Cytochrome P450 mutant enzyme for catalyzing hydroxylation of steroid compound 16 alpha and application of cytochrome P450 mutant enzyme

Abstract

The invention belongs to the technical field of biological catalysis, and relates to a cytochrome P450 mutant enzyme for catalyzing hydroxylation of a steroid compound 16 alpha and application of the cytochrome P450 mutant enzyme. The invention relates to a cytochrome P450 mutant enzyme for catalyzing hydroxylation of a steroid compound 16 alpha. The mutant enzyme is obtained by mutation of at least one site of a 92 site, a 114 site, a 248 site and a 282 site of CYP154C5 in a cytochrome P450 family. Through semi-rational design of CYP154C5, a site-specific mutagenesis method is utilized, a dominant mutant of 16 alpha hydroxylation of the steroid compound is screened, a small and precise mutant library is constructed, the screening efficiency is improved, a high-yield dominant mutant enzyme of the 16 alpha hydroxylation product of the steroid compound is obtained under an H2O2-dependent CYP154C5 peroxygenase system, and compared with a wild type, the dominant mutant enzyme of the 16 alpha hydroxylation product of the steroid compound has the advantages that the mutation efficiency is improved, and compared with a wild type, the mutation enzyme of the 16 alpha hydroxylation product of the steroid compound is greatly improved. The catalytic activity of the catalyst is improved by 12-43 times, and the catalyst has a wider substrate range.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology and relates to a cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds and its applications. Background Technology

[0002] Steroids are ubiquitous in living systems and constitute the second largest class of marketed medical products after antibiotics, playing a vital role in endocrinology, oncology, rheumatology, and gynecology. Hydroxylation is one of the most important reactions regulating the function of steroids. Compared to their less polar non-hydroxylated analogs, hydroxylated steroids typically exhibit higher biological activity. However, the chemical synthesis of hydroxylated steroid variants is challenging. Due to the difficulty in selectively oxidizing the CH bonds in these complex structures, the common method for synthesizing steroids involves cyclization reactions with oxygen functional groups already at the desired positions. This requires cumbersome multi-step synthetic pathways with low overall yields and high costs. Compared to chemical catalysis, biocatalysis offers advantages such as high reaction specificity and stereo- and regioselectivity. In 2003, Szczebara et al. introduced three cytochrome P450 enzymes into Saccharomyces cerevisiae for the hydroxylation modification of steroid nuclei, artificially constructing a self-sufficient biosynthetic pathway. This marked the first time that the entire biosynthesis from glucose to hydrocortisone was achieved in Saccharomyces cerevisiae, laying a solid foundation for the green biomanufacturing of steroidal compounds.

[0003] Cytochrome P450 monooxygenases (P450 or CYP) are a superfamily of iron-containing oxidases with cysteine ​​as the axial ligand and heme as the active site. They play crucial roles in the biosynthesis of natural products, xenobiotic degradation, steroid biosynthesis, and drug metabolism. To date, bioinformatics has identified many cytochrome P450 enzymes involved in the biosynthesis of steroid compounds from various sources (e.g., the CYP102, CYP106, CYP109, CYP154, CYP260, CYP2, and CYP11 families). CYP154C5 from *Nocardia farcinica* IFM 10152 exhibits high regioselectivity and stereoselectivity (16α-hydroxylation) in the hydroxylation of various steroid compounds and is highly expressed in *Escherichia coli*, making it a promising catalyst. However, its ability to perform hydroxylation reactions of steroidal compounds is highly dependent on expensive NADH and Pdx and PdR from *Pseudomonas putida* for continuous electron donation, and the reaction has a low TON (only 0.82-3.33), which is a major factor limiting its industrial application. Studies have shown that a hydrogen peroxide pathway (H2O2shunt) has been discovered in the P450 catalytic cycle, which eliminates the need for cofactor NAD(P)H and reduction chaperone proteins (enzymes), greatly simplifying the catalytic pathway and improving catalytic efficiency. Furthermore, hydrogen peroxide is inexpensive and produces only water as a byproduct, making it environmentally friendly. In conclusion, constructing an H2O2-dependent cytochrome P450 peroxygenase system for catalyzing the hydroxylation of steroidal compounds has significant value and application prospects for improving the hydroxylation of steroidal compounds catalyzed by biological enzymes.

[0004] Chinese patent application No. 202211511297.9 discloses a method for improving the hydrogen peroxide catalytic activity of engineered P450 peroxygenase and achieving the hydroxylation of lauric acid and 4-methoxybenzoic acid. This method modulates the H2O / H2O2 throughput by designing key amino acids at the bottleneck / inlet of the P450 enzyme's water tunnel, allowing H2O2 to enter the internal heme active site more efficiently and enhancing the catalytic activity of P450 peroxygenase. This method was used to engineer CYP154C5 proteins. However, during the modification process, it was found that not all amino acid residue mutations at the bottleneck / inlet of the water tunnel were effective because some amino acids are located far from the active site and the protein surface. Therefore, further research is needed to develop specific properties for different substances. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds, wherein the mutant enzyme is obtained by mutation at least one of the following sites: 92, 114, 248, and 282 of CYP154C5 in the cytochrome P450 family.

[0008] The amino acid at each of the specified sites is mutated to alanine, aspartic acid, or glycine.

[0009] The phenylalanine at position 92 is mutated to alanine, i.e., F92A; the arginine at position 114 is mutated to alanine, i.e., R114A; the arginine at position 248 is mutated to aspartic acid or glycine, i.e., T248D or T248G; and the glutamic acid at position 282 is mutated to alanine, i.e., E282A.

[0010] The mutant enzymes are: CYP154C5-F92A mutant enzyme, CYP154C5-R114A mutant enzyme, CYP154C5-T248G mutant enzyme, CYP154C5-T248D mutant enzyme, CYP154C5-E282A mutant enzyme, CYP154C5-F92A-R114A mutant enzyme, and CYP154C5-F92A-T248G mutant enzyme. Mutant enzymes, including CYP154C5-F92A-T248D, CYP154C5-F92A-E282A, CYP154C5-R114A-T248G, CYP154C5-R114A-T248D, CYP154C5-R114A-E282A, and CYP154C5-T248G-E282... Mutant enzyme A, CYP154C5-F92A-R114A-T248G, CYP154C5-F92A-R114A-T248D, CYP154C5-F92A-R114A-E282A, CYP154C5-F92A-T248G-E282A, CYP154C5-F92A-T248D -E282A mutant enzyme, CYP154C5-R114A-T248G-E282A mutant enzyme, CYP154C5-R114A-T248D-E282A mutant enzyme, CYP154C5-F92A-R114A-T248G-E282A mutant enzyme or CYP154C5-F92A-R114A-T248D-E282A mutant enzyme.

[0011] More preferably, the CYP154C5 mutant enzyme is: CYP154C5-F92A mutant enzyme, CYP154C5-F92A-R114A mutant enzyme, CYP154C5-F92A-T248G mutant enzyme, CYP154C5-F92A-T248D mutant enzyme, CYP154C5-F92A-E282A mutant enzyme, CYP154C5-F92A-R114A-T248G mutant enzyme, CYP154C5-F92A -R114A-T248D mutant enzyme, CYP154C5-F92A-R114A-E282A mutant enzyme, CYP154C5-F92A-T248G-E282A mutant enzyme, CYP154C5-F92A-T248D-E282A mutant enzyme, CYP154C5-F92A-R114A-T248G-E282A mutant enzyme, CYP154C5-F92A-R114A-T248D-E282A mutant enzyme.

[0012] More preferably, the CYP154C5 mutant enzyme is a CYP154C5-F92A-R114A-T248D-E282A mutant enzyme. The amino acid sequence of this mutant enzyme is shown in SEQ ID NO.3.

[0013] More preferably, the CYP154C5 mutant enzyme is a CYP154C5-F92A-R114A-T248D-E282A mutant enzyme. The nucleotide sequence of this mutant enzyme is shown in SEQ ID NO.4.

[0014] A recombinant expression vector comprising the cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of the steroidal compound.

[0015] A recombinant bacterial strain containing the cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds. The host strain is *Escherichia coli* BL21(DE3) expression host.

[0016] The preparation method of the above-mentioned CYP154C5 mutant enzyme includes the following steps:

[0017] Recombinant bacteria containing the CYP154C5 mutant enzyme were cultured to OD. 600 When the concentration of the bacterial cell is 0.6-0.8, FeCl3 and 5'-ALA are added and the culture is carried out for 15-30 min. Then IPTG is added to induce expression. After 15-20 h of induction culture, the bacterial cells are collected, resuspended in buffer, and subjected to ultrasonic disruption and purification to prepare CYP154C5 mutant enzyme.

[0018] The preferred preparation method includes the following steps:

[0019] The CYP154C5 mutant enzyme was transformed into *E. coli* BL21(DE3) expression host, plated on LB agar plates containing 50 μg / mL Kana, and incubated overnight at 37°C. Single colonies were picked and activated overnight in 50 mL of LB medium containing 50 μg / mL Kana. 10 mL of the bacterial culture was then transferred to 500 mL of LB medium containing 50 μg / mL Kana and incubated at 37°C and 200 rpm for 2 hours until OD was achieved. 600 When the concentration of CYP154C5 was 0.6-0.8, FeCl3 and 5'-ALA were added to a final concentration of 0.5 mM, and the mixture was incubated at 30°C for 20 min. IPTG was added to a final concentration of 1 mM to induce expression. The mixture was then placed in a shaker at 30°C and 200 rpm for 18 hours. The cells were collected by centrifugation, resuspended in buffer, and then sonicated and purified to obtain the CYP154C5 mutant enzyme.

[0020] The application of the mutant enzyme, recombinant expression vector, or recombinant strain in catalyzing the hydroxylation of steroidal compounds.

[0021] A method for catalytic hydroxylation of steroidal compounds involves reacting the CYP154C5 mutant enzyme, a steroidal compound solution, H2O2, and PBS buffer solution to obtain a 16α-hydroxylated product of the steroidal compound.

[0022] The final concentration of CYP154C5 mutant enzyme in the reaction system was 1 μM, the final concentration of steroidal compounds was 0.2-1 mM, and the final concentration of H2O2 was 60 mM.

[0023] The steroidal compound solution is prepared by dissolving the steroidal compound in an organic solvent, such as DMSO or methanol, and the steroidal compound may be testosterone, nandrolone, androstenedione, progesterone, adrenocorticosteroid, etc.

[0024] The PBS buffer solution is a PBS buffer solution with a pH of 8.0.

[0025] Beneficial effects of the present invention

[0026] This invention utilizes a semi-rational design of CYP154C5 and site-directed mutagenesis to screen for dominant mutants of 16α-hydroxylated steroidal compounds, constructing a small and efficient mutant library that improves screening efficiency. Under an H2O2-dependent CYP154C5 peroxygenase system, a high-yield dominant mutant enzyme of 16α-hydroxylated steroidal compounds is obtained, exhibiting 12-43 times higher catalytic activity and a broader substrate range compared to the wild type. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Source of materials

[0029] Both *E. coli* DH10B and *E. coli* BL21(DE3) used for protein expression and purification were commercially available products (Thermo Fisher Scientific, Shanghai Sangon Biotech Co., Ltd.).

[0030] The expression host of E. coli BL21(DE3) with catalase genes (katE and katG) knocked out was obtained using routine experimental techniques.

[0031] Testosterone standards were purchased from Aladdin Company;

[0032] The standard nandrolone products were purchased from McLean.

[0033] The androstenedione standard was purchased from McLean Company;

[0034] The conventional solvents and reagents used were of analytical grade and purchased from Shanghai Sinopharm Co., Ltd.

[0035] Example 1: Design and Screening Steps for Mutants

[0036] The nucleotide sequence of the wild-type CYP154C5 is SEQ ID No: 1, and the amino acid sequence is SEQ ID NO: 2. Primer sequences for the mutation sites were designed based on the pET28a-CYP154C5 vector containing the wild-type CYP154C5 gene. Then, using the wild-type (or single mutant, double mutant, etc.) as a template, PCR amplification was performed using primer sequences for different mutation sites to obtain single mutants; then, using the single mutant as a template, double mutants were obtained, and so on, to obtain multiple mutants. The specific procedure for obtaining single mutants is described below.

[0037] Table 1 Primers for mutant amplification

[0038]

[0039]

[0040] Table 2 PCR System

[0041] name volume Primestar mix 25μL Forward primer (F) 1μL Reverse primer (R) 1μL template 1μL <![CDATA[ddH2O]]> 22μL Total volume 50μL

[0042] Table 3 PCR reaction conditions

[0043] step Temperature (℃) time 1 94 5min 2 94 15s 3 57 15s 4 72 4min 5 Steps 2-4 27 cycles 6 72 10min

[0044] After PCR, agarose gel electrophoresis was performed to confirm the presence of the target band.

[0045] The ends of the sequences of different mutants in the PCR system were digested with DPNI to eliminate the template sequence. The DPNI digestion system is shown in Table 3.

[0046] Table 4 DPNI Digestion System

[0047] name volume DPNI 0.5μL buffer 4.5μL PCR system 40μL Total volume 45μL

[0048] Table 5 DPNI digestion reaction conditions

[0049] step Temperature (℃) time 1 37 12min 2 70 15min

[0050] The sequences of different mutants after DPNI digestion were ligated, and the ligation system is shown in Table 5. Ligation was performed at 30℃ for 40 min.

[0051] Table 6 Connection System

[0052]

[0053]

[0054] Transform 10 μL of the ligation system into E. coli DH5α competent cells, mix gently, and incubate on ice for 30 min; heat shock at 42℃ for 90 s, then incubate on ice for 5 min; add 600 μL of fresh LB liquid medium, and incubate at 37℃ with shaking at 200 rpm for 1 h; spread 200 μL of the bacterial culture onto an LB plate containing 50 μg / ml Kana, and incubate overnight at 37℃.

[0055] Once a single colony has grown on the plate, its gene sequence is determined and analyzed.

[0056] The plasmids that were successfully sequenced were different CYP154C5 mutants.

[0057] Taking the CYP154C5-F92A-R114A-T248D-E282A mutant enzyme as an example:

[0058] Using the wild type as a template, a single mutant CYP154C5-F92A was obtained by PCR amplification using the designed F92A primer sequence. Then, using CYP154C5-F92A as a template, a double mutant CYP154C5-F92A-R114A was obtained by PCR amplification using the designed R114A primer sequence. Then, using CYP154C5-F92A-R114A as a template, a triple mutant CYP154C5-F92A-R114A-E282A was obtained by PCR amplification using the designed E282A primer sequence. Finally, using CYP154C5-F92A-R114A-E282A as a template, a tetra mutant CYP154C5-F92A-R114A-T248D-E282A was obtained by PCR amplification using the designed T248D primer sequence.

[0059] Example 2: Experiments using different mutation catalyzed testosterone 16α-hydroxylation reactions

[0060] Using the different CYP154C5 mutant enzymes obtained above, the 16α-hydroxylation of testosterone was catalyzed by H2O2, taking CYP154C5-F92A-R114A-T248D-E282A as an example.

[0061] The reaction system is as follows: 1 mM testosterone (dissolved with DMSO), 1 μM enzyme

[0062] CYP154C5-F92A-R114A-T248D-E282A (F92A-R114A-T248D-E282A) was reacted with 60 mM H2O2 in 1 mL of 100 mM pH 8.0 PBS. The mixture was reacted in a 30°C water bath for 60 min, followed by extraction with 1 mL dichloromethane for 2 min. The organic phase was then passed through a 0.22 μm membrane, dehydrated with anhydrous sodium sulfate, and after complete evaporation of the dichloromethane, redissolved in acetonitrile. HPLC analysis was performed under the following conditions: C18 column; column temperature 30°C; acetonitrile:water = 60:40 (v / v); flow rate 1 mL / min; analysis time 30 min. A control reaction without H2O2 was also included.

[0063] The hydroxylation reaction formula for testosterone is as follows:

[0064]

[0065] The reaction results are as follows:

[0066] Table 7

[0067]

[0068]

[0069] As shown in Table 7, the addition of H2O2 can achieve the 16α-hydroxylation of testosterone by different CYP154C5 mutant enzymes. Example 3: Experiment on the 16α-hydroxylation of nandrolone catalyzed by CYP154C5-F92A-R114A-T248D-E282A.

[0070] The CYP154C5 mutant enzyme obtained above was used to catalyze the 16α-hydroxylation of nandrolone using H2O2.

[0071] The reaction system was as follows: 1 mM nandrolone (dissolved with DMSO), 1 μM enzyme F92A-R114A-T248D-E282A, and 60 mM H2O2 were added to 1 mL of pH 8.0 PBS (100 mM). The mixture was reacted in a 30°C water bath for 60 min, then extracted with 1 mL of dichloromethane for 2 min. The organic phase was passed through a 0.22 μm membrane, dehydrated with anhydrous sodium sulfate, and after complete evaporation of the dichloromethane, redissolved in acetonitrile. The solution was then analyzed by HPLC under the following conditions: C18 column; column temperature 30°C; acetonitrile:water = 60:40; flow rate 1 mL / min; analysis time 30 min. A control reaction without H2O2 was also included.

[0072] The hydroxylation reaction formula for nandrolone is as follows:

[0073]

[0074] The reaction results are as follows:

[0075] Table 8

[0076]

[0077] As shown in Table 8, the addition of H2O2 can achieve the 16α-hydroxylation reaction of nandrolone by F92A-R114A-T248D-E282A.

[0078] Example 4: Experiment on the 16α-hydroxylation of androstenedione catalyzed by CYP154C5-F92A-R114A-T248D-E282A

[0079] The CYP154C5 mutant enzyme obtained above was used to catalyze the 16α-hydroxylation of androstenedione using H2O2.

[0080] The reaction system was as follows: 200 μM androstenedione (DMSO for dissolution), 1 μM enzyme F92A-R114A-T248D-E282A, and 60 mM H2O2 were added to 1 mL of pH 8.0 PBS (100 mM). The mixture was reacted in a 30°C water bath for 60 min, then extracted with 1 mL of dichloromethane for 2 min. The organic phase was passed through a 0.22 μm membrane, dehydrated with anhydrous sodium sulfate, and after complete evaporation of the dichloromethane, redissolved in acetonitrile. The solution was then analyzed by HPLC. HPLC conditions: C18 column; column temperature 30°C; acetonitrile:water = 60:40; flow rate 1 mL / min; analysis time 30 min. A control reaction without H2O2 was also included.

[0081] The hydroxylation reaction formula for nandrolone is as follows:

[0082]

[0083] The reaction results are as follows:

[0084] Table 9

[0085]

[0086] As shown in Table 9, the addition of H2O2 can achieve the 16α-hydroxylation of androstenedione by F92A-R114A-T248D-E282A.

[0087] The amino acid sequence of CYP154C5 is SEQ ID NO.1.

[0088] MGSSHHHHHHSSGLVPRGSHMNACPHSDTLTIDPMITDLAGETSRLRAAGPLTRI

[0089] DLLGVPALAVTGHTLARQLLTDTRLVKDINAWSLWQSGTVTRQWPLIGMIDVD

[0090] RSMFTVDGPEHRRLRIKTTQALTRRRLDALKPTIERYVAELLDDLERAGADGAV

[0091] VDLKSVFAYPLPMRVISALMGVPSEDQEQLLTWYKAFFSILTPQDERLRVIDEMH

[0092] GYFTEMVRRKTAEPGDDLTSALIYATDGETPLTEEEVIGNLQALVAAGHETTVSLI

[0093] LTAVRALLSHPEQLRLVRDGEIGWETAIEETLRWDGPVIHLLMRFATEDIDLGDA

[0094] VIPRGEGVVMSYRAIGRDITVHGADADDFDITRATAARHISFGHGPHICPGAALA

[0095] DNA sequence of RLEAAIALPALFTRFPHLHPALPLDQIPNLPVLTQNDLSHFPIHLGRCYP154C5, SEQ ID NO.2

[0096] CCATGGGCAGTAGCCATCATCATCATCACCATAGTAGTGGTCTGGTTCCGCGT

[0097] GGCAGTCATATGAATGCCTGCCCGCATAGCGATACCCTGACCATTGATCCGAT

[0098] GATTACCGATCTGGCCGGTGAAACCAGCCGCCTGCGCGCAGCAGGTCCGTTA

[0099] ACCCGTATTGATCTGCTGGGCGTGCCGGCACTGGCAGTTACCGGTCATACCCT

[0100] GGCACGCCAGCTGCTGACCGATACCCGTCTGGTTAAAGATATTAATGCCTGGA

[0101] GCCTGTGGCAGAGTGGCACCGTTACCCGCCAGTGGCCGCTGATTGGTATGATT

[0102] GATGTTGATCGCAGTATGTTTACCGTTGATGGTCCGGAACATCGTCGTCTGCG

[0103] TATTAAGACCACCCAGGCCCTGACCCGCCGCCGTTTAGATGCACTGAAACCG

[0104] ACCATTGAACGCTATGTTGCAGAACTGCTGGATGATCTGGAACGCGCCGGCG

[0105] CAGATGGCGCAGTTGTTGATCTGAAAAGCGTTTTTGCCTATCCGCTGCCGATG

[0106] CGTGTGATTAGTGCCCTGATGGGCGTGCCGAGCGAAGATCAGGAACAGCTGC

[0107] TGACATGGTATAAAGCATTTTTCAGTATTCTGACCCCGCAGGATGAACGTCTG

[0108] CGTGTTATTGATGAAATGCATGGCTATTTTACCGAAATGGTTCGTCGTAAAACC

[0109] GCCGAACCGGGTGACGATCTGACCAGCGCCCTGATCTATGCCACCGATGGCG

[0110] AAACCCCGCTGACCGAAGAAGAAGTTATTGGTAATCTGCAGGCACTGGTGGC

[0111] CGCCGGTCATGAAACCACCGTTAGCCTGATTCTGACCGCAGTTCGTGCACTG

[0112] CTGAGTCATCCGGAACAGCTGCGTCTGGTGCGTGATGGCGAAATTGGTTGGG

[0113] AAACCGCAATTGAAGAAACCCTGCGCTGGGATGGTCCGGTTATTCATCTGCTG

[0114] ATGCGCTTTGCCACCGAAGATATTGATCTGGGCGATGCCGTGATTCCGCGTGG

[0115] CGAAGGTGTTGTTATGAGTTATCGTGCAATTGGTCGCGATATTACCGTGCATG

[0116] GTGCAGATGCAGATGATTTTGATATTACCCGTGCAACCGCCGCCCGCCATATTA

[0117] GTTTTGGTCATGGCCCGCATATTTGCCCGGGTGCAGCACTGGCCCGTCTGGAA

[0118] GCCGCAATTGCCCTGCCGGCCCTGTTTACCCGTTTTCCGCATCTGCATCCGGC

[0119] CCTGCCGCTGGATCAGATTCCGAATCTGCCGGTGCTGACCCAGAATGATCTGA

[0120] GTCATTTTCCGATTCATCTGGGCCGCCTCGAG CYP154C5 - F92A - R114A - T248D - E282A mutant CYP154C5 amino acid sequence SEQ ID NO.3 MNACPHSDTLTIDPMITDLAGETSRLRAAGPLTRIDLLGVPALAVTGHTLARQLLTDTRLVKDINAWSLWQSGTVTRQWPLIGMIDVDRSMATVDGPEHRRLRIKTTQALTRRALDALKPTIERYVAELLDDLERAGADGAVVDLKSVFAYPLPMRVISALMGVPSEDQEQLLTWYKAFFSILTPQDERLRVIDEMHGYFTEMVRRKTAEPGDDLTSALIYATDGETPLTEEEVIGNLQALVAAGHEDTVSLILTAVRALLSHPEQLRLVRDGEIGWETAIAETLRWDGPVIHLLMRFATEDIDLGDAVIPRGEGVVMSYRAIGRDITVHGADADDFDITRATAARHISFGHGPHICPGAALARLEAAIALPALFTRFPHLHPALPLDQIPNLPVLTQNDLSHFPIHLGRCYP154C5 - F92A - R114A - T248D - E282A mutant DNA sequence SEQ ID NO.4

[0121] CCATGGGCAGTAGCCATCATCATCATCACCATAGTAGTGGTCTGGTTCCGCGTGGC

[0122] AGTCATATGAATGCCTGCCCGCATAGCGATACCCTGACCATTGATCCGATGATTACC

[0123] GATCTGGCCGGTGAAACCAGCCGCCTGCGCGCAGCAGGTCCGTTAACCCGTATTG

[0124] ATCTGCTGGGCGTGCCGGCACTGGCAGTTACCGGTCATACCCTGGCACGCCAGCTG

[0125] CTGACCGATACCCGTCTGGTTAAAGATATTAATGCCTGGAGCCTGTGGCAGAGTGG

[0126] CACCGTTACCCGCCAGTGGCCGCTGATTGGTATGATTGATGTTGATCGCAGTATGGC

[0127] AACCGTTGATGGTCCGGAACATCGTCGTCTGCGTATTAAGACCACCCAGGCCCTGA

[0128] CCCGCCGCGCATTAGATGCACTGAAACCGACCATTGAACGCTATGTTGCAGAACTG

[0129] CTGGATGATCTGGAACGCGCCGGCGCAGATGGCGCAGTTGTTGATCTGAAAAGCG

[0130] TTTTTGCCTATCCGCTGCCGATGCGTGTGATTAGTGCCCTGATGGGCGTGCCGAGC

[0131] GAAGATCAGGAACAGCTGCTGACATGGTATAAAGCATTTTTCAGTATTCTGACCCC

[0132] GCAGGATGAACGTCTGCGTGTTATTGATGAAATGCATGGCTATTTTACCGAAATGGT

[0133] TCGTCGTAAAACCGCCGAACCGGGTGACGATCTGACCAGCGCCCTGATCTATGCCA

[0134] CCGATGGCGAAACCCCGCTGACCGAAGAAGAAGTTATTGGTAATCTGCAGGCACT

[0135] GGTGGCCGCCGGTCATGAAGATACCGTTAGCCTGATTCTGACCGCAGTTCGTGCAC

[0136] TGCTGAGTCATCCGGAACAGCTGCGTCTGGTGCGTGATGGCGAAATTGGTTGGGA

[0137] AACCGCAATTGCAGAAACCCTGCGCTGGGATGGTCCGGTTATTCATCTGCTGATGC

[0138] GCTTTGCCACCGAAGATATTGATCTGGGCGATGCCGTGATTCCGCGTGGCGAAGGT

[0139] GTTGTTATGAGTTATCGTGCAATTGGTCGCGATATTACCGTGCATGGTGCAGATGCA

[0140] GATGATTTTGATATTACCCGTGCAACCGCCGCCCGCCATATTAGTTTTGGTCATGGC

[0141] CCGCATATTTGCCCGGGTGCAGCACTGGCCCGTCTGGAAGCCGCAATTGCCCTGCC

[0142] GGCCCTGTTTACCCGTTTTCCGCATCTGCATCCGGCCCTGCCGCTGGATCAGATTCC

[0143] GAATCTGCCGGTGCTGACCCAGAATGATCTGAGTCATTTTCCGATTCATCTGGGCC

[0144] GCTAACTCGAG

[0145] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds, characterized in that, The mutant enzyme is obtained by mutation at least one of the following sites: position 92, position 114, position 248, and position 282 of CYP154C5 in the cytochrome P450 family.

2. The cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds according to claim 1, characterized in that, The amino acid at each of the specified sites is mutated to alanine, aspartic acid, or glycine.

3. The cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds according to claim 2, characterized in that, The phenylalanine at position 92 is mutated to alanine, i.e., F92A; the arginine at position 114 is mutated to alanine, i.e., R114A; the arginine at position 248 is mutated to aspartic acid or glycine, i.e., T248D or T248G; and the glutamic acid at position 282 is mutated to alanine, i.e., E282A.

4. The cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds according to any one of claims 1-3, characterized in that, The mutant enzymes are: CYP154C5-F92A mutant enzyme, CYP154C5-R114A mutant enzyme, CYP154C5-T248G mutant enzyme, CYP154C5-T248D mutant enzyme, CYP154C5-E282A mutant enzyme, CYP154C5-F92A-R114A mutant enzyme, and CYP154C5-F92A-T248G mutant enzyme. Mutant enzymes, including CYP154C5-F92A-T248D, CYP154C5-F92A-E282A, CYP154C5-R114A-T248G, CYP154C5-R114A-T248D, CYP154C5-R114A-E282A, and CYP154C5-T248G-E282... Mutant enzyme A, CYP154C5-F92A-R114A-T248G, CYP154C5-F92A-R114A-T248D, CYP154C5-F92A-R114A-E282A, CYP154C5-F92A-T248G-E282A, CYP154C5-F92A-T248D -E282A mutant enzyme, CYP154C5-R114A-T248G-E282A mutant enzyme, CYP154C5-R114A-T248D-E282A mutant enzyme, CYP154C5-F92A-R114A-T248G-E282A mutant enzyme or CYP154C5-F92A-R114A-T248D-E282A mutant enzyme.

5. A recombinant expression vector comprising the cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds as described in claim 1.

6. A recombinant strain comprising the cytochrome P450 mutant enzyme that catalyzes the 16α-hydroxylation of steroidal compounds as described in claim 1.

7. An application as described in claim 1, 5, or 6, characterized in that: The application of the mutant enzyme, recombinant expression vector, or recombinant strain in catalyzing the hydroxylation of steroidal compounds.

8. A method for catalytic hydroxylation of steroidal compounds, characterized in that, The CYP154C5 mutant enzyme described in claim 1, a steroidal compound solution, H2O2, and PBS buffer solution were added to react and obtain the 16α-hydroxylated product of the steroidal compound.

9. The method for catalytic hydroxylation of steroidal compounds according to claim 8, characterized in that, The final concentration of CYP154C5 mutant enzyme in the reaction system was 1 μM, the final concentration of steroidal compounds was 0.2-1 mM, and the final concentration of H2O2 was 60 mM.

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