Cyclohexanone monooxygenase mutant and its application in the preparation of podophyllotoxin precursor

Abstract

This invention belongs to the field of bioengineering technology, and specifically relates to a cyclohexanone monooxygenase mutant, a nucleic acid encoding the cyclohexanone monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, and the application of the cyclohexanone monooxygenase mutant in the preparation of the anticancer drug podophyllotoxin precursor (R)-3-piperylbutyrolactone. Compared with the prior art, the cyclohexanone monooxygenase mutant provided by this invention has advantages such as mild reaction conditions, high economy, high product stereoselectivity, and high yield, and therefore has a promising application in the preparation of the anticancer drug podophyllotoxin precursor.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a cyclohexanone monooxygenase mutant, a nucleic acid encoding the cyclohexanone monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, and the application of the cyclohexanone monooxygenase mutant in the preparation of the anticancer drug podophyllotoxin precursor (R)-3-piperylbutyrolactone. Background Technology

[0002] Podophyllotoxin (PTOX) is a lignan extracted from the roots and stems of plants in the Podophyllum genus. It binds to tubulin, causing cell arrest during metaphase of mitosis, leading to cell death and thus exhibiting antitumor activity. Its glycosylated derivatives, such as etoposide (VP-16) and teniposide (VM-26), can reduce toxicity while maintaining their antitumor efficacy to avoid or mitigate damage to the human body. Since the 1980s, dozens of podophyllotoxin-based drugs targeting various human cancers have entered Phase I to III clinical trials.

[0003] Currently, podophyllotoxin is mainly extracted from plants of the Podophyllum genus. This method has low yield and causes serious environmental damage, which is inconsistent with the concept of sustainable development. In recent years, the biosynthetic pathway of podophyllotoxin and its derivatives has become clearer, and researchers have been able to synthesize some intermediates in this pathway using biocatalysis and methods combined with chemical methods. Based on this, the efficient and highly stereoselective synthesis of chiral (R)-3-piperylbutyrolactone has become an urgent problem to be solved. Currently, the poor stereoselectivity and low yield of (R)-3-piperylbutyrolactone in chemical synthesis cannot be avoided, and the chemical reaction conditions are relatively harsh. In response to the call for green living and green manufacturing, biosynthesis, with its advantages of good catalytic selectivity, mild reaction conditions, and environmentally friendly reaction systems, is gradually becoming a beneficial supplement to chemical synthesis.

[0004] Using chiral 3-piperylcyclobutanone as a substrate and Baeyer-Villiger monooxygenases (BVMOs) as catalysts for its asymmetric oxidation to obtain optically pure (R)-3-piperylbutyrolactone is the most efficient biosynthetic method. Currently reported biocatalysts for the synthesis of 3-piperylbutyrolactone include: *Acinetobacter calcoaceticus* NCIMB 9871, the fungus *Cunninghamella echinulata* NRRL3655, and CHMOs derived from *Rhodococcus*. RhodoCHMO derived from Brevibacterium sp. HCU1 Brevi1 and CHMO Brevi2 CHMO is derived from Xanthobacter sp.ZL5 and CHMO is derived from Polaromonas sp.strain JS666. JS666 However, the vast majority of catalytic products are S-configured, and only a few BVMOs can catalyze the formation of R-configured products. However, problems such as low stereoselectivity, insufficient stability, and low substrate loading still exist, making it difficult to meet the requirements of catalytic production. Summary of the Invention

[0005] Targeting cyclohexanone monooxygenase CHMO Brevi1 To address the problems of low stereoselectivity, insufficient stability, and low substrate loading in the catalytic synthesis of (R)-3-piperylbutyrolactone, this invention provides a cyclohexanone monooxygenase mutant and its application in the preparation of podophyllotoxin precursors.

[0006] Specifically, this invention utilizes protein engineering techniques to modify CHMO. Brevi1 Molecular modification was performed to provide a cyclohexanone monooxygenase mutant with significantly enhanced stereoselectivity and stability, a nucleic acid encoding the cyclohexanone monooxygenase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, and the application of the cyclohexanone monooxygenase mutant in the preparation of the anticancer drug podophyllotoxin precursor (R)-3-piperylbutyrolactone.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] One of the technical solutions is that the present invention provides a cyclohexanone monooxygenase mutant with significantly improved stereoselectivity and stability, which is a derived protein with a new amino acid sequence formed by replacing one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2, such as Leu at position 145, Phe at position 246, Asn at position 282, Met at position 385, and Leu at position 438, with other amino acid residues.

[0009] The derived protein exhibits enhanced stereoselectivity in catalyzing the synthesis of (R)-3-piperylbutyrolactone compared to the cyclohexanone monooxygenase corresponding to the amino acid sequence shown in SEQ ID No. 2.

[0010] The enhanced stereoselectivity mainly refers to the fact that the conversion rate of (R)-3-piperylbutyrolactone catalyzed by the cyclohexanone monooxygenase mutant is increased by more than 50%, preferably by more than 60%, and further preferably by more than 100% compared with the conversion rate of (R)-3-piperylbutyrolactone catalyzed by the cyclohexanone monooxygenase corresponding to the amino acid sequence shown in SEQ ID No. 2.

[0011] This invention uses the wild-type (WT) cyclohexanone monooxygenase with the amino acid sequence shown in SEQ ID No. 2 to construct a small and refined mutant library using the focused rational iterative site-specific mutation (FRISM) method, from which mutants with significantly improved stereoselectivity and stability of cyclohexanone monooxygenase are screened.

[0012] In one embodiment of the present invention, preferably, the amino acid sequence of the cyclohexanone monooxygenase mutant is one of the following:

[0013] (1) Replace Leu at position 145 of the amino acid sequence shown in SEQ ID No. 2 with Ala;

[0014] (2) Replace Phe at position 246 of the amino acid sequence shown in SEQ ID No. 2 with Leu;

[0015] (3) Replace Asn at position 282 of the amino acid sequence shown in SEQ ID No. 2 with Leu;

[0016] (4) Replace Met at position 385 of the amino acid sequence shown in SEQ ID No. 2 with Ala;

[0017] (5) Replace Leu at position 438 of the amino acid sequence shown in SEQ ID No.2 with Phe.

[0018] The second technical solution of the present invention provides a gene encoding the cyclohexanone monooxygenase mutant.

[0019] The gene is capable of expressing the cyclohexanone monooxygenase mutant as described in technical solution one, which is derived from cloning the gene sequences of the series of cyclohexanone monooxygenase mutants described in technical solution one through genetic engineering technology.

[0020] The third technical solution of the present invention provides a recombinant expression vector containing the gene described in the second technical solution of the present invention.

[0021] The recombinant expression vector can be constructed by linking the nucleotide sequence of the cyclohexanone monooxygenase gene of the present invention to various commercially available empty vectors using conventional methods in the art.

[0022] For example, the recombinant expression vector of the present invention can be prepared by the following method: using a plasmid carrying the cyclohexanone monooxygenase gene as a template, the whole plasmid is amplified by PCR using appropriate primers, and the PCR product is digested with Dpn I restriction enzyme to obtain a recombinant expression vector containing the nucleic acid molecule encoding the cyclohexanone monooxygenase.

[0023] The fourth technical solution of the present invention provides a recombinant expression transformant containing the gene (cyclohexanone monooxygenase mutant gene) described in the second technical solution of the present invention.

[0024] The recombinant expression transformant can be transformed into Escherichia coli host cells using conventional techniques in the art.

[0025] The fifth technical solution of the present invention provides a recombinant cyclohexanone monooxygenase mutant catalyst, wherein the recombinant cyclohexanone monooxygenase mutant catalyst is any one of the following forms:

[0026] (1) Cultivate the recombinant expression transformant and isolate the transformant cells containing the cyclohexanone monooxygenase mutant;

[0027] (2) Cultivate the recombinant expression transformant and isolate the crude enzyme solution containing the cyclohexanone monooxygenase mutant;

[0028] (3) Cultivate the recombinant expression transformant, separate the crude enzyme solution containing the cyclohexanone monooxygenase mutant, and freeze-dry the crude enzyme solution of the cyclohexanone monooxygenase mutant to obtain crude enzyme powder.

[0029] The culture methods and conditions for the recombinant expression transformants and the protein purification methods described herein are conventional methods and conditions in the art.

[0030] For recombinant *E. coli*, the preferred culture medium is TB medium: 5 g / L glycerol, 12 g / L peptone, 24 g / L yeast extract, and potassium phosphate buffer at a final concentration of 100 mM, pH 7.0. The preferred culture method is as follows: the recombinant *E. coli* is inoculated into TB medium containing kanamycin and cultured at 37°C. When the optical density OD of the culture medium... 600When the cytokinase concentration reaches 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1-0.5 mmol / L as an inducer, and continue culturing at 16-25℃ for 20-24 h. After culturing, centrifuge the culture medium at high speed, collect the precipitate, and wash twice with potassium phosphate buffer to obtain recombinant expression transformant cells. Resuspend the harvested recombinant cells in 5-10 volumes (v / w) of potassium phosphate buffer, sonicate to disrupt, centrifuge, and collect the supernatant to obtain the crude enzyme solution of the cyclohexanone monooxygenase mutant. Freeze the harvested crude enzyme solution at -80℃, and then freeze-dry it at low temperature using a vacuum freeze dryer to obtain crude enzyme powder.

[0031] The sixth technical solution of the present invention provides the application of the recombinant cyclohexanone monooxygenase mutant catalyst as described in technical solution one or the recombinant cyclohexanone monooxygenase mutant catalyst as described in technical solution five in the preparation of podophyllotoxin precursor (R)-3-piperylbutyrolactone. The recombinant cyclohexanone monooxygenase mutant catalyst as described in technical solution one or the recombinant cyclohexanone monooxygenase mutant catalyst as described in technical solution five, coupled with formate dehydrogenase, is used to catalyze the asymmetric oxidation of the 3-piperylcyclobutanone substrate to prepare podophyllotoxin precursor (R)-3-piperylbutyrolactone.

[0032] The seventh technical solution of the present invention provides a method for the enzymatic conversion preparation of (R)-3-piperylbutyrolactone, using 3-piperylcyclobutanone as a substrate, and in the presence of the coenzyme NADPH, using a recombinant cyclohexanone monooxygenase mutant as described in technical solution one or a recombinant cyclohexanone monooxygenase mutant as described in technical solution five as a catalyst to catalyze the asymmetric oxidation synthesis of (R)-3-piperylbutyrolactone, while NADPH is oxidized to generate NADP. + .

[0033] In some embodiments of the present invention, a formate dehydrogenase-catalyzed formate dehydrogenation reaction is coupled to convert NADP... + Enzymatic reduction and regeneration to NADPH.

[0034] In some embodiments of the present invention, the enzymatic reaction is carried out in a buffer solution at pH 6.0–9.0 at 15–40°C. The reaction system includes 3-piperylcyclobutanone at a final concentration of 0.5–50 mM, 5–20% (v / v) methanol as a co-solvent, 5–200 mM sodium formate, and 0.1–1 mM NADP+, 10–100 U / L of a recombinant cyclohexanone monooxygenase mutant as described in one of the technical solutions or a recombinant cyclohexanone monooxygenase mutant as described in technical solution five, and 10–200 U / L of formate dehydrogenase catalyst.

[0035] Preferably, the reaction is carried out under aeration and stirring, and the reaction time is determined by the complete conversion of the substrate or the product concentration no longer increasing.

[0036] During the reaction intervals, a 0.5 mL sample of the reaction solution was taken, and 0.5 mL of ethyl acetate was added for extraction. After centrifugation, the upper 0.3 mL extract was dried over anhydrous sodium sulfate to remove the ethyl acetate. Then, 0.3 mL of n-hexane was added to dissolve the extract. After filtration through a 0.22 μm pore size filter membrane, liquid chromatography analysis was performed to determine the substrate conversion rate and the ee value of the product. The specific analytical conditions are as follows:

[0037] The chromatographic column was a Daicel Chiralpak AD-H normal phase column, the mobile phase was n-hexane:isopropanol = 95:5, the flow rate was 1 ml / min, the column temperature was 40℃, and the detection wavelength was 254 nm.

[0038] Compared with the prior art, the technical effects of the present invention are mainly reflected in the following aspects:

[0039] The cyclohexanone monooxygenase mutant provided by this invention has the advantages of simple preparation, high stereoselectivity of catalytic products, good stability, and high substrate loading capacity, and has a promising application in the preparation of podophyllotoxin precursors for the anticancer drug. Detailed Implementation

[0040] The reaction or detection conditions described in this invention can be combined or modified based on common knowledge in the art, and can be verified experimentally. The technical solutions and effects of this invention will be clearly and completely described below with reference to specific embodiments. However, the scope of protection of this invention is not limited to these embodiments; all changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0041] The materials used in the following embodiments are sourced from:

[0042] The parent recombinant plasmid pET28a-CHMO contains the nucleic acid sequence shown in SEQ ID No. 1 of the sequence listing. It was constructed by the inventor and can be prepared by those skilled in the art using conventional techniques based on the provided nucleic acid sequence shown in SEQ ID No. 1 of the sequence listing.

[0043] The plasmid extraction kit, PCR purification kit, and nucleic acid gel recovery kit were all purchased from Beijing Adley Biochemical Co., Ltd.

[0044] Unless otherwise stated, the specific experiments in the following examples were performed according to conventional methods and conditions in the art, or in accordance with the product instructions for the kit.

[0045] Example 1: Construction of the CHMO mutant library

[0046] The three-dimensional spatial structure of the CHMO of the amino acid sequence shown in SEQ ID No. 2 was obtained through AlphaFold2 homology modeling, and docking with the substrate molecule was completed using AutoDock Tools. Near the substrate... The amino acid residues within the range include: aspartic acid at position 58, serine at position 59, tyrosine at position 64, leucine at position 145, phenylalanine at position 246, glycine at position 247, valine at position 248, phenylalanine at position 278, and asparagine at position 282. Site-directed mutagenesis was used to mutate these amino acid residues. Based on the open reading frame of the CHMO, the upstream and downstream primers were designed as follows:

[0047] The upstream primer is shown in SEQ ID NO.3:

[0048] ccgggtgtcc gtacggcttc tgagtttcac tac 33

[0049] Downstream primer, as shown in SEQ ID NO.4:

[0050] gtagtgaaac tcagaagccg tacggacacc cgg 33

[0051] Using the pET-28a(+) plasmid carrying the CHMO gene as a template, the whole plasmid was amplified by PCR using site-directed mutagenesis to construct a mutant library. PCR system (20 μL): 50 ng CHMO plasmid, 0.5 μL each of forward and reverse primers (10 μM), 10 μL 2×Prime Star DNA polymerase, 0.5 μL DMSO, and sterile water to a final volume of 20 μL. PCR reaction procedure: (1) 95℃ pre-denaturation for 3 min; (2) 98℃ denaturation for 10 s; (3) 55℃ annealing for 15 s; (4) 72℃ extension for 7.5 min; steps (2) to (4) were performed for a total of 20 cycles; the final extension was performed at 72℃ for 5 min, and the product was stored at 4℃. After obtaining the PCR product, it was incubated with Dpn I at 37℃ for 3-5 h to digest the template plasmid.

[0052] The digested PCR product was transformed into E. coli BL21(DE3) competent cells and evenly spread onto LB agar plates containing 50 μg / mL kanamycin. The plates were incubated overnight at 37°C. Single colonies were picked from the plates and transferred to test tubes containing 4 mL of LB medium containing 50 μg / mL kanamycin. The cultures were incubated at 37°C with a shaker at 200 rpm for 10–12 h until the culture became turbid. Plasmids were extracted using a kit and sent to Qingke Biotechnology Co., Ltd. for sequencing analysis.

[0053] After successful construction of the recombinant plasmid was verified by sequencing, 10 μL of glycerol bacterial culture was inoculated into a test tube containing 4 mL of LB medium with 50 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. Then, 1 mL of the bacterial culture from the test tube was inoculated into a shake flask containing 100 mL of the above medium and cultured at 37°C and 200 rpm until OD500 reached. 600 Once the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.2 mM, adjust the shaker temperature to 16℃, and continue culturing for 20-24 h to induce protein expression.

[0054] Example 2: Screening of the CHMO mutant library

[0055] After the constructed mutant strain completed protein expression, the cells were collected by centrifugation and subjected to a catalytic reaction in whole cells in 2 mL centrifuge tubes. The reaction system consisted of 0.5 mL of potassium phosphate buffer (100 mM, pH 7.0-9.0), 3-piperylcyclobutanone substrate at a concentration of 0.2-1.0 mmol / L, NADPH to substrate molar ratio of 1.0-2.0, and the cyclohexanone monooxygenase mutant catalyst at a concentration of 10-100 U / L. The reaction temperature was 20-40 °C, and the reaction was carried out with shaking in a high-speed shaker for 3 h. After the reaction was completed, 0.5 mL of ethyl acetate was added to the reaction system, and the mixture was shaken and extracted for 5 min. After centrifugation, 300 μL of the upper organic phase was transferred to another 2 mL centrifuge tube, an appropriate amount of anhydrous sodium sulfate was added to dry the cells, and the organic phase was volatilized in a fume hood. The substrate was redissolved in 300 μL of n-hexane, filtered through a 0.22 μm pore size membrane, and then analyzed by high-performance liquid chromatography (HPLC) to determine the conversion rate of the substrate and the ee value of the product.

[0056] Through screening, it was found that the stereoselectivity of the catalytic products of mutants with the following substitutions were significantly improved or reversed: leucine at position 145 was replaced with alanine (L145A), phenylalanine at position 246 was replaced with leucine (F246L), asparagine at position 282 was replaced with leucine (N282L), methionine at position 385 was replaced with alanine (M385A), and leucine at position 438 was replaced with phenylalanine (L438F). The sequences of these mutants, as well as the conversion rate and enantiomeric excess (ee) of the catalytic reaction, are listed in Table 1.

[0057] Table 1: Sequences and catalytic results of CHMO mutant cyclohexanone monooxygenase

[0058]

[0059] Example 3: Purification and Activity Assay of Cyclohexanone Monooxygenase

[0060] After inducing the expression of the target protein in *E. coli*, the bacterial culture was collected in 50 mL centrifuge tubes and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was discarded, and each gram of bacterial precipitate was resuspended in 20 mL of potassium phosphate buffer (100 mM, pH 7.0). The centrifuge tubes were then placed in a cup containing an ice-water mixture and disrupted using an ultrasonic cell disruptor at 400 W for 15 min, with a 1-second operation followed by a 1-second pause. After disruption, the supernatant was collected by centrifugation and purified using a nickel column.

[0061] The following are the formulations of the purification buffer: Solution A: 500mM NaCl, 2mM β-mercaptoethanol, 50mM KPB, pH 7.0; Solution B: 500mM NaCl, 300mM imidazole, 2mM β-mercaptoethanol, 50mM KPB, pH 7.0; Solution C: 150mM NaCl, 1mM dithiothreitol (DTT), 50mM KPB, pH 7.0.

[0062] The crude enzyme solution expressing cyclohexanone monooxygenase was added to a nickel column. First, contaminating proteins were eluted with solution A. Then, mixtures of solutions A and B with different imidazole concentrations were added sequentially for gradient elution. Samples from each gradient were collected and analyzed by SDS-PAGE to determine the appropriate elution concentration. The combined eluent of the target protein was collected and added to an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The solution was then concentrated by centrifugation at 3500 rpm at 4°C. Solution C was repeatedly used to repeatedly replace the eluent to thoroughly reduce the imidazole concentration. Finally, the purified enzyme solution was aliquoted into 1.5 mL centrifuge tubes, flash-frozen in liquid nitrogen, and stored at -80°C.

[0063] The purified enzyme obtained from nickel column chromatography was diluted to an appropriate concentration using potassium phosphate buffer (100 mM, pH 7.0). 10 μL of the enzyme solution was added to 1 mL of the activity assay system (930 μL potassium phosphate buffer, 0.2 mM NADPH, 0.2 mM 3-piperylcyclobutanone). After thorough mixing, the mixture was quickly placed in a spectrophotometer, and the change in NADPH absorbance was measured at 30 °C for 1 min. The calculated enzyme activity of the parent cyclohexanone monooxygenase was 270 mU / mg of purified enzyme, and the enzyme activity of M1 was 22 mU / mg of purified enzyme.

[0064] Example 4 pH effect on cyclohexanone monooxygenase CHMO M1 Effect of catalytic activity

[0065] The effect of pH on cyclohexanone monooxygenase (CHMO) was determined according to the method in Example 3 within the pH range of 6.0–9.0. M1Effect on catalytic activity. The buffer solutions were 100 mM potassium phosphate buffer (pH 6.0–8.0) and 100 mM Tris-HCl buffer (pH 8.0–9.0).

[0066] Add 0.2 mM NADPH and 0.2 mM 3-piperylcyclobutanone to 1 mL of the above buffer system, preheat to 30 °C, and then add an appropriate amount of cyclohexanone monooxygenase (CHMO). M1 Mix thoroughly and incubate at 30°C. Detect the change in NADPH absorbance at 340 nm within 1 minute using a spectrophotometer. Determine the concentration of cyclohexanone monooxygenase (CHMO) in buffer solutions of different pH values. M1 The differences in activity are shown in Table 2. The preferred pH range for the enzymatic reaction is 7.0–8.0, more preferably pH 7.0.

[0067] Table 2: pH effect on CHMO M1 Effect of catalytic activity on the asymmetric oxidation activity of 3-piperylcyclobutanone

[0068] pH Relative vitality (%) 6.0 44.3 6.5 79.4 7.0 100.0 7.5 96.6 8.0 90.3 8.5 21.1 9.0 3.0

[0069] Example 5 Temperature effect on cyclohexanone monooxygenase CHMO M1 Effect of catalytic activity

[0070] In a 1 mL potassium phosphate buffer (100 mM, pH 7.0) system, add 0.2 mM NADPH and 0.2 mM 3-piperylcyclobutanone, preheat at 20–50 °C for 2 min, and then add an appropriate amount of cyclohexanone monooxygenase (CHMO). M1 The mixture was thoroughly mixed and kept at the same temperature as the preheating temperature for the reaction. The change in NADPH absorbance at 340 nm within 1 minute was measured using a spectrophotometer. The activity of cyclohexanone monooxygenase (CHMO) was determined under different temperature conditions. M1 The differences in activity were shown in Table 3. The preferred temperature range for the enzymatic reaction is 30–40 °C.

[0071] Table 3: Temperature effect on CHMO M1 Effect of catalytic activity on the asymmetric oxidation activity of 3-piperylcyclobutanone

[0072] temperature Relative vitality (%) 20 74.6 25 77.8 30 81.0 35 100.0 40 92.2 45 75.9 50 20.3

[0073] Example 6: Preparation of lyophilized cyclohexanone monooxygenase powder

[0074] Cyclohexanone monooxygenase-expressing *Escherichia coli* glycerol culture was streaked onto kanamycin-resistant plates. Single colonies were picked and inoculated into test tubes containing 4 mL of 50 μg / mL kanamycin LB medium and incubated overnight at 37°C with a shaker at 200 rpm. At a 1% inoculation ratio, the culture was transferred to shake flasks containing 600 mL of TB liquid medium and incubated at 37°C with a shaker at 200 rpm until OD500 reached. 600 Once the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.2 mM, adjust the shaker temperature to 16℃, and continue culturing for 20-24 h to induce protein expression.

[0075] Pour the bacterial culture into a centrifuge cup and centrifuge at 4℃ and 6000 rpm for 10 min. Discard the supernatant. Resuspend each gram of bacterial precipitate in 10 mL of potassium phosphate buffer (100 mM, pH 7.0) and centrifuge for 10 min. Repeat this step once. After resuspending, filter the resuspended cells through a 120-mesh sieve to remove solid particles. Set the pressure of the high-pressure homogenizer to 800 bar and homogenize the cells twice. Centrifuge the homogenate at 4℃ and 6000 rpm for 30 min to remove cell debris. Pour the supernatant crude enzyme solution into a clean iron tray and freeze it at -80℃ for one day. After removal, place it in a freeze dryer for low-temperature vacuum drying for about 2-3 days. Remove the dried enzyme powder and store it at 4℃.

[0076] Example 7: Preparation of Formate Dehydrogenase Lyophilized Enzyme Powder

[0077] Formate dehydrogenase-expressing *Escherichia coli* glycerol culture was streaked onto ampicillin-resistant plates. Single colonies were picked and inoculated into test tubes containing 4 mL of 50 μg / mL ampicillin in LB medium and incubated overnight at 37°C with a shaker at 200 rpm. At a 1% inoculation ratio, the culture was transferred to shake flasks containing 600 mL of TB liquid medium and incubated at 37°C with a shaker at 200 rpm until OD... 600 Once the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.2 mM, adjust the shaker temperature to 16℃, and continue culturing for 20-24 h to induce protein expression.

[0078] The subsequent freeze-drying process is the same as in Example 4.

[0079] Example 8 CHMO M1 Whole cell or lyophilized enzyme powder catalyzes the asymmetric oxidation of 30mM 3-piperylcyclobutanone

[0080] At a reaction scale of 100 mL, 20 U / L CHMO was added. M1 Whole cells or lyophilized enzyme powder, 40 U / L formate dehydrogenase lyophilized enzyme powder, 0.5 mM NADP +The reaction mixture consisted of 50 mM sodium formate and 30 mM 3-piperylcyclobutanone substrate. Oxygen was continuously introduced into the reaction system using an oxygen bulb. The reaction was carried out at 25 °C with magnetic stirring. The conversion rate was monitored intermittently. After 24 h of reaction, the conversion rate was found to be greater than 99%, and the ee value of the product was 96.8%. To avoid emulsification during extraction, the reaction solution was centrifuged to precipitate cells and proteins. The supernatant was collected, ethyl acetate was added, and the mixture was thoroughly shaken and mixed through a separatory funnel. After standing, the upper organic phase was collected, and this step was repeated three times to collect as much product as possible. The extracts were combined and collected, and dried with anhydrous sodium sulfate. Solid particles were removed by filtration, and the organic phase was concentrated by rotary evaporation to obtain the desired crude product.

[0081] The crude product was added to the upper layer of a silica gel column and separated by column chromatography using a mobile phase (petroleum ether: ethyl acetate = 10:1). The eluents were sequentially collected into test tubes, and the product elution range was determined by thin-plate chromatography. The eluents were combined and collected, and then concentrated by rotary evaporation to obtain 0.48 g of the desired pure product, with a separation yield of 72.7%.

[0082] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

[0083] The sequences involved in this invention are as follows:

[0084] The nucleotide sequence of the gene encoding cyclohexanone monooxygenase (CHMO) (SEQ ID NO.1)

[0085]

[0086]

[0087] Amino acid sequence of cyclohexanone monooxygenase (CHMO) (SEQ ID NO.2)

[0088]

[0089]

[0090] Upstream primer (SEQ ID NO.3)

[0091] ccggggtgtcc gtacggcttc tgagtttcac tac 33(SEQ ID NO.4);

[0092] gtagtgaaac tcagaagccg tacggacacc cgg

Claims

1. A cyclohexanone monooxygenase mutant, characterized in that, Its amino acid sequence is obtained by replacing Leu at position 145 of the amino acid sequence shown in SEQ ID No. 2 with Ala.

2. The gene encoding the cyclohexanone monooxygenase mutant of claim 1.

3. A recombinant expression vector containing the gene of claim 2.

4. A recombinant expression transformant containing the recombinant expression vector of claim 3.

5. A recombinant cyclohexanone monooxygenase mutant catalyst, characterized in that, The recombinant cyclohexanone monooxygenase mutant catalyst is any one of the following forms: (1) Cultivate the recombinant expression transformant of claim 4 and isolate the transformant cells containing the cyclohexanone monooxygenase mutant of claim 1; (2) Cultivate the recombinant expression transformant of claim 4 and separate the crude enzyme solution containing the cyclohexanone monooxygenase mutant of claim 1; (3) Cultivate the recombinant expression transformant according to claim 4, separate the crude enzyme solution containing the cyclohexanone monooxygenase mutant according to claim 1, and freeze-dry the crude enzyme solution of the cyclohexanone monooxygenase mutant to obtain crude enzyme powder.

6. The recombinant cyclohexanone monooxygenase mutant catalyst of claim 1 or the recombinant cyclohexanone monooxygenase mutant catalyst of claim 5 in the preparation of podophyllotoxin precursor ( R Application of 3-piperylbutyrolactone.

7. An enzymatic conversion method for preparing ( R The method for producing 3-piperylbutyrolactone is characterized by, Using 3-piperylcyclobutanone as a substrate, in the presence of the coenzyme NADPH, asymmetric oxidation synthesis was catalyzed by the recombinant cyclohexanone monooxygenase mutant of claim 1 or the recombinant cyclohexanone monooxygenase mutant of claim 5. R )-3-piperylbutyrolactone, while NADPH is oxidized to NADP. + ; The formate dehydrogenation reaction catalyzed by formate dehydrogenase is coupled to convert NADP... + Enzymatic reduction and regeneration to NADPH.

8. The method according to claim 7, characterized in that, The reaction conditions were as follows: the concentration of the substrate 3-piperylcyclobutanone was 0.5–50 mmol / L, the concentration of the cosolvent was 5–20% (v / v), the concentration of the cosubstrate sodium formate was 5–200 mmol / L, and NADP... + The addition amount is 0.1~1 mmol / L, pH 7.0~8.0, and temperature is 30~40 °C.

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