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

Abstract

The application discloses a kind of generation (4R) 4-hydroxyisophorone monooxygenase mutant and its preparation method, the monooxygenase is derived from cytochrome P450 of bacillus megaterium BM3 Monooxygenase. The cytochrome P450 BM3 Monooxygenase mutant has high stereoselectivity and high reactivity to catalyze isophorone hydroxylation, mutant A74G / V78A / F87V / L188Q / T327A / A328F catalyzes isophorone to obtain (4R) The diastereomeric excess of 4-hydroxyisophorone can reach 99%, and the yield can reach 98%; the cytochrome P450 BM3 Monooxygenase mutant catalyzes isophorone synthesis (4R) 4-hydroxyisophorone production process is simple, reaction condition is mild, production process is environmental protection, is conducive to industrial production, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to a monooxygenase mutant and its preparation method, particularly to a method for generating... (4R) -4-hydroxyisophorone monooxygenase mutant and its preparation method. Background Technology

[0002] Cytochrome P450 monooxygenases (CYPs) are a class of heme-dependent monooxygenases capable of regio- and stereoselective hydroxylation of C–H bonds under mild conditions, serving as 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. The two 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 cycloenone compound widely used in pharmaceuticals, fragrances, and functional materials. This molecule contains a chiral center, which, when located at P450... BM3 When hydroxylation occurs at the C4 position under catalysis, a second chiral center is introduced, resulting in a 4-hydroxyisophorone derivative with bichiral centers. These compounds have significant potential applications in the development of chiral drug lead structures.

[0004] However, the existing P450 BM3 Catalytic systems for the C4 oxidation of isophorone generally suffer from insufficient selectivity. This is due to two reasons: firstly, the enzyme's substrate-binding pocket easily leads to competitive hydroxylation at multiple sites; secondly, C4 hydroxylation involves conformational control of two chiral centers simultaneously, making it difficult to simultaneously achieve regioselectivity and diastereoselectivity. Therefore, it is currently difficult to achieve targeted hydroxylation at the C4 position. (4R) Efficient and specific synthesis of 4-hydroxyisophorone. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for efficient and specific synthesis (4R) 4-Hydroxyisophorone cytochrome P450 BM3 Monooxygenase mutants are provided, along with the nucleic acid encoding the mutant, the recombinant vector containing the mutant, recombinant cells and products, and their preparation methods and applications.

[0006] Technical solution: The monooxygenase mutant is obtained by amino acid mutation of the sequence shown in SEQ ID NO.1, wherein the mutation is at least one of A74G, V78A, F87V, L188Q, T327A, and A328F, and the mutation is counted starting from the second position of the sequence.

[0007] The wild-type cytochrome P450 used in this invention BM3 The amino acid sequence of the monooxygenase is shown in SEQ ID NO.1. The starting amino acid of this sequence is methionine, and the mutation site in the mutant is counted starting from the second position of this sequence. This invention utilizes the disclosed cytochrome P450. BM3 The sequence and structural information of monooxygenases were obtained through a non-redundant search of databases such as NCBI. Based on principles such as protein structural similarity, conserved site analysis, and host origin diversity, potential enzyme genes were screened. These genes were functionally expressed in an *E. coli* expression system and subsequently purified to obtain purified cytochrome P450. BM3 Monooxygenase mutants. Specifically, semi-rational design was used to target the aforementioned cytochrome P450. BM3 Directed evolutionary modification of monooxygenase genes yielded a highly efficient stereoselective catalytic reaction using isophorone as a substrate to produce... (4R) 4-Hydroxyisophorone cytochrome P450 BM3 Monooxygenase mutants, preferably A74G / F87V / L188Q, A74G / F87V / L188Q / A328F, A74G / V78A / F87V / L188Q / A328F, and A74G / V78A / F87V / L188Q / T327A / A328F, whose amino acid sequences correspond to SEQ ID NO.2-5, respectively.

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

[0009] The present invention 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 any vector conventional in the art, such as various plasmids, bacteriophages, or viral vectors; preferably, PET series expression vectors, such as the pET28a(+) plasmid.

[0010] The present invention 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] This invention also provides a method for preparing the monooxygenase mutant: constructing and culturing the recombinant cells, and inducing expression of the monooxygenase mutant. Preferably, the method for constructing the recombinant cells involves: using whole-plasmid PCR to mutate the wild-type monooxygenase gene to obtain the target mutant gene and constructing a recombinant vector; then transforming the recombinant vector into competent cells to obtain the recombinant cells. After expression is completed, the cells are preferably collected; or the cells are lysed to collect crude enzyme solution or pure enzyme. Preferably, the collected cells or pure enzyme are prepared into immobilized cells or immobilized enzyme using immobilization technology.

[0012] The present invention also provides a method for generating (4R) Products containing -4-hydroxyisophorone may include the monooxygenase mutant, the nucleic acid, the recombinant vector, or the recombinant cell.

[0013] This invention also provides a method for the catalytic production of isophorone using the product. (4R) Application in 4-hydroxyisophorone. The catalysis can be carried out in a potassium phosphate buffer solution (dicarboxylate-potassium dihydrogen phosphate). The reaction conditions for the catalysis include a temperature of 20–35 °C and a pH of 5.0–10.0 for 8–20 h; preferably, the temperature is 25 °C, the pH is 7.5, and the reaction time is 16 h.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0015] 1. The cytochrome P450 provided by this invention BM3 Monooxygenase mutants exhibit high stereoselectivity for catalyzing the hydroxylation of isophorone. The mutants A74G / V78A / F87V / L188Q / T327A / A328F catalyze the... (4R) The diastereomeric excess of 4-hydroxyisophorone can reach 99%, which is far superior to that of the wild type;

[0016] 2. The cytochrome P450 provided by this invention BM3 Monooxygenase mutants exhibit high reactivity for catalyzing the hydroxylation of isophorone. The mutants A74G / V78A / F87V / L188Q / T327A / A328F catalyze the hydroxylation of isophorone. (4R) The yield of 4-hydroxyisophorone can reach 98% (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 (4R) 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 (4R) The reaction of -4-hydroxyisophorone;

[0019] Figure 2 It is cytochrome P450 BM3 Graphs showing the yield and diastereoselectivity of isophorone to (4R)-4-hydroxyisophorone catalyzed by wild-type and mutant monooxygenases (M1-M4 correspond to mutants A74G / F87V / L188Q, A74G / F87V / L188Q / A328F, A74G / V78A / F87V / L188Q / A328F, and A74G / V78A / F87V / L188Q / T327A / A328F, respectively).

[0020] Figure 3 Cytochrome P450 at different pH values BM3 Comparison of catalytic effects of monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F;

[0021] Figure 4 Cytochrome P450 at different temperatures BM3 Comparison of catalytic effects of monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F;

[0022] Figure 5 Cytochrome P450 at different reaction times BM3 Comparison of catalytic effects of monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F. 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 is 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 aureus ( 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. coliBL21(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 P450 BM3 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. BM3 Monooxygenase mutants: A74G / F87V / L188Q, A74G / F87V / L188Q / A328F, A74G / V78A / F87V / L188Q / A328F, A74G / V78A / F87V / L188Q / T327A / A328F. Cytochrome P450 was obtained using a plasmid extraction kit. BM3 Recombinant mutant plasmid strains of monooxygenase mutants and corresponding mutant plasmids.

[0042] 2. Construct the encoding of the cytochrome P450. BM3 The nucleic acid of the monooxygenase mutant and the expression vector encoding the above nucleic acid.

[0043] With cytochrome P450 BM3 Using the pET-28a(+) plasmid of the monooxygenase gene as a template, gene mutation was performed on it using whole plasmid PCR to obtain the target mutant gene (i.e., encoding cytochrome P450). BM3 The nucleic acid of the monooxygenase mutant was constructed and stored in the pET-28a(+) plasmid (i.e., the expression vector encoding the above nucleic acid). The PCR primers encoding the mutant nucleic acid are shown in Table 1.

[0044] Table 1

[0045] .

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

[0047] Table 2

[0048] Composition volume 10×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 of KOD-Plus- 1 μL ddH2O up to 25 μL

[0049] The PCR reaction procedure (32 cycles) for the whole plasmid PCR is shown in Table 3.

[0050] Table 3

[0051] .

[0052] After PCR amplification of the target fragment, the amplification products were detected by 1% agarose gel electrophoresis. The results showed that the amplification products were single bands, each approximately 6000 bp in size. The amplification products were then purified and recovered using a DNA gel purification kit.

[0053] Finally, cytochrome P450 was obtained. BM3 The pET28a(+) plasmid of the monooxygenase mutant, i.e., cytochrome P450. BM3 Expression vector for monooxygenase mutants.

[0054] 3. Construct host cells for transfection with expression vectors.

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

[0056] Specifically, the purified gene fragment was digested with EasyCut endonuclease-DpnI to remove the template, and then recombined with recombinase. The recombinant product was transformed into... E. coli DH5α competent cells were plated on LB agar plates containing 30 mg / mL kanamycin sulfate and incubated at 37°C for 16 h. Single colonies were then transferred to LB broth containing 30 mg / mL kanamycin sulfate and cultured at 37°C with shaking at 220 rpm for 16 h. After incubation, a portion of the culture was diluted with sterile glycerol to a final concentration of 15–20%, numbered, and stored at 80°C for later use to obtain recombinant mutant plasmid clones. E. coli DH5α / pET28a(+)-P450 BM3 A portion of the bacterial culture was centrifuged at 8,000 rpm for 3 min and the cells were collected. The cells were then extracted using a high-purity plasmid miniprep kit. E. coli DH5α / pET28a(+)-P450 BM3 Plasmids were extracted and the correctness of the mutation sites was verified by sequencing.

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

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

[0059] The successfully sequenced pET28a(+) plasmid was transformed into Escherichia coli BL21(DE3) strain (hereinafter referred to as...). E. coli BL21(DE3)). E. coli Using BL21(DE3) as the expression host (i.e., host cell), a recombinant mutant protein expression strain was constructed. E. coli BL21(DE3) / pET28a(+)-P450 BM3 .

[0060] Specifically, the successfully constructed recombinant mutant plasmid will be transformed into... E. coli BL21(DE3) competent cells were plated onto plates containing kanamycin sulfate at a final concentration of 30 mg / mL. Single colonies were then picked and inoculated into culture tubes containing 10 mL of LB broth containing 30 mg / mL kanamycin sulfate. The cells were incubated at 37°C and 220 rpm with shaking for 16 h to obtain the recombinant mutant protein expression strain. E. coli BL21(DE3) / pET28a(+)-P450 BM3 Inoculate 8–10 mL of the culture medium 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. In OD... 600 When the concentration reaches approximately 0.6, add a final concentration of 0.03 g / 10 mL tryptone, 0.04 μM PTG (isopropyl-β-D-thiogalactopyranoside), 0.3 μM δ-AlA, and 1 mL 10 mM FeCl3. Induce at 18°C ​​for approximately 36–48 h to obtain cytochrome P450. BM3 Culture of monooxygenase mutant.

[0061] After induction, the cells were obtained by centrifugation, resuspended in buffer, and sonicated under ice bath conditions (2 s intervals, 5 s intervals, 30 min total). The cells were then centrifuged at 12,000 rpm / min for 20 min at 4°C, and the supernatant was collected to obtain cytochrome P450. BM3 Crude enzyme solution of monooxygenase mutant.

[0062] Furthermore, based on cytochrome P450 BM3 The amino acid sequence of the monooxygenase was used to calculate the molar absorptivity of the protein. The purified protein was then analyzed using A... 450 The absorbance of the protein can be measured to calculate the protein content (wild-type cytochrome P450). BM3 Monooxygenases and cytochrome P450 BM3 The concentration of monooxygenase mutants.

[0063] Example 2

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

[0065] Based on wild-type cytochrome P450 prepared in Example 1 BM3 Monooxygenase and the crude enzyme solution catalyst corresponding to the preferred mutants A74G / F87V / L188Q, A74G / F87V / L188Q / A328F, A74G / V78A / F87V / L188Q / A328F, and A74G / V78A / F87V / L188Q / T327A / A328F, at a protein concentration of 4 μM, a substrate isophorone concentration of 20 mM, and a coenzyme concentration of 10 mM. ls The final concentration of GDH was 5 mg / ml, and the cofactor NADP was... + Phosphoric acid with a final concentration of 0.8 mM, a final glucose concentration of 50 mM, and a pH of 7.5.

[0066] In a reaction system composed of potassium buffer solution, 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 potassium buffer was obtained. (4R) 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.

[0067] 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.

[0068] 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. BM3 The monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F exhibit significantly improved catalytic performance, with a catalytic yield of 98% and a diastereomeric over-exchange rate of 99%, which is also superior to the wild-type enzyme and is conducive to industrial production.

[0069] Example 3

[0070] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3 Catalytic effects of monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F at different pH values.

[0071] Cytochrome P450 BM3 Using the monooxygenase mutant A74G / V78A / F87V / L188Q / T327A / A328F as a catalyst, based on the reaction system of Example 2, the concentration of the substrate isophorone was adjusted to 30 mM. The mixture was 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), Tris-hydrochloric acid buffer solution at pH 8.5 (0.1 M), and glycine-sodium hydroxide buffer solution at pH 9.0 and 10.0 (0.1 M) overnight at 30 °C (approximately 16 h) at 400 rpm. After the reaction, a product containing... (4R) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0072] like Figure 3 As shown, cytochrome P450 BM3 The monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F exhibit good catalytic efficiency within 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.

[0073] Example 4

[0074] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3 Catalytic effects of monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F at different temperatures.

[0075] Cytochrome P450 BM3 Using the monooxygenase mutant A74G / V78A / F87V / L188Q / T327A / A328F as a catalyst, the reaction system of Example 2 was further modified by reacting overnight (approximately 16 h) at 20 °C, 25 °C, 30 °C, and 35 °C at 400 rpm. After the reaction, a product containing [catalyst name missing] was obtained. (4R) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0076] like Figure 4 As shown, temperature has a significant effect on the catalytic activity of the monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F. The catalytic efficiency is better in the range of 25~35 °C, while the enzyme activity is significantly reduced below 25 °C.

[0077] Example 5

[0078] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3 Catalytic effects of monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F at different reaction times.

[0079] Cytochrome P450 BM3 Using the monooxygenase mutant A74G / V78A / F87V / L188Q / T327A / A328F 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... (4R) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0080] 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.

[0081] Example 6

[0082] This embodiment investigates the cytochrome P450 with the best yield and diastereoselectivity in Example 2. BM3 Catalytic effects of monooxygenase mutants A74G / V78A / F87V / L188Q / T327A / A328F at different protein and substrate concentrations.

[0083] Cytochrome P450 BM3 Using the monooxygenase mutant A74G / V78A / F87V / L188Q / T327A / A328F 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 protein concentrations of 4 μM, 6 μM, and 8 μM, and substrate concentrations of 15 mM, 20 mM, and 25 mM, respectively. After the reaction, a product containing... (4R) The reaction solution for 4-hydroxyisophorone. The detection method is the same as in Example 2.

[0084] The test results are shown in Table 4.

[0085] Table 4

[0086] .

Claims

1. A method of generation (4R) -4-hydroxyisophorone monooxygenase mutants catalyze the production of isophorone (4R) Its application in 4-hydroxyisophorone is characterized by... The mutant was obtained by amino acid mutation of the sequence shown in SEQ ID NO.

1. The mutation is A74G / V78A / F87V / L188Q / T327A / A328F. The mutation is counted starting from the second position of the sequence. The amino acid sequence of the mutant corresponds to SEQ ID NO.5.

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