Monooxygenases and their use in the synthesis of (s)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone

Abstract

The present application relates to monooxygenase and its application in synthesis of (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone. Specifically, the present application provides monooxygenase derived from Bradyrhizobium sp. 200, mutants thereof, genes thereof, recombinant expression vectors containing the genes and recombinant expression transformants, and application of the monooxygenase or mutants thereof or recombinant expression transformants as catalysts to catalyze asymmetric hydroxylation of 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid methyl ester, and then prepare indoxacarb active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydr-oxyl-1-indanone. Based on the scheme of the present application, the process for preparing indoxacarb active intermediate (S)-5-chloro-2-methoxycar-bonyl-2-hydroxy-1-indanone is simple, the reaction conditions are mild, the conversion rate is high, and the optical purity and enantioselectivity of the product are significantly improved. The green synthesis path based on enzyme catalysis significantly reduces the environmental burden, and has superior industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a monooxygenase derived from Bradyrhizobium sp. 200, its mutants, its gene, a recombinant expression vector containing the gene, and a recombinant expression transformant, as well as the application of using the monooxygenase or its mutants or recombinant expression transformants as catalysts to catalyze the asymmetric hydroxylation reaction of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid, thereby preparing the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene of indoxacarb. Background Technology

[0002] Indoxacarb is a novel oxadiazine insecticide developed by DuPont, and a representative compound among sodium ion channel inhibitors. It exhibits broad-spectrum and highly effective insecticidal activity, particularly against lepidopteran pests. Indoxacarb's key advantages include: high insecticidal activity, excellent environmental compatibility, extremely low toxicity to mammals, and safety for birds, fish, and beneficial organisms, making it an ideal alternative to highly toxic organophosphate insecticides. It is important to emphasize that the insecticidal activity of indoxacarb is provided solely by its (S)-isomer, while the (R)-isomer lacks biological activity. Therefore, preparing indoxacarb via the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indoxaone is a understood method for obtaining indoxacarb.

[0003] Chinese patents CN102924278B and CN115896188B both disclose methods for chemically synthesizing the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indanone of indoxacarb. However, chemical synthesis methods require the use of organic reagents, have relatively harsh reaction conditions, and also suffer from problems such as low product selectivity and insufficient stereoselectivity.

[0004] Enzymes, as highly efficient biocatalysts, can significantly accelerate biological and chemical reactions in industrial production. Their catalytic processes have significant advantages such as mild reaction conditions, low pollution, high safety, and low energy consumption, thus showing broad application prospects in the preparation of indoxacarb active intermediates.

[0005] Therefore, developing efficient bio-enzymes to catalyze the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid is of great practical significance and application value for improving the greening level of industrial production and the stereoselectivity of the reaction. Summary of the Invention

[0006] The purpose of this invention is to develop suitable enzymes for the preparation of indoxacarb active intermediates. Based on this, this invention provides a monooxygenase derived from Bradyrhizobium sp. 200, its mutant, its gene, a recombinant expression vector containing the gene, and a recombinant expression transformant, as well as the application of using the monooxygenase mutant or recombinant expression transformant as a catalyst to catalyze the asymmetric hydroxylation reaction of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid, thereby preparing the indoxacarb active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene.

[0007] This invention, through gene mining, discovered a highly active and selective monooxygenase derived from *Bradyrhizobium* sp. 200. Furthermore, this invention provides a recombinant expression vector and recombinant expression transformant containing the gene of this monooxygenase or its mutant form, and the application of using this monooxygenase mutant or recombinant expression transformant as a catalyst to catalyze the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid, thereby preparing the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indeneone for indoxacarb.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] One of the technical solutions of the present invention is to provide a monooxygenase, which is a protein of the following (a) or (b):

[0010] Protein (a): A protein consisting of the amino acid sequence shown in SEQ ID No. 2;

[0011] Protein (b): A protein derived from (a) with higher catalytic activity than (a) by substitution, deletion or addition of several amino acids in the amino acid sequence shown in SEQ ID No. 2 and capable of catalyzing the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid to prepare the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene.

[0012] Furthermore, the monooxygenase described in this invention is derived from a rhizobium (Bradyrhizobium sp.200), and its sequence information is obtained from GenBank WP_247832616.

[0013] This invention also provides a method for obtaining the monooxygenase:

[0014] Through large-scale sequence alignment and screening of microorganisms in nature and those preserved in the laboratory, a monooxygenase sequence was discovered in *Bradyrhizobium* sp. 200. The monooxygenase catalyzing the corresponding reaction in *Bradyrhizobium* sp. 200 was synthesized and named monooxygenase BjHAPMO. The amino acid sequence of this monooxygenase is shown in SEQ ID No. 2.

[0015] The gene synthesis mentioned in this invention can be achieved using conventional biotechnology methods in the field.

[0016] The monooxygenase described in this invention has the following properties: it can catalyze the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid to prepare the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene, which is used as an indoxacarb.

[0017] The second technical solution of the present invention is to provide an isolated nucleic acid, wherein the nucleic acid encodes the monooxygenase.

[0018] In one embodiment of the present invention, a monooxygenase gene BjHAPMO is provided, the nucleotide sequence of which is shown in SEQ ID No. 1, and is 1965 nucleotides in length. Its coding sequence (CDS) extends from the first base to the 1965th base, with a start codon of ATG and a stop codon of TAA, and contains no introns. The amino acid sequence of the protein encoded by this gene is shown in SEQ ID No. 2.

[0019] The present invention also provides sources of the encoding DNA of the monooxygenase BjHAPMO, including: obtaining the encoding DNA of the monooxygenase BjHAPMO through gene cloning technology, or obtaining the encoding DNA of the monooxygenase BjHAPMO through artificial full-sequence synthesis.

[0020] In one embodiment of the present invention, the monooxygenase BjHAPMO gene of the present invention is derived from rhizobia. The specific method for preparing its encoding DNA includes: using the genomic DNA of rhizobia as a template, synthesizing the complete DNA sequence of the monooxygenase BjHAPMO, and obtaining the complete DNA sequence encoding the monooxygenase BjHAPMO using conventional techniques in the art (such as polymerase chain reaction, PCR).

[0021] The preferred synthetic primers involved are shown in SEQ ID No. 3 (upstream primer) and SEQ ID No. 4 (downstream primer):

[0022] Upstream primer: 5'-GGGAATTC CATATG ACCGTTCAAGAACTCA-3', the underlined sequence is the restriction site of restriction endonuclease Nde I;

[0023] Downstream primer: 5'-CCG CTCGAG ACGTTCGGTCGCGGTAGAAAC-3', the underlined sequence is the restriction enzyme site of Xho I.

[0024] The third technical solution of the present invention is to provide a recombinant expression vector containing the nucleic acid of the monooxygenase gene.

[0025] The recombinant expression vector can be constructed by cloning the monooxygenase (preferably BjHAPMO) gene into various expression vectors using conventional methods in the art.

[0026] The expression vectors preferably include various plasmid vectors conventional in the art, with pET28a plasmid being the most preferred.

[0027] Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the DNA fragment of the monooxygenase BjHAPMO gene sequence obtained by PCR amplification is digested with restriction endonucleases Nde I and Xho I, and the empty vector plasmid pET28a is digested with restriction endonucleases Nde I and Xho I. The digested monooxygenase BjHAPMO gene DNA fragment and pET28a plasmid are recovered and ligated using T4 DNA ligase to construct the recombinant expression vector pET28a-BjHAPMO containing the monooxygenase BjHAPMO gene.

[0028] The fourth technical solution of the present invention is to provide a recombinant expression transformant containing the recombinant expression vector.

[0029] The recombinant expression transformant can be prepared by transforming the above-mentioned recombinant expression vector into host cells.

[0030] In some embodiments of the present invention, the host cell is a conventional host cell in the art, as long as the recombinant expression vector can stably replicate itself and the gene for the monooxygenase BjHAPMO carried by it can be effectively expressed.

[0031] In some embodiments of the present invention, the host cell is preferably Escherichia coli, more preferably Escherichia coli BL21(DE3) or Escherichia coli DH5α.

[0032] The preferred genetically engineered strain of the present invention can be obtained by transforming the recombinant expression vector into Escherichia coli BL21(DE3). For example, transforming the recombinant expression vector pET28a-BjHAPMO into Escherichia coli BL21(DE3) yields recombinant E. coli BL21(DE3) / pET28a-BjHAPMO.

[0033] Fifth technical solution of the present invention: providing a monooxygenase catalyst, selected from any of the following forms:

[0034] (1) Culturing the recombinant expression transformant and isolating the transformant cells containing the monooxygenase;

[0035] (2) Cultivate the recombinant expression transformant, isolate the transformant cells containing the monooxygenase, and break the transformant cells containing the monooxygenase to obtain the cell lysate;

[0036] (3) Cultivate the recombinant expression transformant, isolate the transformant cells containing the monooxygenase, break the transformant cells containing the monooxygenase, obtain the cell lysate, and freeze-dry the cell lysate of the monooxygenase to obtain lyophilized enzyme powder.

[0037] (4) The monooxygenase BjHAPMO or a mutant of monooxygenase BjHAPMO, wherein the mutant of monooxygenase BjHAPMO refers to a protein whose amino acid sequence shown in SEQ ID No. 2 has been substituted, deleted or added with several amino acids and can catalyze the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid to prepare the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene, while having improved catalytic activity.

[0038] The present invention also provides a method for preparing the monooxygenase catalyst.

[0039] The preferred method for preparing the recombinant monooxygenase BjHAPMO of the present invention is as follows: culturing the recombinant expression transformant as described above, and isolating the recombinant expressed monooxygenase BjHAPMO. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art that can enable the transformant to grow and produce the recombinant monooxygenase of the present invention. The culture medium is preferably LB medium, with the following formula: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0. There are no special limitations on the culture method and conditions; appropriate selection can be made according to the host cell type and culture method, based on conventional knowledge in the art, as long as the transformant can grow and produce the monooxygenase BjHAPMO. The specific operation of culturing the recombinant expression transformant can be performed according to conventional operations in the art. Preferably, the recombinant Escherichia coli of the present invention, such as E. coli BL21(DE3) / pET28a-BjHAPMO, is inoculated into LB medium containing kanamycin and cultured at 37°C. When the optical density OD of the culture medium reaches a certain value... 600 When the concentration reaches 0.5–1.0 (preferably 0.6), isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1–1.0 mmol / L (preferably 0.5 mmol / L) to induce enzyme production. After culturing at 16°C for 24 h, the monooxygenase BjHAPMO described in this invention can be efficiently expressed. After culturing, the precipitated bacterial cells are collected by centrifugation; these are the resting cells of the recombinant expression transformant. The harvested cells are suspended in PBS buffer (100 mM, pH 6.0), sonicated, and the lysate is centrifuged. The supernatant is collected to obtain the crude enzyme solution of the recombinant monooxygenase BjHAPMO. The centrifuged cell pellet is freeze-dried to obtain frozen stem cells, which are beneficial for long-term storage and convenient for future use.

[0040] Assay for the activity of monooxygenase BjHAPMO: A 1 ml reaction system (50 mmol / L sodium phosphate buffer, pH 8.0) containing 2 mmol / L p-methylacetophenone and 0.2 mmol / L NADPH was preheated to 30 °C. Then, an appropriate amount of crude monooxygenase BjHAPMO was added, mixed thoroughly, and incubated at 30 °C. The absorbance change of NADPH at 340 nm was detected on a spectrophotometer, and the absorbance change value was recorded over a certain period of time.

[0041] Enzyme activity can be calculated using the following formula:

[0042] Enzyme activity (U) = EW × V × 10 3 / (6220×l)

[0043] In the formula, EW is the change in absorbance at 340 nm within 1 minute; V is the volume of the reaction solution in ml; 6220 is the molar extinction coefficient of NADPH in L / (mol·cm); and l is the optical path distance in cm. One unit of enzyme activity (U) corresponds to the amount of enzyme required to oxidize 1 μmol of NADPH per minute under the above conditions.

[0044] The sixth technical solution of the present invention: providing the application of the monooxygenase in the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid.

[0045] The chemical structure of the methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid is shown below:

[0046]

[0047] Furthermore, the monooxygenase catalyst is provided to catalyze the asymmetric hydroxylation of 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid, thereby preparing the active intermediate of indoxacarb (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene.

[0048] In one embodiment of the present invention, the asymmetric hydroxylation of 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid can be carried out according to the following exemplary method: in a phosphate buffer at pH 7-8, with glucose dehydrogenase, glucose, and NADP... + In the presence of the monooxygenase catalyst, the asymmetric hydroxylation reaction of 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid is catalyzed to obtain the active intermediate of indoxacarb (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene.

[0049] In this application, the concentration of the substrate in the reaction solution can be 0.1–20 mmol / L. Depending on the reaction system used, the amount of the monooxygenase can be 1–200 U / L. During the enzymatic asymmetric hydroxylation of 5-chloro-1-oxo-2,3-dihydro-1H-inden-2-carboxylic acid, the coenzyme NADPH is oxidized to NADP. + To facilitate the cyclic regeneration of the coenzyme NADPH, glucose and glucose dehydrogenase from Bacillus megaterium were added to the reaction system (JInd Microb Biotechnol, 2011, 38:633-641). Depending on the reaction system, the activity units of glucose dehydrogenase could be equal to those of the monooxygenase. The molar ratio of glucose to substrate could be 1.0–1.5, and the added NADP... +The dosage can be 0–1.0 mmol / L. The buffer solution is a sodium phosphate buffer, preferably with a pH range of 6.5–8.5, more preferably pH 8.0. The concentration of the phosphate buffer solution can be 0.05–0.2 mol / L. The temperature of the enzymatic asymmetric reduction reaction can be 25–40 °C, preferably 30 °C. During the reaction, the reaction conversion rate is measured intermittently, and the reaction time is based on the time when the substrate is completely converted or the reaction conversion rate stops increasing, generally 1–24 hours.

[0050] The reaction conversion rate was analyzed by liquid chromatography. Preferably, a ZORBAX RR EclipseXDB-C18 column (4.6 mm × 250 mm × 5 μm) was used for conversion analysis, with a constant column temperature of 30 °C and a detection wavelength of 210 nm. The mobile phase consisted of 0.1% trifluoroacetic acid (A) and 0.1% trifluoroacetic acid (acetonitrile) (B); gradient elution was used: 10% B (0.01 min), 100% B (12.5 min), 100% B (14 min), 10% B (15 min), and stop (20 min); the flow rate was 0.8 ml / min.

[0051] The reaction products can be analyzed by liquid chromatography. Preferably, an enantiomeric excess (ee) analysis is performed using a Chiralpak AD-H column (25cm × 4.6mm × 5μm) at a detection wavelength of 210nm. The mobile phase is hexane / ethanol = 90:10, and the flow rate is 0.8ml / min.

[0052] Compared with the prior art, the positive and progressive effects of the present invention are as follows:

[0053] The monooxygenase BjHAPMO provided by this invention can stereoselectively catalyze the asymmetric hydroxylation of 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid to prepare the active intermediate (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene. The reaction conditions are mild, the conversion rate is high, the product has good optical purity, and the ee value can be higher than 90%, which has good prospects for industrial application. Detailed Implementation

[0054] The reaction or detection conditions described in the invention can be combined or modified based on common knowledge in the art, and can be verified experimentally. The invention is further illustrated below by way of examples. It should be understood that although the listed examples illustrate preferred embodiments of the invention, the specific examples are given only to better explain the invention and are not intended to limit the invention to the scope of the described examples.

[0055] The materials used in the following embodiments are sourced from:

[0056] The rhizobium (Bradyrhizobium sp.200), the sequence information of which is from GenBank WP_247832616.

[0057] The expression plasmid pET28a was purchased from Novagen.

[0058] E. coli DH5α and E. coli BL21(DE3) competent cells, 2×Taq PCR MasterMix, and agarose gel DNA recovery kit were all purchased from Beijing Tiangen Biotech Co., Ltd.

[0059] The restriction endonucleases Nde I and Xho I are both commercially available products from New England Biolabs (NEB).

[0060] 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.

[0061] Example 1: Gene Cloning of Monooxygenase BjHAPMO

[0062] Using the genomic DNA of rhizobium (Bradyrhizobium sp. 200) as a template, the monooxygenase sequence was determined by sequence alignment and codon optimization was performed to synthesize the complete DNA sequence of the monooxygenase BjHAPMO. The complete DNA sequence encoding the monooxygenase BjHAPMO was obtained using conventional techniques in the field (such as polymerase chain reaction, PCR).

[0063] The preferred synthetic primers involved are shown in SEQ ID No. 3 (upstream primer) and SEQ ID No. 4 (downstream primer):

[0064] Upstream primer: 5'-GGGAATTC CATATG ACCGTTCAAGAACTCA-3'

[0065] Downstream primer: 5'-CCG CTCGAG ACGTTCGGTCGCGGTAGAAAC-3'

[0066] The underlined portion of the upstream primer represents the restriction enzyme Nde I cleavage site, while the underlined portion of the downstream primer represents the restriction enzyme Xho I cleavage site.

[0067] The PCR system consisted of 25 μl of 2×Taq PCR Master Mix, 2.5 μl each of upstream and downstream primers (10 ng / μl), 1 μl of genomic DNA of monooxygenase BjHAPMO (100 ng / μl), and 19 μl of ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 minutes, followed by 32 cycles of the following: 94℃ denaturation for 30 seconds, 50℃ annealing for 30 seconds, and 72℃ extension for 5 minutes; after each cycle, a final extension at 72℃ for 10 minutes was performed. The PCR amplification product was purified by gel electrophoresis, and the target fragment was recovered using a DNA recovery kit. DNA sequencing revealed that the open reading frame encoded by this sequence was 1965 bp in length, and its base sequence is shown in SEQ ID No. 1.

[0068] Example 2: Preparation of monooxygenase BjHAPMO expression plasmid and recombinant expression transformant

[0069] The target DNA fragment obtained by PCR amplification in Example 1 and the empty pET28a plasmid were simultaneously digested overnight with restriction endonucleases Nde I and Xho I, then purified by agarose gel electrophoresis and recovered using a DNA kit. The recovered digested target fragment and the empty vector were ligated at 4°C for 12 hours using T4 DNA ligase to obtain the recombinant plasmid pET28a-BjHAPMO.

[0070] The obtained recombinant plasmid was transformed into E. coli DH5α, plated on LB agar plates containing 50 μg / ml kanamycin, and incubated at 37°C for 8 hours. Colony PCR was performed to verify the growth, and positive clones that successfully amplified the target band were selected. After sequencing verification, the corresponding plasmid was extracted and further transformed into E. coli BL21(DE3). Positive clones were selected to obtain the recombinant expression transformant E. coli BL21(DE3) / pET28a-BjHAPMO.

[0071] Example 3: Induced expression of monooxygenase BjHAPMO

[0072] The recombinant expression transformant E. coli BL21(DE3) / pET28a-BjHAPMO obtained in Example 2 was inoculated into LB medium containing 50 μg / ml kanamycin and cultured on a shaker at 37°C for 12 hours. Then, at a 1% (v / v) inoculation rate, it was inoculated into 500ml Erlenmeyer flasks containing 100ml of LB medium (containing 50 μg / ml kanamycin) and cultured on a shaker at 37°C and 180 rpm. When the OD of the culture medium... 600When the concentration reached 0.6, IPTG was added to a final concentration of 0.2 mmol / L for induction. After induction at 16°C for 24 hours, the culture medium was centrifuged at 12,000 rpm, the cell pellet was collected, and the cells were washed with physiological saline to obtain resting cells. The cells were then freeze-dried to obtain frozen stem cells.

[0073] Five g of resting cells obtained as described above were suspended in 100 ml of sodium phosphate buffer (100 mM, pH 6.0), and the cells were sonicated in an ice-water bath. The supernatant was collected by centrifugation, which was the crude enzyme solution of recombinant monooxygenase BjHAPMO. Analysis of the crude enzyme solution by polyacrylamide gel electrophoresis showed that recombinant monooxygenase BjHAPMO existed in a soluble form. The obtained crude enzyme solution of recombinant monooxygenase BjHAPMO was freeze-dried to obtain crude enzyme powder of recombinant monooxygenase BjHAPMO.

[0074] Example 4: Effect of pH on the catalytic activity of monooxygenase BjHAPMO

[0075] The effect of pH on the activity of recombinant monooxygenase BjHAPMO was determined using standard methods within the pH range of 6–9. The buffer solutions were citrate-sodium citrate buffer (5.0–6.0), sodium phosphate buffer (6.0–8.0), Tris-HCl buffer (7.0–9.0), and glycine-NaOH buffer (8.0–11).

[0076] In 1 ml of the above buffer system, p-nitroacetophenone and NADPH were added to a final concentration of 2 mmol / L and 0.2 mmol / L, respectively. The mixture was preheated to 30°C, and then an appropriate amount of monooxygenase was added. After mixing thoroughly, the mixture was incubated at 30°C. The absorbance change of NADPH at 340 nm was detected using a spectrophotometer. The activity differences of monooxygenase BjHAPMO in buffer solutions with different pH values ​​were measured, and the results are shown in Table 1. The preferred pH range for the enzymatic reaction is 7.5–8.5, and more preferably pH 8.0.

[0077] Table 1 Effect of pH on the asymmetric catalytic hydroxylation activity of BjHAPMO

[0078]

[0079]

[0080] Example 5: Effect of temperature on the catalytic activity of monooxygenase BjHAPMO

[0081] In a 1 ml sodium phosphate buffer (50 mM, pH 8.0) system, p-nitroacetophenone and NADPH were added to final concentrations of 2 mmol / L and 0.2 mmol / L, respectively. The mixture was preheated at 25–45 °C for 2 min, then an appropriate amount of monooxygenase was added, mixed thoroughly, and incubated at the same temperature as the preheating. The absorbance change of NADPH at 340 nm was detected using a spectrophotometer. The activity differences of monooxygenase BjHAPMO under different temperature conditions were measured, and the results are shown in Table 2. The preferred temperature range for the enzymatic reaction is 30–40 °C.

[0082] Table 2 Effect of temperature on the asymmetric catalytic hydroxylation activity of BjHAPMO

[0083]

[0084]

[0085] Example 6: Recombinant monooxygenase BjHAPMO-catalyzed asymmetric hydroxylation of 5-chloro-1-oxo-2,3-dihydro-1H-inden-2-carboxylic acid to synthesize (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indenone

[0086] 5 g of crude BjHAPMO enzyme powder as described in Example 3 and 3 U of glucose dehydrogenase lyophilized enzyme powder were added to 1 ml of sodium phosphate buffer (50 mmol / L, pH 8.0). Then, 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid, glucose, and NADP+ were added to final concentrations of 2 mmol / L, 3 mmol / L, and 0.2 mmol / L, respectively. The reaction was carried out at 30°C and 1000 rpm using a shaker. After 12 hours, the substrate conversion rate was >99% as determined by liquid chromatography.

[0087] After the reaction was completed, the product was extracted with 2 times its volume of ethyl acetate. The extracted organic phase was then air-dried in a fume hood. After the sample was dried, the ee value of the product was determined by liquid chromatography to be higher than 90%.

[0088] The sequence information involved in this invention is as follows:

[0089] Nucleotide sequence of the monooxygenase gene BjHAPMO (SEQ ID No. 1)

[0090]

[0091] Amino acid sequence of monooxygenase BjHAPMO of SEQ ID No. 2

[0092] MTVQELKTLQASGPQRGTSHGAELQHHIQCGADLPTLLMTTAHTTGDLSVLRNGWRPVDVLGVAQCNVSDEEKALIREECYRRLADHSKRGGQPPVRPTYDLLRGIGEWFLGSSIEPLIPLLAEELIFDGHDLRQPQWNKETIAPDRPFHVAIIGAGESGIIAAVRFKQAGIPFTIYEKNGDVGGTWLENHYPGCRVDINSFVYSYASAPRVWHDYFGLRNETLSYLQKVARDNGLYEHTKFGAEIAEAVWSDTEQVWRLTINSAGKTETVSPNMIVFAVGQLNRPKLPEIAGIDRFKGESFHSAQWNHNVTFEGKRIGVIGTGASACQFIPQIANVAAKVTVFARTATWLLPTPNLHERVEGSERWLFENLPGYAQWYRGSLLMLQTPGILEYVIVDPNYAASEQAVSESNNFVRQELQQWIEAQIAERPDLRDALIPNSPVGSKRILRDNGTWAKTLKRDNVAVVREKISEIITDGIRCADGNSHEFDVIVYGTGFHASKFLFPIKVRGANGCSLQDAWKDGARAYLGMTIPQFPNMFCMYGPNTNLVVHGASIVMFSELTAKYIVDAVRVMLEKGAATMDVREEVFSGYDRRVDEANRARAWGYSKVNSWYKDANGRVGQNYPFTATEFYQRTNAVVAADYRFGPVSTATER

[0093] Forward primer of SEQ ID No. 3

[0094] gggaattc catatg accgttcaagaactca 30

[0095] Reverse primer of SEQ ID No. 4

[0096] ccg ctcgag acgttcggtcgcggtagaaac 30

[0097] 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.

Claims

1. The application of a monooxygenase catalyst in the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-inden-2-carboxylic acid to prepare optically active (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indenone, characterized in that, The monooxygenase is a protein composed of the amino acid sequence shown in SEQ ID No.

2.

2. The application according to claim 1, characterized in that, The monooxygenase catalyst is selected from any of the following forms: (1) Culture the recombinant expression transformant and isolate the transformant cells containing monooxygenase; (2) Culture the recombinant expression transformant, isolate the transformant cells containing monooxygenase, and break the transformant cells containing the monooxygenase to obtain the cell lysate; (3) Culture the recombinant expression transformant, isolate the transformant cells containing monooxygenase, break the transformant cells containing the monooxygenase, obtain the cell lysate, and freeze-dry the cell lysate of the monooxygenase to obtain lyophilized enzyme powder. The recombinant expression transformant comprises a recombinant expression vector containing nucleic acid encoding a monooxygenase.

3. The application according to claim 1, characterized in that, In glucose dehydrogenase, glucose and NADP + In the presence of the monooxygenase catalyst, the asymmetric hydroxylation of methyl 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid ester was catalyzed to prepare optically active (S)-5-chloro-2-methoxycarbonyl-2-hydroxy-1-indene.

4. The application according to claim 3, characterized in that, The concentration of the substrate in the reaction solution is 0.1–20 mmol / L, the amount of monooxygenase in the monooxygenase catalyst is 0.1–200 U / L, the molar ratio of glucose to substrate is 1.0–1.5, and additional NADP is added. + The dosage is 0.2–1.0 mmol / L; the pH range of the reaction system is 6.0–10.0; and the temperature of the asymmetric hydroxylation reaction is 25–40°C.

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