gene were resistant to 50 μg / mL kanamycin sulfate and 25 μg / mL chloramphenicol, and the crude enzyme solution, supernatant, and precipitate were prepared and detected by SDS-PAGE electrophoresis under the same above conditions and methods.

[0043] By SDS-PAGE electrophoresis detection ( Figure 1 ), the target gene CYP5150AP3 was contained in the bacterial suspension, supernatant, and precipitate prepared from the above 4 host bacteria. The protein gel map was scanned by gray scale using ImageJ, the peak value of gray scale scanning was recorded, and the soluble percentage was calculated. The results are shown in Table 1 and Figure 4 as shown, the results show that the expression level of the target gene in the host bacterium BL21-Codon plus(DE3)-RIPL is the highest, reaching 26.93%.

[0044] Soluble protein percentage = protein content in supernatant / protein content in whole cells

[0045] Table 1. Effects of CYP5150AP3 expression in different host cells on soluble protein content

[0046]

[0047] Example 2: Optimization of the N-terminal Loop Structure of the CYP5150AP3 Target Gene Encoded Protein

[0048] Using the recombinant plasmid pET-28a-CYP5150AP3 in Example 1 as a template, PCR amplification was carried out under the action of the primers in Table 2. The N-terminal Loop structure of the amino acid sequence of CYP5150AP3 shown in SEQ ID NO.2 was excised by 5 - 38 amino acids (-5AA, -10AA, -15AA, -20AA, 2 - 5AA, -30AA, -35AA,

[0049] -38AA).

[0050] Table 2 Primers

[0051]

[0052] PCR amplification system: 2×Phanta Max Buffer (Novizan, Nanjing) 25 μL, Phanta Max Super-Fidelity DNA Polymerase (Novizan, Nanjing) 1 μL, dTNP (Novizan, Nanjing) 1 μL, each primer pair 2 μL, template 1 μL, ddH2O 18 μL.

[0053] PCR amplification program: Pre-denaturation at 95°C for 1 min; denaturation at 95°C for 30 s, annealing at 65°C for 90 s, extension at 72°C for 1 min (a total of 30 cycles); extension at 72°C for 10 min.

[0054] Transfer the PCR amplification product into the BL21-Codon plus(DE3)-RIPL competent cells in Example 2, coat it on an LB plate containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and incubate it upside down at 37°C overnight to select positive clones.

[0055] Express and detect using the method in Example 1, and the results are shown in Table 3, Figure 2 and Figure 4 . The results show that after excising 35 amino acids from the N-terminal Loop of CYP5150AP3, the proportion of the expressed soluble protein is the highest.

[0056] Table 3 Effects of Different N-terminal Truncated Mutants on the Soluble Protein Content of CYP5150AP3

[0057]

[0058]

[0059] Example 3: Effects of Molecular Chaperones on the Soluble Expression of CYP5150AP3

[0060] Plasmids pTf16, pKJE7, pGro7, pG-Tf2, and pG-KJE8 carry different chaperone systems, as shown in Table 4.

[0061] Table 4 Chaperone structures

[0062]

[0063] After removing 35 amino acids from the N-terminal Loop of CYP5150AP3, the recombinant plasmid pET-28a-CYP5150AP3-35 was constructed according to the method of Example 1. It was co-transformed into Escherichia coli BL21-Codon plus(DE3)-RIPL competent cells with plasmids pTf16, pKJE7, pGro7, pG-Tf2, and pG-KJE8 respectively to construct recombinant genetically engineered bacteria. Using 50 μg / mL kanamycin and 25 μg / mL chloramphenicol as screening resistances, induction expression and detection were carried out by the method of Example 1. The recombinant Escherichia coli BL21-Codon plus(DE3)-RIPL / pET-28a-CYP5150AP3-35 without chaperone was used as a control. The results are shown in Table 5. Figure 3 and Figure 4 , and the results showed that pGro7 was the best chaperone, and the optimal expression system engineering bacteria was the recombinant Escherichia coli BL21-Codon plus(DE3)-RIPL / pET-28a-CYP5150AP3-35 / pGro7.

[0064] Table 5 Effects of different chaperones on the soluble protein content of CYP5150AP3

[0065]

[0066]

[0067] Example 4. Screening of culture conditions

[0068] The inducer concentration (0.025, 0.05, 0.1, 0.15, 0.2 mM), induction time (12, 14, 16, 18, 20 h), and induction temperature (18, 25, 28, 32 °C) of the recombinant Escherichia coli BL21-Codonplus(DE3)-RIPL / pET-28a-CYP5150AP3-35 / pGro7 screened in Example 3 were optimized. The specific operations are as follows:

[0069] 1. Inducer concentration

[0070] Inoculate the above recombinant Escherichia coli into LB liquid medium containing 50 μg / mL kanamycin antibiotic, and culture it at 37°C and 220 rpm for 8 h; take 4 mL of the bacterial solution and add it to 200 mL of TB medium containing 50 μg / mL kanamycin, and culture it at 37°C and 220 rpm until the cell concentration OD 600 = 0.6 - 0.8, add IPTG with different final concentrations (0.025, 0.05, 0.1, 0.15, 0.2 mM), 0.5 mM 5-ALA, 0.5 mM FeSO4, and induce and culture at 25°C and 180 rpm for 14 h. Finally, centrifuge at 8000 rpm for 10 min at 4°C to collect the wet cells. The wet cells are prepared into a cell suspension with a cell concentration of 10 g / L using 100 mM phosphate buffer solution with a pH of 6.0, and broken in an ultrasonic crusher at 300 W for 20 min (broken for 2 s, paused for 1 s). The obtained crude enzyme solution is centrifuged at 12000 rpm for 10 min, the supernatant is aspirated, and the precipitate is mixed with the same volume of 100 mM phosphate buffer solution with a pH of 6.0, and the percentage of soluble protein is calculated using the method of Example 1.

[0071] (2) Induction time

[0072] Fix the inducer concentration in the above (1) at 0.1 mM, and change the induction culture time to 12, 14, 16, 18, 20 h respectively, with the other operations being the same.

[0073] (3) Induction temperature

[0074] Fix the inducer concentration in the above (1) at 0.1 mM, change the induction culture time to 16 h, and change the induction temperature to 18, 25, 28, 32°C respectively, with the other operations being the same.

[0075] Finally, it is found that when the concentration of the inducer IPTG is 0.1 mM, the induction time is 16 h, and the induction temperature is 28°C, the content of soluble protein in the expressed protein is the highest, increasing from 92.34% to 96.22%.

[0076] The above embodiments are for illustrating the implementation schemes disclosed in the present invention and should not be construed as limitations on the present invention. In addition, various modifications and changes to the methods and conditions in the present invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A method for improving the efficiency of heterologous soluble expression of cytochrome P450, characterized in that: The method comprises the following steps: a plasmid containing a gene encoding a truncated protein of cytochrome P450 of fungal origin and a plasmid containing a molecular chaperone are co-transferred into a host bacterium to obtain a recombinant genetic engineering bacterium capable of improving the heterologous soluble expression efficiency of cytochrome P450; the host bacterium comprises one of Rosetta (DE3), BL21-Codon plus (DE3)-RIPL, Super Arctic Express (DE3) RP and BL21 (DE3); the plasmid containing a molecular chaperone comprises one of pTf16, pKJE7, pGro7, pG-Tf2 and pG-KJE8; and the gene encoding the truncated protein of cytochrome P450 is a gene obtained by removing 5 to 38 amino acids from the N-terminus of cytochrome P450.

2. The method according to claim 1, characterized in that The amino acid sequence of cytochrome P450 is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that The host bacteria is Escherichia coli BL21-Codon plus (DE3)-RIPL.

4. The method according to claim 1, characterized in that The plasmid containing the molecular chaperone is pGro7.

5. The method according to claim 1, characterized in that 5, 10, 15, 20, 25, 30, 35 or 38 amino acids are cleaved from the N-terminus of the cytochrome P450.

6. The method according to claim 1, characterized in that The N-terminal 35 amino acids of cytochrome P450 are cleaved.

7. The method according to claim 1, characterized in that The recombinant genetic engineering bacteria are constructed according to the following steps: 35 amino acids are removed from the N-terminus of cytochrome P450 and then inserted into the BamHI site of the pET-28a expression vector to construct a recombinant plasmid pET-28a-CYP5150AP3-35; the recombinant plasmid pET-28a-CYP5150AP3-35 and the plasmid pGro7 are co-transformed into the competent Escherichia coli BL21-Codon plus (DE3)-RIPL, spread on an LB plate containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, cultured inverted at 37°C overnight, and positive clones are selected to obtain the recombinant genetically engineered bacteria.

8. The method according to claim 1, characterized in that The recombinant genetically engineered bacteria are induced to express in the following manner: the recombinant genetically engineered bacteria are inoculated into an LB medium containing 50 μg / mL kanamycin or 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37°C, 180-220 rpm for 8 hours; the bacterial solution is inoculated into a TB medium containing 50 μg / mL kanamycin or 50 μg / mL kanamycin and 25 μg / mL chloramphenicol at a volume concentration of 1-3%, and cultured at 37°C, 180-220 rpm until the bacterial concentration OD 600 =0.6~0.8, add IPTG with a final concentration of 0.025-0.2mM, 0.5mM 5-aminolevulinic acid hydrochloride, 0.5mM FeSO4, and induce culture at 18-25℃, 180rpm for 12-20h; centrifuge at 8000rpm for 10min at 4℃ to collect wet bacteria; the wet bacteria are prepared into a bacterial suspension with a bacterial concentration of 5-15g / L with 100mM, pH 6.0 phosphate buffer solution, and crushed for 20min in an ultrasonic crusher at 300W, crushing for 2s, and pausing for 1s; the obtained crude enzyme solution is centrifuged at 12000rpm for 10min to obtain a supernatant containing cytochrome P450 soluble protein.

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