A monooxygenase mutant producing (4s)-4-hydroxyisophorone and a method for preparing the same

Abstract

The application discloses a kind of generation (4S) 4-hydroxyisophorone monooxygenase mutant and preparation method thereof, the monooxygenase is cytochrome P450 BM3 Monooxygenase from bacillus megaterium. The cytochrome P450 BM3 Monooxygenase mutant has high stereoselectivity and high reaction activity to catalyze isophorone hydroxylation, mutant R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL catalyzes isophorone to obtain (4S) 4-hydroxyisophorone diastereomeric excess rate can reach 95%, yield can reach 96%; the cytochrome P450 BM3 Monooxygenase mutant catalyzes isophorone synthesis (4S) 4-hydroxyisophorone process is simple, environmental protection, and industrialization prospect is good.

Description

TECHNICAL FIELD

[0001] The present application relates to a monooxygenase mutant and a preparation method thereof, in particular to a monooxygenase mutant for producing (4S) 4-hydroxyisophorone and a preparation method thereof. BACKGROUND

[0002] Cytochrome P450 monooxygenases (CYPs) are a class of heme-dependent monooxygenases that can perform regio- and stereoselective hydroxylation of C-H bonds under mild conditions, and are green catalytic tools for constructing complex chiral molecules. Among them, Cytochrome P450 BM3 (CYP102A1) has a naturally fused reductase domain (containing FAD and FMN) and an oxygenase domain, which synergistically work together to achieve precise oxidative modification of substrate molecules by providing electrons through NADPH.

[0003] Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one) is an important cycloalkenone compound, which is widely used in the fields of medicine, perfume and functional materials. The molecule itself contains a chiral center, and when it is hydroxylated at the C4 position under the catalysis of P450 BM3 , a second chiral center will be introduced, thereby generating 4-hydroxyisophorone derivatives with double chiral centers. Such compounds have important application potential in the development of chiral drug lead structures.

[0004] However, the existing P450 BM3 catalytic system generally has the problem of insufficient selectivity in the oxidation process at the C4 position of isophorone. The reasons are twofold: on the one hand, the substrate binding pocket of the enzyme easily leads to multi-site competitive hydroxylation; on the other hand, C4 hydroxylation involves the control of the configuration of two chiral centers, and the regioselectivity and diastereoselectivity are difficult to balance. Therefore, it is currently difficult to achieve efficient and specific synthesis of (4S) 4-hydroxyisophorone. SUMMARY

[0005] The purpose of the present application is to provide a cytochrome P450 BM3 monooxygenase mutant capable of efficiently and specifically synthesizing 4-hydroxyisophorone, and to provide a nucleic acid encoding the mutant, a recombinant vector containing the mutant, a recombinant cell and a product, as well as a preparation method and application thereof. (4S)

[0006] ​The monooxygenase mutant is obtained by mutating the sequence shown in SEQ ID NO. 1, and the mutation is at least one of R47L, Y51F, F81W, A191T, N239H, I259V, A276T, A328I, A330S, L353I, I401L, L437VL, and L437LL, and the mutation is counted from the 2nd amino acid of the sequence.

[0007] The wild-type cytochrome P450 used in the application is shown in SEQ ID NO. 1. BM3 The amino acid sequence of the monooxygenase is shown in SEQ ID NO. 1. The starting amino acid of the sequence is methionine, and the mutation site of the mutant is counted from the 2nd amino acid of the sequence. L437VL and L437LL both represent the insertion of an amino acid before the leucine at position 437. The application uses the published cytochrome P450 BM3 The sequence and structure information of the monooxygenase are obtained by non-redundant search in databases such as NCBI, and some potential enzyme genes are screened according to the principles of protein structure similarity, conserved site analysis, and host source diversity. These genes are functionally expressed in an E. coli expression system, and then purified to obtain purified cytochrome P450 BM3 The monooxygenase mutant is obtained by mutating the sequence shown in SEQ ID NO. 1, and the mutation is at least one of R47L, Y51F, F81W, A191T, N239H, I259V, A276T, A328I, A330S, L353I, I401L, L437VL, and L437LL, and the mutation is counted from the 2nd amino acid of the sequence. BM3 The monooxygenase gene is subjected to directed evolution modification to obtain a cytochrome P450 (4S) The cytochrome P450 BM3 The monooxygenase mutant, preferably R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / L437VL, R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / I401L / L437VL, R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437VL, and R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL, has an amino acid sequence corresponding to SEQ ID NO. 2-5, respectively.

[0008] The application also provides a nucleic acid encoding the monooxygenase mutant.

[0009] The present application also provides a recombinant vector comprising the nucleic acid. The recombinant vector can maintain its replication or autonomous replication ability in various host cells such as prokaryotic and / or eukaryotic cells, thereby amplifying or expressing the nucleic acid. The recombinant vector can be various vectors in the art, such as various plasmids, bacteriophages or viral vectors, etc. Preferably, the PET system expression vector, such as pET28a(+) plasmid.

[0010] The present application also provides a recombinant cell comprising the recombinant vector. The recombinant cell is preferably Escherichia coli, such as Escherichia coli C43 or Escherichia coli BL21.

[0011] The present application also provides a method for preparing the monooxygenase mutant: constructing and culturing the recombinant cell, and inducing expression of the monooxygenase mutant. The method for constructing the recombinant cell is preferably: using whole plasmid PCR to mutate the wild-type monooxygenase gene to obtain the target mutant gene and construct the recombinant vector; and then transforming the recombinant vector into a competent cell to obtain the recombinant cell. After the expression is completed, the cells are preferably collected; or the cells are broken, and the crude enzyme solution or pure enzyme is collected. The collected cells or pure enzyme are preferably prepared into immobilized cells or immobilized enzyme by using immobilization technology.

[0012] The present application also provides a product for generating (4S) -4-hydroxyisophorone, comprising the monooxygenase mutant, or the nucleic acid, or the recombinant vector, or the recombinant cell.

[0013] The present application also provides a use of the product in catalyzing isophorone to generate (4S) -4-hydroxyisophorone. The catalysis can be carried out in a potassium phosphate buffer solution (dipotassium hydrogen phosphate-potassium dihydrogen phosphate). The reaction conditions of the catalysis include that the reaction is carried out at a temperature of 20-35 ℃, a pH of 5.0-10.0, and for 8-20 h; preferably, the temperature is 25 ℃, the pH is 7.5, and the reaction time is 16 h.

[0014] Advantages: Compared with the prior art, the present application has the following obvious advantages:

[0015] 1. The cytochrome P450 BM3 monooxygenase mutant has high stereoselectivity for catalyzing isophorone hydroxylation, and the mutant R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL catalyzes to obtain (4S) -4-hydroxyisophorone with a diastereomeric excess rate of up to 95%, which is much better than the wild type;

[0016] 2. The cytochrome P450BM3 Monooxygenase mutants exhibit high reactivity for catalyzing the hydroxylation of isophorone. The mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL catalyze the production of... (4S) The yield of 4-hydroxyisophorone can reach 96% (20mM), which is far superior to that of wild type;

[0017] 3. The cytochrome P450 provided by this invention BM3 Monooxygenase mutants catalyze isophorone synthesis (4S) The production process of 4-hydroxyisophorone is simple, the reaction conditions are mild, and the production process is environmentally friendly, which is conducive to industrial production and has broad application prospects. Attached Figure Description

[0018] Figure 1 Cytochrome P450 BM3 Monooxygenase-catalyzed isophorone synthesis (4S) The reaction of -4-hydroxyisophorone;

[0019] Figure 2 It is cytochrome P450 BM3 The yield and diastereoselectivity of isophorone to (4S)-4-hydroxyisophorone catalyzed by wild-type and mutant monooxygenases are shown in the figure (M1-M4 correspond to mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / L437VL, respectively). L353I / I401L / L437VL, mutant R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437VL, mutant R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL);

[0020] Figure 3 Cytochrome P450 at different pH values BM3 Comparison of catalytic effects of monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL;

[0021] Figure 4Cytochrome P450 at different temperatures BM3 Comparison of catalytic effects of monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL;

[0022] Figure 5 Cytochrome P450 at different reaction times BM3 Comparison of catalytic effects of monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Wild-type cytochrome P450 used in the examples BM3 The amino acid sequence of the monooxygenase is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.6. It can use isophorone as a substrate and NADPH as a cofactor to donate electrons in glucose-glucose dehydrogenase (GPH). ls Under a GDH electron cycle system, catalytic synthesis of 4-hydroxyisophorone was performed, including... (4R) -4-hydroxyisophorone and (4S) -4-hydroxyisophorone is one of the enantiomers. The reaction process is as follows: Figure 1 As shown.

[0025] The acquisition methods of the materials used in the embodiments:

[0026] strains and plasmids

[0027] plasmid pET-28a(+), E. coli BL21(DE3), E. coli All DH5α samples are held in the applicant's collection and originated from commercial sources. Wild-type cytochrome P450 BM3 Monooxygenases are derived from Bacillus megaterium ( Bacillus megaterium ), ls GDH comes from Staphylococcus oxidans ( Gluconobacter oxydans The encoding genes for both enzymes were synthesized by Genewiz (Suzhou) Co., Ltd. The site-directed mutagenesis sequences of the enzymes were obtained by PCR and constructed by the applicant.

[0028] Reagents and culture media

[0029] The DNA gel extraction kit and plasmid extraction kit are both from Sangon Biotech (Shanghai) Co., Ltd.

[0030] The LB liquid culture medium consisted of 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride.

[0031] The LB solid medium consists of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0032] The FB liquid culture medium consists of 14.66 g / L dipotassium hydrogen phosphate, 2.48 g / L sodium dihydrogen phosphate, 2.5 g / L ammonium sulfate, 2 g / L anhydrous sodium sulfate, 1.2 g / L trisodium citrate dihydrate, 0.5 g / L ammonium chloride, and 5 ml / L glycerol.

[0033] The TB liquid culture medium consisted of 12 g / L yeast extract, 12 g / L tryptone, 4 ml / L glycerol, 12.5 g / L dipotassium hydrogen phosphate, and 2.3 g / L potassium dihydrogen phosphate.

[0034] ls Obtaining GDH powder: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] ls The pET-22b plasmid containing the GDH gene was transformed into... E. coli BL21(DE3) host bacteria were plated on the surface of LB solid medium containing 100 μg / mL ampicillin and incubated at 37 °C for 8 h. Individual *E. coli* colonies were inoculated into 3 mL of LB liquid medium containing 100 μg / mL ampicillin and incubated overnight at 37 °C as seed culture. The seed culture was inoculated at a 5% inoculation rate into a 250 mL Erlenmeyer flask containing 50 mL of TB medium and incubated at 37 °C and 200 rpm. After 8 h of incubation, IPTG was added to a final concentration of 0.5 mM, and the incubation temperature was set to 18 °C for another 16 h. The fermentation broth was centrifuged, the cells were collected, and resuspended in 200 mM phosphate buffer (pH 7.5). The resulting crude enzyme solution was sonicated, centrifuged, and the supernatant was collected and lyophilized to obtain... ls GDH powder.

[0035] Example 1

[0036] This embodiment provides the cytochrome P450 obtained by screening according to the present invention. BM3 Methods for monooxygenase mutants, and providing the cytochrome P450 BM3 Method for preparing monooxygenase mutants.

[0037] 1. Construction of cytochrome P450 by ARTP mutagenesis in Escherichia coli BM3 The nucleic acid of the monooxygenase mutant and the host cell of the expression vector.

[0038] Will contain wild-type cytochrome P450BM3 Using Escherichia coli monooxygenase as a template strain, gene mutations were performed on it using the ARTP mutagenesis method to obtain strains carrying the mutated gene (i.e., those carrying cytochrome P450). BM3 E. coli with monooxygenase mutant nucleic acid was obtained through screening and sequencing to obtain cytochrome P450. BM3 Nucleic acid of monooxygenase mutant.

[0039] Specifically, it will contain wild-type cytochrome P450 BM3 Escherichia coli with monooxygenase was inoculated from a glycerol tube and placed in a solution containing 30 mg / mL kanamycin sulfate (… Kanamycin sulfate Streak the bacteria on LB solid medium and incubate overnight at 37°C. Pick a single colony and incubate in LB broth containing 30 mg / mL kanamycin sulfate at 37°C for 12 hours. Take a sample and dilute to OD500. 600 =0.8, add glycerol to a final concentration of 5% to obtain the bacterial suspension required for ARTP mutagenesis.

[0040] 10 μL of the obtained bacterial suspension was transferred to a sterile iron plate and mutagenesis was induced using the Tianmu Bio-ARTP-M plasma mutagenesis breeding system. The voltage was set to 100V, and the running time was 60s to 240s. After mutagenesis, the iron plate was placed in 0.5 mL of LB liquid medium and incubated at 37℃ for 30 min. 10 μL of the medium was then spread onto LB solid medium containing 30 mg / mL kanamycin sulfate and incubated overnight at 37℃. 500 single colonies from the medium were picked and transferred to 96-well plates containing 0.5 mL of LB liquid medium containing 30 mg / mL kanamycin sulfate per well. The plates were then shaken and incubated at 37℃ and 220 rpm for 16 h to obtain the seed culture. 100 μL of the seed culture was inoculated into 2 mL of FB liquid medium containing a final concentration of 30 mg / mL kanamycin sulfate, 2 mM MgSO4, 0.01% w / v vitamin B1, and trace elements. 600 When the concentration reaches approximately 0.6, add a final concentration of 0.03 g / 10 mL of tryptone, 0.04 μM IPTG (isopropyl-β-D-thiogalactopyranoside), 0.3 μM δ-AlA, and 1 mL of 10 mM FeCl3. Induce at 18°C ​​for approximately 36–48 h to obtain cytochrome P450. BM3 Cultures of different mutants of monooxygenase.

[0041] Finally, through catalytic performance screening and sequencing, four cytochrome P450 molecules were identified. BM3Monooxygenase mutants: R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / L437VL, R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / I401L / L437VL, R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437VL, R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL. And the cytochrome P450 was obtained by plasmid extraction kit BM3 Recombinant mutant plasmid strains of monooxygenase mutants and corresponding mutant plasmids.

[0042] 2, Constructing the expression vector encoding the cytochrome P450 BM3 Nucleic acid of monooxygenase mutants and expression vector encoding the above nucleic acid.

[0043] With the cytochrome P450 BM3 The pET-28a(+) plasmid of monooxygenase gene was used as a template to perform gene mutation by whole plasmid PCR method, and the target mutant gene (i.e. nucleic acid encoding cytochrome P450 BM3 Monooxygenase mutants) was obtained, and was constructed on the pET-28a(+) plasmid (i.e. expression vector encoding the above nucleic acid). The PCR primer encoding the mutant nucleic acid is shown in Table 1.

[0044] Table 1

[0045] .

[0046] The PCR reaction system of the whole plasmid PCR is shown in Table 2.

[0047] Table 2

[0048] Composition Volume 10 x Buffer for KOD-Plus- 2.5 μL 2 mM dNTP 2.5 μL 25 mM MgSO4 1.5 μL DMSO 1 μL 10 pmol / μL Forward Primer 0.75 μL 10 pmol / μL Reverse Primer 0.75 μL DNA template < 100 ng KOD-Plus- 1 μL ddH2O up to 25 μL

[0049] The PCR reaction program (cycle 32 times) of the whole plasmid PCR is shown in Table 3.

[0050] Table 3

[0051] .

[0052] After the target fragment PCR amplification, the amplification product was detected by 1% agarose gel electrophoresis, and the results showed that the amplification product was a single band with a size of about 6000 bp. The amplification product was purified and recovered by using a DNA gel recovery and purification kit set.

[0053] The final cytochrome P450 BM3 The pET28a(+) plasmid of monooxygenase mutant, i.e., cytochrome P450 BM3 The expression vector of monooxygenase mutant.

[0054] 3. Constructing a host cell transfected with an expression vector.

[0055] The purified gene fragment was transferred into Escherichia coli DH5α strain (hereinafter referred to as E. coli DH5α), and a recombinant mutant expression strain E. coli DH5α / pET28a(+)-P450 BM3 was constructed.

[0056] Specifically, the purified gene fragment was digested with EasyCut endonuclease-DpnI to remove the template, and then recombined with recombinase. The recombination product was transformed into E. coli DH5α competent cells, spread on the surface of LB solid medium containing 30 mg / mL kanamycin sulfate, and cultured at 37°C for 16 h. Single colonies were picked into LB liquid culture containing 30 mg / mL kanamycin sulfate and cultured at 37°C with 220 rpm shaking for 16 h. After the culture, a part of the bacterial solution was added with sterile glycerol to a final glycerol concentration of 15-20%, numbered, and stored at 80°C for standby, obtaining the recombinant mutant plasmid clone strain E. coli DH5α / pET28a(+)-P450 BM3 ; a part of the bacterial solution was centrifuged at 8,000 rpm for 3 min and the cells were collected, and the plasmid was extracted from E. coli DH5α / pET28a(+)-P450 BM3 using a high-purity plasmid extraction kit, and the correctness of the mutation site was verified by sequencing.

[0057] The final cytochrome P450 BM3 The recombinant mutant plasmid clone strain of monooxygenase mutant and the corresponding recombinant mutant plasmid, i.e., the transfected cytochrome P450 BM3 monooxygenase mutant expression vector host cell.

[0058] 4. Constructing a recombinant mutant protein expression strain to obtain a crude enzyme solution containing cytochrome P450 BM3 monooxygenase mutant.

[0059] The sequencing successful pET28a(+) plasmid was transformed into E. coli BL21(DE3) strain (hereinafter referred to as E. coli BL21(DE3)), E. coli BL21(DE3) is an expression host (i.e. host cell), and a recombinant mutant protein expression strain is constructed E. coli BL21(DE3) / pET28a(+)-P450 BM3 .

[0060] Specifically, the successfully constructed recombinant mutant plasmid is transformed into E. coli BL21(DE3) competent cells, spread on a kanamycin sulfate plate with a final concentration of 30 mg / mL, and then a single colony is inoculated into a culture tube containing 10 mL of LB liquid medium containing 30 mg / mL of kanamycin sulfate, and cultured at 37°C, 220 rpm for 16 h, thereby obtaining a recombinant mutant protein expression strain E. coli BL21(DE3) / pET28a(+)-P450 BM3 . The inoculation amount is 8-10 mL, which is transferred into 1 L of FB liquid medium containing a final concentration of 30 mg / mL kanamycin sulfate, 2 mM MgSO4, 0.01% w / v vitamin B1 and trace elements, and when the OD 600 reaches about 0.6, a final concentration of 0.03 g / 10 mL of tryptone, 0.04 μM IPTG (i.e. isopropyl-β-D-thiogalactopyranoside), 0.3 μM δ-AlA, 1 mL of 10 mM FeCl3 is added, and induced at 18°C for about 36-48 h, thereby obtaining a culture containing cytochrome P450 BM3 monooxygenase mutants.

[0061] After induction, the bacterial cells are centrifuged, resuspended with buffer, and the cells are broken by ultrasonic treatment under ice bath conditions (working for 2 s, interval for 5 s, working time for 30 min), centrifuged at 12,000 rpm / min for 20 min at 4°C, and the supernatant is collected, thereby obtaining a crude enzyme solution of cytochrome P450 BM3 monooxygenase mutants.

[0062] Further, according to the amino acid sequence of cytochrome P450 BM3 monooxygenase, the molar absorption coefficient of the protein is calculated, and the absorbance of the purified protein is measured by A 450 method, and the concentration of the protein (wild-type cytochrome P450 BM3 monooxygenase and cytochrome P450 BM3 monooxygenase mutants) can be calculated.

[0063] Example 2

[0064] This example compares wild-type cytochrome P450. BM3 Monooxygenase and the preferred mutant of Example 1 catalyze the generation of (4S) The ability to improve the yield and diastereoselectivity of 4-hydroxyisophorone.

[0065] Based on the wild-type cytochrome P450 prepared in Example 1 BM3 Monooxygenase and preferred mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / L437VL, R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / I401L / L437VL, R47L / Y51F / F8 The crude enzyme solutions corresponding to 1W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437VL and R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL were used as catalysts at a protein concentration of 4 μM, a substrate isophorone concentration of 20 mM, and a coenzyme concentration of 1W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL. ls The final concentration of GDH was 5 mg / ml, and the cofactor NADP was... + In a reaction system consisting of a potassium phosphate buffer solution with a final concentration of 0.8 mM, a final glucose concentration of 50 mM, and a pH of 7.5, the reaction was carried out overnight (approximately 16 h) at 30 °C with a speed of 400 rpm. After the reaction was completed, a solution containing... (4S) The reaction solution of 4-hydroxyisophorone was extracted with 500 µL of ethyl acetate and centrifuged at 1,2000 rpm for 1 min. 300 µL of the supernatant was then analyzed by gas chromatography.

[0066] Gas chromatography conditions: Agilent CYCCOSIL-B column. Gas chromatography program: injection pressure 23 psi, flow rate 2.5 ml / min, column temperature increased from 60 °C for 1 min, then increased to 160 °C at a rate of 20 °C / min and held for 6 min, then increased to 240 °C at a rate of 20 °C / min and held for 2 min. A single sample was sampled for a total of 18 min.

[0067] Test results are as follows Figure 2 As shown. Cytochrome P450 BM3 The yields of the preferred mutants of monooxygenases were all higher than those of the wild-type enzymes, with cytochrome P450 being the most abundant. BM3The monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL all showed higher yields than the wild-type enzymes. Among them, the cytochrome P450BM3 monooxygenase mutant exhibited significantly enhanced catalytic performance, with a catalytic yield of 96% and a diastereomeric overcompensation rate of 95%, which was also superior to the wild-type enzymes, thus facilitating industrial production.

[0068] Example 3

[0069] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3 Catalytic effects of monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL at different pH values.

[0070] Using the mutant as a catalyst, based on the reaction system of Example 2, the concentration of the substrate isophorone was adjusted to 30 mM, and reacted with citrate-sodium citrate buffer solution at pH=5.0 and 6.0 (0.1 M), dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution at pH=7.0, 7.5, and 8.0 (0.1 M), and glycine-sodium hydroxide buffer solution at pH=9.0 and 10.0 (0.1 M), respectively, overnight (approximately 16 h) at 30 °C with 400 rpm. After the reaction, a product containing... (4S) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0071] like Figure 3 As shown, the cytochrome P450 BM3 The monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL exhibit good catalytic efficiency in the pH range of 7.0–8.0. However, the catalytic activity of this enzyme is relatively limited under acidic buffer conditions (pH = 5.0–7.0). When the buffer pH > 8.0, the enzyme activity gradually decreases.

[0072] Example 4

[0073] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3Catalytic effects of monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL at different temperatures.

[0074] Using the mutant as a catalyst, the reaction system of Example 2 was further modified by reacting the mutant at 400 rpm overnight at 20°C, 25°C, 30°C, and 35°C (approximately 16 h). After the reaction, a product containing [missing information] was obtained. (4S) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0075] like Figure 4 As shown, temperature has a significant effect on the catalytic activity of the mutant. The catalytic efficiency is better in the range of 25-35 °C, while the enzyme activity is significantly reduced below 25 °C.

[0076] Example 5

[0077] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3 Catalytic effects of monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL at different reaction times.

[0078] Using the mutant as a catalyst, and employing the same reaction system as in Example 2, the reaction was carried out at 400 rpm and 30 °C for 1 h, 2 h, 4 h, 8 h, and 16 h, respectively. After the reaction, a product containing [missing information] was obtained. (4S) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0079] like Figure 5 As shown, the optimal reaction time for this enzyme is 12–16 h. The catalytic yield gradually increases with increasing reaction time, and then gradually plateaus after 12 h.

[0080] Example 6

[0081] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3 Catalytic effects of monooxygenase mutants R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL at different protein and substrate concentrations.

[0082] Using the mutant as a catalyst, based on the reaction system of Example 2, the reaction was carried out overnight (approximately 16 h) at 30 °C at 400 rpm under the conditions of protein concentrations of 4 μM, 6 μM, and 8 μM, and substrate concentrations of 15 mM, 20 mM, and 25 mM. After the reaction, a product containing [missing information] was obtained. (4S) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0083] The test results are shown in Table 4.

[0084] Table 4

[0085] .

Claims

1. A process for the production of (4S) - a monooxygenase mutant of 4-hydroxyisophorone, characterized in that, obtained by mutating the sequence shown in SEQ ID NO. 1, the mutation being R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / L353I / I401L / L437VL, R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437VL or R47L / Y51F / F81W / A191T / N239H / I259V / A276T / A328I / A330S / L353I / I401L / L437LL, the amino acid sequence of which corresponds to SEQ ID NO. 3-5 respectively, the mutation being counted from the 2nd position of the corresponding sequence.

2. A nucleic acid, characterized in that, The monooxygenase mutant of claim 1.

3. A recombinant vector, characterized in that, The nucleic acid of claim 2.

4. A recombinant cell, characterized in that, The recombinant vector of claim 3.

5. A method for preparing the monooxygenase mutant according to claim 1, characterized in that, The recombinant cell of claim 4 is constructed and cultured, and the monooxygenase mutant is induced to express.

6. The production method according to claim 5, characterized by, The method for constructing the recombinant cell is to obtain the target mutant gene by mutating the wild-type monooxygenase gene using whole-plasmid PCR, and then to construct a recombinant vector; and then to transform the recombinant vector into a competent cell to obtain the recombinant cell.

7. The preparation method according to claim 5, characterized in that, After the expression is completed, the cells are collected; or the cells are broken, and the crude enzyme solution or the pure enzyme is collected.

8. The preparation method according to claim 7, characterized in that, The collected cells or the pure enzyme are prepared into immobilized cells or immobilized enzyme using immobilization technology.

9. A product generated by the process of claim 1. (4S) -4-hydroxyisophorone, characterized in that The product comprises the monooxygenase mutant of claim 1, or the nucleic acid of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4.

10. Use of the product of claim 9 in the catalytic production of 4-hydroxyisophorone. (4S) -4-hydroxyisophorone.​

Images