CN120505287A

CN120505287A - Application of monooxygenase and mutant thereof in preparation of indoxacarb chiral intermediate

Info

Publication number: CN120505287A
Application number: CN202510496135.XA
Authority: CN
Inventors: 罗志波; 吴彦; 卢夏锋; 杜峰; 黄培斌
Original assignee: Hangzhou Weiyuan Biotechnology Co ltd
Current assignee: Hangzhou Weiyuan Biotechnology Co ltd
Priority date: 2025-04-21
Filing date: 2025-04-21
Publication date: 2025-08-19

Abstract

本发明公开了多种BVMO或其突变体，这些BVMO或其突变体能够有效催化5‑氯‑1‑氧代‑2,3‑二氢‑1H‑茚‑2‑羧酸甲酯生成(S)‑5‑氯‑2,3‑二氢‑2‑羟基‑1‑氧代‑1H‑茚‑2‑羧酸甲酯，其中(S)‑5‑氯‑2,3‑二氢‑2‑羟基‑1‑氧代‑1H‑茚‑2‑羧酸甲酯的转化率和光学纯度均能达到99％，展现出极佳的催化效果和转化效率。本发明采用生物催化法制备茚虫威中间体，能够极大地降低生产成本并避免环境污染问题，具有广阔的应用前景与不菲的市场价值。The present invention discloses a variety of BVMOs or mutants thereof, which can effectively catalyze the conversion of 5-chloro-1-oxo-2,3-dihydro-1H-indene-2-carboxylic acid methyl ester to produce (S)-5-chloro-2,3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester. The conversion rate and optical purity of (S)-5-chloro-2,3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester can both reach 99%, demonstrating excellent catalytic effect and conversion efficiency. The present invention uses a biocatalytic method to prepare indoxacarb intermediates, which can greatly reduce production costs and avoid environmental pollution problems, and has broad application prospects and considerable market value.

Description

Application of monooxygenase and mutant thereof in preparation of indoxacarb chiral intermediate

Technical Field

The invention relates to the technical field of biosynthesis, in particular to application of monooxygenase and a mutant thereof in preparation of indoxacarb chiral intermediates.

Background

Indoxacarb (Indoxacarb) is an organic compound, has a molecular formula of C ₂₂H₁₇ClF₃N₃O₇, is a broad-spectrum oxadiazine pesticide, can effectively prevent and treat various pests on crops such as grains, cotton, fruits, vegetables and the like by blocking sodium ion channels in nerve cells of insects to make the nerve cells lose functions. Indoxacarb is widely accepted in the market due to high efficiency, low toxicity and difficulty in generating interactive resistance.

The (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester is a key chiral intermediate for synthesizing indoxacarb, and the intermediate can be further chemically synthesized to obtain a target product indoxacarb. As to the preparation method of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester, WO2003040083A1 discloses a method for preparing (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester by using cinchona alkaloid as a catalyst and tert-butyl hydroperoxide as an oxidant, however, the productivity of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester is only about 60%, and the ee value is only about 60%. CN105152958 discloses an improved process for cinchona alkaloid catalysts, the conversion can reach 99%, the ee value can reach 87%, but the results are still not ideal, and cinchona alkaloid is very expensive. In addition, the applicable metal complex catalyst is expensive and causes environmental pollution, which is unfavorable for industrial production.

In view of this, there is a need to develop a new biosynthesis method to solve the problems of low yield, low stereoselectivity, high cost and environmental pollution of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester in the existing method.

Disclosure of Invention

The present invention aims to solve at least one of the above technical problems in the prior art. To this end, the object of the present invention is to provide monooxygenases, in particular Baeyer-Villiger monooxygenases (Baeyer-Villiger monooxygenase, BVMO) and the use of mutants thereof for the preparation of indoxacarb chiral intermediates. In the invention, various BVMO and mutants thereof are developed and can be effectively used as catalytic enzyme for synthesizing indoxacarb chiral intermediates. The indoxacarb chiral intermediate synthesis based on the BVMO and the mutant thereof can solve the technical problems of low yield and low stereoselectivity of the indoxacarb chiral intermediate in the prior art, and the method has the advantages of less enzyme consumption, low preparation cost, environmental friendliness and extremely high practical value.

In a first aspect of the invention there is provided a Baeyer-Villiger monooxygenase (Baeyer-Villiger monooxygenase, BVMO) or a mutant thereof, said BVMO comprising an active fragment.

In some embodiments of the invention, the active fragment has the amino acid sequence of Genbank accession number WP_248592649.1, WP_179644013.1, WP_344596053.1, HLU96674.1 and U5S003.1.

In some embodiments of the invention, the active fragment has the amino acid sequence of Genbank accession number HLU96674.1.

In some embodiments of the invention, the BVMO further optionally comprises a tag sequence.

In some embodiments of the invention, the tag sequences include a Flag tag, a GST tag, a His tag, an HA tag, a C-Myc tag, and mCherry.

In some embodiments of the invention, the tag sequence is a His6 tag.

In some embodiments of the invention, the BVMO has the sequence shown in SEQ ID NO. 2.

In some embodiments of the invention, the BVMO is a sequence as set forth in SEQ ID NO. 2.

In some embodiments of the invention, the BVMO mutant comprises:

on the basis of the BVMO sequence described in the above aspect, mutants with catalytic activity are maintained after 1-5 amino acid substitutions, deletions or additions.

In the present invention, the phrase "mutant retaining catalytic activity" refers to a mutant retaining catalytic activity of introducing an oxygen atom beside a carbonyl group.

In some embodiments of the invention, a "catalytically active mutant" refers to a mutant that retains activity for the catalytic conversion of methyl 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylate to indoxacarb intermediate.

In some embodiments of the invention, the indoxacarb intermediate is a chiral intermediate. In some embodiments of the invention, the indoxacarb intermediate is (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester.

In some embodiments of the invention, the mutant has a conversion of greater than or equal to 40% of 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester.

In some embodiments of the invention, the mutant has a conversion of greater than or equal to 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester.

In some embodiments of the invention, the BVMO mutant is one that retains catalytic activity after 1-3 amino acid substitutions, deletions or additions based on the BVMO sequences described in the above aspects.

In some embodiments of the invention, the BVMO mutant is one that retains catalytic activity after 1-3 amino acid substitutions, deletions or additions based on SEQ ID NO. 2.

In some embodiments of the invention, the BVMO mutant is one that retains catalytic activity after 1-3 amino acid substitutions based on the BVMO sequences described in the above aspects.

In some embodiments of the invention, the BVMO mutant is one that retains catalytic activity after 1-3 amino acid substitutions based on SEQ ID NO. 2.

In some embodiments of the invention, the BVMO mutant is an amino acid substitution at least one of isoleucine (I) at position 145, phenylalanine (F) at position 417 and leucine (L) at position 500 of SEQ ID NO. 2.

In some embodiments of the invention, the BVMO mutant is one in which an amino acid substitution occurs at positions 1-3 of isoleucine (I) at position 145, phenylalanine (F) at position 417 and leucine (L) at position 500 of SEQ ID NO. 2.

In some embodiments of the invention, when the BVMO mutant is an amino acid substitution at position 1 in SEQ ID NO.2, it comprises an isoleucine (I) substitution at position 145 with phenylalanine (F), a phenylalanine (F) substitution at position 417 with threonine (T), and a leucine (L) substitution at position 500 with proline (P).

In some embodiments of the invention, when the BVMO mutant is an amino acid substitution at position 2 in SEQ ID NO. 2, it comprises an isoleucine (I) substitution at position 145 with phenylalanine (F) and a phenylalanine (F) substitution at position 417 with threonine (T), an isoleucine (I) substitution at position 145 with phenylalanine (F) and a leucine (L) substitution at position 500 with proline (P), and a phenylalanine (F) substitution at position 417 with threonine (T) and a leucine (L) substitution at position 500 with proline (P).

In some embodiments of the invention, when the BVMO mutant is an amino acid substitution at position 3 in SEQ ID NO.2, it comprises an isoleucine (I) substitution at position 145 with phenylalanine (F), a phenylalanine (F) substitution at position 417 with threonine (T) and a leucine (L) substitution at position 500 with proline (P).

In some embodiments of the invention, when the BVMO mutant is one having the sequence set forth in SEQ ID NO 9, SEQ ID NO 10, or SEQ ID NO 11, the amino acid substitution occurs at the 1 position in SEQ ID NO 2.

In some embodiments of the invention, when the BVMO mutant is one having the sequence shown in SEQ ID NO. 12, an amino acid substitution occurs at the 3 position in SEQ ID NO. 2.

In some embodiments of the invention, the BVMO or mutant thereof is further modified, including but not limited to, glycosylation, phosphorylation, acetylation, methylation, ubiquitination, or lipidation, or the introduction of unnatural amino acids (e.g., alpha-amino keto acids or beta-amino keto acids).

In a second aspect of the invention, there is provided a biologic comprising:

(1) A nucleic acid molecule encoding a BVMO or a mutant thereof as described in the above aspects;

(2) An expression vector comprising the nucleic acid molecule of (1);

(3) A transformant containing the nucleic acid molecule according to (1);

(4) A transformant containing the expression cassette of (2);

in some embodiments of the invention, the expressors include viruses, plasmids, and phages.

In some embodiments of the invention, the plasmid comprises a plasmid of the pET28a vector.

In the present invention, the term "expressor" refers to a vector or an expression system for integrating or inserting a desired foreign gene.

In some embodiments of the invention, the transformant comprises bacterial, fungal and animal and plant cells.

In the present invention, the term "transformant" refers to a recipient cell into which a novel genetic marker is obtained after the incorporation or introduction of an exogenous gene.

In some embodiments of the invention, the transformant is not related to animal and plant propagation material.

In some embodiments of the invention, the construction of the expression and transformant may be accomplished based on any means conventional in the art.

In a third aspect of the invention there is provided the use of BVMO or a mutant or biological thereof as described in the above aspects in an enzyme catalysed reaction.

In some embodiments of the invention, the enzyme-catalyzed reaction comprises a BVMO catalyzed substrate undergoing a Baeyer-Villiger rearrangement reaction, a formate dehydrogenase catalyzing the oxidation of formate and the reduction of a coenzyme.

In a fourth aspect, the invention provides the use of BVMO or a mutant or biological thereof as described in the preceding aspects in the preparation of indoxacarb intermediates and/or indoxacarb.

In some embodiments of the invention, the indoxacarb intermediate is a chiral intermediate.

In some embodiments of the invention, the indoxacarb intermediate is (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester.

In a fourth aspect of the invention, there is provided a process for the preparation of indoxacarb intermediate comprising the steps of:

5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester is taken as a substrate, the BVMO or a mutant or biological product thereof is added, then formic acid dehydrogenase or biological material with the functions of expressing, secreting or producing the formic acid dehydrogenase, ammonium formate and coenzyme are added, and the mixture reacts for 2 to 24 hours at a certain catalytic temperature, thus obtaining the indoxacarb intermediate.

In some embodiments of the invention, the catalytic temperature ranges from 20 to 45 ℃.

In some embodiments of the invention, the catalytic temperature ranges from 30 to 40 ℃.

In some embodiments of the invention, the substrate further comprises a solvent and/or a co-solvent.

In some embodiments of the invention, the solvent and/or co-solvent comprises at least one of ethyl acetate, isooctane, petroleum ether, methyl tert-butyl ether, methanol, n-butanol, and cyclohexane.

In some embodiments of the invention, the solvent and/or co-solvent is cyclohexane.

In some embodiments of the invention, the coenzyme comprises at least one of reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), nicotinamide adenine dinucleotide phosphate (NADP ⁺) or a salt thereof, reduced Nicotinamide Adenine Dinucleotide (NADH), and nicotinamide adenine dinucleotide (NAD ⁺) or a salt thereof.

In some embodiments of the invention, the salts of nicotinamide adenine dinucleotide phosphate (NADP ⁺) include NADP ⁺ sodium salt and NADP ⁺ potassium salt.

In some embodiments of the invention, the salt of nicotinamide adenine dinucleotide (NAD ⁺) comprises a sodium salt of NAD ⁺ and a potassium salt of NAD ⁺.

In some embodiments of the invention, a buffer is also added to the method.

In some embodiments of the invention, the buffer comprises at least one of PB buffer, tris-HCl buffer, and TEA buffer.

In some embodiments of the invention, the buffer is at least one of a PB buffer or a TEA buffer.

In some embodiments of the invention, the pH of the buffer is between 6 and 10.

In some embodiments of the invention, the pH of the buffer is 7-9.

In some embodiments of the invention, the BVMO or mutant or biologic thereof is present in the system at a concentration of 20 to 300g/L.

In some embodiments of the invention, the BVMO or mutant or biologic thereof is present in the system at a concentration of 40 to 200g/L.

In some embodiments of the invention, the substrate is present in the system at a concentration of 10 to 120g/L.

In some embodiments of the invention, the substrate is present in the system at a concentration of 20 to 100g/L.

In some embodiments of the invention, the concentration ratio of the substrate to BVMO or mutant thereof, or biologic in the system is from 2:1 to 1:2.

In some embodiments of the invention, the concentration ratio of the substrate to BVMO or mutant thereof, or biologic in the system is 1:2.

In some embodiments of the invention, the formate dehydrogenase or biological material having the ability to express, secrete or produce formate dehydrogenase is present at a concentration of 20-50g/L in the system.

In some embodiments of the invention, the ammonium formate is present in the system at a concentration of 300 to 700mM.

In some embodiments of the invention, the concentration of the coenzyme in the system is from 0.1 to 0.8g/L.

In some embodiments of the invention, the solvent and/or co-solvent is used in an amount of 5-40% (v/v) by volume in the system.

In some embodiments of the invention, the solvent and/or co-solvent is used in an amount of 10-50% (v/v) by volume in the system.

In some embodiments of the invention, the method further comprises extracting and purifying the product.

In some embodiments of the invention, the method comprises adding BVMO or mutant thereof, or biological product, formate dehydrogenase or biological material with expression, secretion or production of formate dehydrogenase, buffer solution, substrate, cosolvent, ammonium formate and coenzyme into a reaction vessel, reacting for 2-24h at a certain catalytic temperature, extracting, separating, and steaming to obtain indoxacarb chiral intermediate.

In some embodiments of the invention, the extracting comprises extracting with an organic solvent to obtain an organic phase, the organic solvent comprising ethyl acetate and toluene.

In some embodiments of the invention, the 1L reaction system comprises 40-200g of recombinant bacterium wet bacteria expressing BVMO or mutants thereof, 20-50g of recombinant bacterium wet bacteria expressing formate dehydrogenase, 20-100g of substrate, 50-400mL of cosolvent, 300-700mmol of ammonium formate, 0.125-1mmoL of coenzyme and the balance of buffer.

Wherein, the recombinant fungus wet fungus for expressing formate dehydrogenase, ammonium formate and coenzyme construct a coenzyme circulation system.

In the present invention, BVMO or a mutant thereof may be used in the form of an enzyme in a free form (for example, in the form of an enzyme powder) or in the form of a cell expressing BVMO or a mutant thereof (for example, in the form of a wet cell). Or in other forms, such as the disrupted supernatant of cells expressing the BVMO or mutants thereof, or whole cell immobilization, or immobilization of free enzyme powder.

In some embodiments of the invention, the enzyme protein expression host microorganism used may be E.coli ESCHERICHIA COLI BL (DE 3), and the recombinant plasmid may be a pET28 plasmid, such as pET28a+. The molecular biology operation involved in the operation steps is a routine experimental operation method well known in the biological field, and comprises the steps of gene acquisition (PCR), splicing of plasmids and target genes (i.e. vector construction), introduction of plasmids containing target gene fragments into bacterial cells (i.e. transformation), culture of bacterial cells in a culture medium and enzyme production (i.e. fermentation).

In a fifth aspect of the invention there is provided a process for the preparation of indoxacarb comprising preparing indoxacarb intermediate using the process described in the preceding aspect and then preparing indoxacarb.

The beneficial effects of the invention are as follows:

1. In the invention, various BVMOs or mutants thereof are developed, and the BVMOs or mutants thereof can effectively catalyze 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester to generate (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester, wherein the conversion rate and optical purity of the (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester can reach 99 percent, and the excellent catalytic effect and conversion efficiency are shown.

2. The indoxacarb intermediate prepared by the biocatalysis method can greatly reduce the production cost and avoid the problem of environmental pollution, and has wide application prospect and great market value.

Drawings

FIG. 1 is a plasmid map of a Baeyer-Villiger monooxygenase plasmid vector in an embodiment of the present invention.

Fig. 2 is a technical roadmap of the invention.

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester.

FIG. 4 is an HPLC detection chart of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The starting materials, reagents or apparatus used in the examples and comparative examples were either commercially available from conventional sources or may be obtained by prior art methods unless specifically indicated. Unless otherwise indicated, assays or testing methods are routine in the art.

In the examples described below, the molecular biological procedures involved are all routine experimental procedures well known in the biological arts, including gene acquisition (PCR), splicing of plasmids and genes of interest (i.e., vector construction), introduction of plasmids containing fragments of genes of interest into cells of the bacteria (i.e., transformation), cultivation of the bacteria in a medium, and enzyme production (i.e., fermentation).

In the following examples, the LB liquid medium used was prepared by dissolving 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl in water as a solvent and sterilizing at high temperature.

The LB solid culture medium is prepared by dissolving 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl and 20g/L agar in water, and sterilizing at high temperature.

Example 1

In this example, recombinant cells expressing a wild type Baeyer-Villiger monooxygenase were prepared, wherein the specific preparation method is as follows:

(1) Preparation of Baeyer-Villiger monooxygenase (Baeyer-Villiger monooxygenase, BVMO) plasmid vector:

The corresponding gene fragment of interest (BVMO nucleotide sequence) was inserted into a commercially available pET28a+ using a commercial plasmid pET28a+ (available from Beijing Optimago technologies Co., ltd.) according to routine procedures in the art to give a BVMO plasmid vector (plasmid map shown in FIG. 1), wherein the cloning sites are NdeI cleavage site and SalI cleavage site.

Wherein the source and amino acid sequence information of the wild-type BVMO are shown in table 1 below.

TABLE 1 Source and amino acid sequence information of wild-type BVMO

BVMO numbering	Source(s)	Amino acid sequence Genbank numbering
			BVMO-1	Thermobifida alba	WP_248592649.1
BVMO-2	Spinactinospora alkalitolerans	WP_179644013.1
			BVMO-3	Actinomadura vinacea	WP_344596053.1
BVMO-4	Thermobifida alba	HLU96674.1
			BVMO-5	Dietzia sp.D5	U5S003.1
BVMO-6	Polaromonas sp.JS666	WP_011485409.1
			BVMO-7	Leptospira biflexa	WP_012388673.1

His tag (His 6, HHHHH, SEQ ID NO: 1) was added to the C-terminal based on the amino acid sequences of BVMO-1 to BVMO-7 as above, respectively.

The amino acid sequence of BVMO-4 is shown in Genbank number HLU96674.1, and based on the sequence, his tag is added at C terminal, the sequence is specifically ：MDVLVVGAGFSGLYALYRLRELGRTAHVIESAGDVGGVWYWNRYPGARCDIESIEYCYSFSEEVVQEWNWSERYAAQPEILRYINFVADKFDLRSGITFDTTVTSAAFDEDSSTWTVETDRGDRIRTRHLVMASGQLSVAQLPDIPGLRDFAGEFYHTGNWPHEPVDFSGKRVGVIGTGSSGIQVSPQIAKQAAELFVFQRTPHFAMPARNAPLDPDFLADLKTRYAEYREEARNSPGGTHRYQGPKSALEVSEDELVETLERYWEKGGPDILAAYRDILRDREANERVAEFVRGKIRSLVRDPEVAERLVPKGYPFGTKRLILEIDYYDMYNRDNVHLVDTLSAPIEEVTPRGVRTSEREYELDSLVLATGFDALTGALFKIDIRGVGGASLKEKWAAGPRTYLGLSTAGFPNLFFIAGPGSPSALSNMLVSIEQHVEWVTDHIEYLFKNGLTRSEAVLEKEDSWVEHVNEVANETLYPAANSWYLGANVPGKPRVFMLYVGGFHRYRKICDEVAANGYEGFVLSHHHHHH(SEQ ID NO：2).

Of course, additional tag sequences may not be introduced as desired.

(2) Obtaining recombinant bacteria expressing BVMO:

Expression of the enzyme protein was performed using E.coli (ESCHERICHIA COLI) BL21 (DE 3) as a transformant. The plasmid vector constructed in the steps is respectively transformed into escherichia coli cells by a conventional method, then inoculated into LB liquid medium containing 50 mug/mL kanamycin, cultured overnight at 37 ℃, then inoculated into LB medium containing 50 mug/mL kanamycin at 2 percent inoculum size (v/v), cultured to the concentration of bacterial cells OD ₆₀₀ = 0.6 at 37 ℃ and 150rpm, added with IPTG with the final concentration of 0.1mM, subjected to induction culture for 12 hours at 28 ℃, centrifuged for 10 minutes at 4 ℃ and 12000rpm, collected, washed with 0.85 percent normal saline, centrifuged again to obtain recombinant bacteria expressing BVMO, and stored at-20 ℃ for standby (namely resting cells for catalytic reaction).

Example 2

Synthesis of indoxacarb chiral intermediates was performed using recombinant E.coli expressing BVMO-1, BVMO-2, BVMO-3, BVMO-4, BVMO-5, BVMO-6, and BVMO-7 prepared in example 1 as a catalyst. The reaction system comprises 18mL of phosphate buffer (PB buffer, 100mM, pH 8.0), 2mL of cyclohexane, 0.6g of methyl 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylate, 1.0g of recombinant E.coli wet cell of BVMO-1, BVMO-2, BVMO-3, BVMO-4, BVMO-5, BVMO-6 or BVMO-7, 0.3g of E.coli wet cell of formate dehydrogenase, 0.3g of ammonium formate, and 0.01g of NADP ⁺ sodium salt. The catalytic reaction is carried out for 12 hours at 35 ℃, and the technical scheme is shown in figure 2. After the reaction was completed, 1mL of the mixture was sampled from the different groups, and then mixed with 1mL of ethyl acetate, centrifuged at 12000rpm for 1min for layering, and after the upper organic phase was dried over anhydrous sodium sulfate, the conversion and ee value of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester were measured by HPLC, and the structure of the product (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester was confirmed by nuclear magnetic hydrogen spectroscopy.

Wherein, the preparation method of the colibacillus wet thalli of the formate dehydrogenase is the same as that of the recombinant bacterium wet thalli for expressing BVMO. The formate dehydrogenase plasmid vector is also constructed based on a commercial plasmid pET28a+, wherein the inserted target gene fragment is a formate dehydrogenase gene sequence with the GenBank number of AXT 18256.1. The transformant was also E.coli BL21 (DE 3).

The conversion rate is calculated as follows:

wherein the substrate is 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester, and the product is (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester.

The nuclear magnetic resonance hydrogen spectrum and HPLC detection results are shown in FIGS. 3-4 and Table 2.

TABLE 2 conversion and e.e. values of BVMO of each group

The results showed that BVMO-1, BVMO-2, BVMO-3, BVMO-4 and BVMO-5 all produced in the S form, and BVMO-4 produced (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester with the highest ee and conversion, and BVMO-5 and BVMO-6 were nearly inactive.

Example 3

Based on the results of example 2, the two-phase reaction system composed of BVMO and the organic phase was optimized using BVMO-4 as a catalyst. The biphasic reaction system comprises 18mL PB buffer (100 mM, pH 8.0) and a total of 20mL of an organic phase selected from isooctane, n-heptane, petroleum ether, methyl tert-butyl ether, ethyl acetate, methanol, cyclohexane and n-butanol, and further comprises 0.6g of a substrate (methyl 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylate), 1.0gBVMO-4 recombinant Escherichia coli wet cell, 0.5g of formate dehydrogenase Escherichia coli wet cell, 0.5g of ammonium formate, and 0.01g of NADP ⁺ sodium salt. The catalytic reaction was carried out at 35℃for 16H, after the reaction was completed, 1mL of the sample from the different groups was mixed with 1mL of ethyl acetate, and then the mixture was centrifuged at 12000rpm for 1min to separate layers, and after the upper organic phase was dried over anhydrous sodium sulfate, the conversion and ee value of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester were measured by HPLC.

The HPLC detection results are shown in Table 3.

TABLE 3 conversion and e.e. values for different biphase systems

Organic phase	Conversion rate	E.e. value
			Acetic acid ethyl ester	21%	99.1% (S type)
Isooctane	16%	98.5% (S type)
			N-heptane	13%	98.7% (S type)
Petroleum ether	45%	99.0% (S type)
			Methyl tert-butyl ether	18%	98.8% (S type)
Methanol	66%	99.1% (S type)
			N-butanol	56%	98.5% (S type)
Cyclohexane	83%	99.0% (S type)

The results show that the conversion of cyclohexane is highest in various organic phases, up to 83% conversion and the ee value of the product is greater than 99%. Cyclohexane was chosen as the organic phase for the subsequent reaction taking into account both the conversion and the ee of the product.

Example 4

Based on the results of example 2 and example 3, the temperature optimization of the catalytic reaction was performed with BVMO-4 as catalyst. The composition and procedure of the reaction system were substantially the same as in example 3, except that cyclohexane was selected for the organic phase, and the catalytic reaction temperatures were set to 20 ℃,25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃ respectively, and the catalytic reaction time was 2 hours.

The HPLC detection results are shown in Table 4.

TABLE 4 conversion and e.e. values for different catalytic temperatures

The results show that the ee value of the product produced by BVMO-4 at different catalytic temperatures does not vary much and that the catalytic reaction has the highest conversion at 35 ℃, so 35 ℃ is chosen as the optimal temperature for the reaction.

Example 5

Based on the results of examples 2-4, the pH optimization of the catalytic reaction was performed with BVMO-4 as catalyst. The composition and procedure of the reaction system were substantially the same as in example 4, except that cyclohexane was selected as the organic phase, the catalytic reaction temperature was selected to be 35 ℃, and the pH of the PB buffer was set to pH 6.0, pH 7.0, pH 8.0, pH 9.0 and pH 10.0, respectively.

The HPLC detection results are shown in Table 5.

TABLE 5 conversion and e.e. values for different pH values

PH of PB buffer	Conversion rate	E.e. value
			pH 6.0	6.5%	98.7% (S type)
pH 7.0	14.6%	99.0% (S type)
			pH 8.0	15.8%	99.0% (S type)
pH 9.0	11.3%	99.0% (S type)
			pH 10.0	4.2%	98.8% (S type)

The results show that BVMO-4 produced products with little variation in ee value at different pH and the catalytic reaction had the highest conversion at pH 8.0, thus pH 8.0 was chosen as the optimal pH for the reaction.

Example 6

In this example, the effect of various BVMO-4 concentrations (wet cell concentration) and substrate (methyl 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylate) concentrations on the conversion and e.e. values were tested separately using BVMO-4 as a catalyst to optimize the wet cell concentration and substrate concentration of the reaction system.

The reaction system comprises 18mL PB buffer (100 mM, pH 8.0), 2.0mL cyclohexane, 20-120g/L substrate (5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester), 10-300g/L BVMO-4 recombinant escherichia coli wet cell, 30g/L formate dehydrogenase escherichia coli wet cell, 30g/L ammonium formate, and 0.5g/L NADP ⁺ sodium salt. Catalytic reaction was carried out for 24H at 35℃and 1mL of the sample was mixed with an equal amount of ethyl acetate, and the mixture was centrifuged at 12000rpm for 1min to separate the layers, and after the upper organic phase was dried over anhydrous sodium sulfate, the conversion of indoxacarb chiral intermediate (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester was measured by HPLC.

The HPLC detection results are shown in Table 6.

TABLE 6 conversion of different substrate and wet cell concentrations

The results showed that the conversion rate was increased from 44.7% to 93.8% in the case where the substrate concentration was increased from 10g/L to 40g/L, and then the reaction of the higher substrate and wet cell concentration was tested at a ratio of substrate: wet cell concentration of 1:2, and the results showed that the substrate in the concentration range of 30-100g/L could be completely reacted, however, as the substrate concentration was increased to 120g/L, the substrate conversion rate was significantly decreased even if the wet cell ratio was greatly increased. Thus, a substrate concentration of 100g/L and a wet cell concentration of 200g/L were selected as reaction conditions for the subsequent product preparation.

Example 7

This example provides an enlarged preparation of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester using BVMO-4 catalyst based on the optimized reaction conditions of examples 2-6. The reaction system comprises 2700mL PB buffer (100 mM, pH 8.0), 300mL cyclohexane, 300g 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester, 600gBVMO-4 recombinant escherichia coli wet cell, 90g escherichia coli wet cell of formate dehydrogenase, 90g ammonium formate, and 0.15g NADP ⁺ sodium salt. Catalytic reaction is carried out for 24 hours at 35 ℃, the conversion rate is 98.4%, toluene with the total volume of 1/2, 1/2 and 1/4 is added for extraction for three times, and the organic phase is subjected to rotary evaporation after the extraction for three times, thus obtaining (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester with the ee value of more than 99 percent, and 274.4g of products are obtained, and the yields are 85.4 percent respectively.

Example 8

Based on the results of the above examples, BVMO-4 mutants were prepared using BVMO-4 as the original template, with the following specific procedures.

(1) Construction of a single mutant library:

The recombinant BVMO-4 strain prepared in the above example was activated to extract plasmid (pET 28a (+) -BVMO-4) therefrom. The PCR was performed using pET28a (+) -BVMO-4 as a template, designing a mutation primer (shown in Table 7 below), and performing a Polymerase Chain Reaction (PCR) in which the PCR amplification system (50. Mu.L) is shown in Table 8 to obtain an amplified product. Then, 20. Mu.L of the PCR product was taken, and the template plasmid DNA was digested with 1. Mu.L of the endonuclease DpnI at 37℃for 3 hours, and inactivated at 65℃for 10 minutes. The recombinant plasmid was transformed into competent cells of E.coli BL21 (DE 3) by heat shock, and the clones were inoculated into 10mL LB plate medium and cultured at 37℃for 12-16h.

TABLE 7 mutant primers

TABLE 8PCR amplification System (50. Mu.L)

Component (A)	Content of
		Upstream primer (100. Mu.M)	1μL
Downstream primer (100. Mu.M)	1μL
		2X Phanta buffer (Nuo Wei Zan)	25μL
DNTP mixture (10 mM each)	1μL
		Plasmid template	0.1ng-1ng
DNA polymerase Phanta (Nuo Wei Zan)	1U
		Ultrapure water	To 50 mu L

PCR amplification procedure was performed for 30 cycles with reference to Phanta Super-FIDELITY DNA polymerase instructions, 95℃pre-denaturation 30s, 95℃denaturation 30s,65℃annealing 30s,72℃extension 6min, then 72℃final extension 7min,16℃incubation.

Positive clones and the original strain on the plates were then randomly selected, inoculated into 10mL of LB liquid medium (50 mg/L kanamycin was added), and cultured in a temperature-controlled shaker at 37℃and 200rpm for 8-10 hours. The culture broth was inoculated in an inoculum size of 1% into 100mL of LB liquid medium (50 mg/L kanamycin was added) and cultured in a temperature-controlled shaker at 37℃and 180rpm for 2-2.5 hours (OD 600 value was 0.6-0.8). Then, the inducer IPTG was added at a final concentration of 0.1mM, and the mixture was placed in a temperature-controlled shaker at 24℃and 180rpm to continue the culture for 12 hours. Then, the cultured bacterial liquid was placed in an 800mL centrifuge cup, centrifuged at 8000rpm and 4℃for 10min in a low-temperature high-speed centrifuge, and the supernatant was discarded to collect bacterial cells and weighed.

The single mutation variants BVMO-4-I145F (SEQ ID NO: 9), BVMO-4-F417T (SEQ ID NO: 10) and BVMO-4-L500P (SEQ ID NO: 11) were obtained.

The effect of each mutant on the conversion of methyl 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylate was tested using the method described in the above examples, with the conversion and the e.e. value as indicators.

(2) Iterative mutation (construction of multiple mutant):

Extracting plasmids from the BVMO-4-I145F, BVMO-4-F417T and BVMO-4-L500P recombinant bacteria obtained by screening in the above steps, carrying out PCR amplification by using the mutation primers shown in Table 7 as templates (the system is the same as Table 8), constructing recombinant bacteria according to the method in the above steps, and screening.

Finally, the three mutant BVMO-4-I145F/F417T/L500P (SEQ ID NO: 12) is obtained.

(3) And (3) detecting catalytic activity:

And (3) taking 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester as a substrate, carrying out catalytic activity detection on the single mutant strain and the triple mutant strain prepared by the steps, and comparing the catalytic activity of each mutant. The method comprises the following specific steps:

The detection method is the same as the above embodiment. Specifically, the reaction system was set up with 18mL of PB buffer (100 mM, pH 8.0), 2mL of cyclohexane, 0.6g of methyl 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylate, 1.0g of each of the recombinant E.coli wet cells of the BVMO-4 mutant, 0.3g of the E.coli wet cell of formate dehydrogenase, 0.3g of ammonium formate, and 0.01g of NADP ⁺ sodium salt. Catalytic reaction was carried out for 6H at 35℃and 1mL of each sample was mixed with 1mL of ethyl acetate, centrifuged at 12000rpm for 1min for delamination, and the upper organic phase was dried over anhydrous sodium sulfate and the ee value and conversion of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester were determined by HPLC. Relative viability of the mutants was calculated using the conversion of the wild-type BVMO-4 as a reference (viability is expressed as conversion, where the viability of the wild-type BVMO-4 is defined as 100%).

The results are shown in Table 9.

TABLE 9 conversion and relative Activity of mutants (%)

BVMO-4 or mutants thereof	Conversion rate	Relative vitality (%)
			BVMO-4	41%	100
BVMO-4-I145F	49.6%	121
			BVMO-4-F417T	54.1%	132
BVMO-4-L500P	52.5%	128
			BVMO-4-I145F/F417T/L500P	81.1%	198

As a result, it was found that methyl (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylate having an ee value of more than 99% could be obtained from each of the 4 mutants. Furthermore, the relative viability of the mutants BVMO-4-I145F, BVMO-4-F417T, BVMO-4-L500P and BVMO-4-I145F/F417T/L500P relative to BVMO-4 was increased by 21%, 32%, 28% and 98%, respectively, over a shorter catalytic reaction time. The catalytic effect of the mutant BVMO-4-I145F/F417T/L500P is optimal, which indicates that the catalytic effect of the mutant BVMO-4-I145F/F417T/L500P is optimal in a short time.

(4) Preparation of (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester using the mutant BVMO-4-I145F/F417T/L500P magnification. The reaction system was set up with 2700mL PB buffer (100 mM, pH 8.0), 300mL cyclohexane, 300g methyl 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylate, 300g BVMO-4-I145F/F417T/L500P recombinant E.coli wet cell, 90g formate dehydrogenase E.coli wet cell, 90g ammonium formate, 0.15g NADP ⁺ sodium salt. Catalytic reaction is carried out for 24 hours at 35 ℃, the conversion rate is 99.1%, toluene with the total volume of 1/2, 1/2 and 1/4 is added for extraction for three times, the organic phase is subjected to rotary evaporation after the extraction for three times, and the (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester with the ee value of more than 99% can be obtained, 281.2g of products are obtained, and the yield is 87.5%. The result shows that the three-mutation mutant BVMO-4-I145F/F417T/L500P can achieve the same catalytic effect under the effect of halving the thallus quantity, and has good application potential.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A Baeyer-Villiger monooxygenase or a mutant thereof, wherein the Baeyer-Villiger monooxygenase comprises an active fragment;

the active fragment has the amino acid sequence of Genbank number as follows:

WP_248592649.1、WP_179644013.1、WP_344596053.1、HLU96674.1、U5S003.1;

The mutant of the Baeyer-Villiger monooxygenase comprises the steps of,

Mutants retaining catalytic activity after 1-5 amino acid substitutions, deletions or additions;

Preferably, the mutant has a conversion of 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester of greater than or equal to 40%, preferably greater than or equal to 50%.

2. The Baeyer-Villiger monooxygenase or mutant thereof of claim 1, wherein the Baeyer-Villiger monooxygenase mutant is a mutant that retains catalytic activity after 1-3 amino acid substitutions, deletions or additions based on the Baeyer-Villiger monooxygenase sequence of claim 1;

preferably, the Baeyer-Villiger monooxygenase mutant is a mutant with catalytic activity after 1-3 amino acid substitutions, deletions or additions on the basis of SEQ ID NO. 2;

preferably, the mutant of Baeyer-Villiger monooxygenase is an amino acid substitution at least one of isoleucine (I) at position 145, phenylalanine (F) at position 417 and leucine (L) at position 500 of SEQ ID NO. 2.

3. A biologic, characterized in that it comprises:

(1) A nucleic acid molecule encoding the Baeyer-Villiger monooxygenase or mutant thereof of any one of claims 1-2;

(2) An expression vector comprising the nucleic acid molecule of (1);

(3) A transformant containing the nucleic acid molecule according to (1);

(4) A transformant containing the expression cassette of (2);

wherein the transformant is not related to animal and plant propagation material;

preferably, the expressors include viruses, plasmids and phages;

Preferably, the transformants include bacterial, fungal and animal and plant cells.

4. Use of a Baeyer-Villiger monooxygenase or mutant thereof according to any one of claims 1-2, or a biologic according to claim 3, in an enzyme catalyzed reaction;

Preferably, the enzyme-catalyzed reaction comprises a Baeyer-Villiger monooxygenase catalyzed substrate undergoing a Baeyer-Villiger rearrangement reaction, a formate dehydrogenase catalyzing the oxidation of formate and the reduction of a coenzyme.

5. Use of a Baeyer-Villiger monooxygenase or mutant thereof as claimed in any one of claims 1-2, or a biological product as claimed in claim 3, in the preparation of indoxacarb intermediate and/or indoxacarb;

preferably, the indoxacarb intermediate is a chiral intermediate;

Preferably, the indoxacarb intermediate is (S) -5-chloro-2, 3-dihydro-2-hydroxy-1-oxo-1H-indene-2-carboxylic acid methyl ester.

6. A process for preparing indoxacarb intermediates comprising the steps of:

Taking 5-chloro-1-oxo-2, 3-dihydro-1H-indene-2-carboxylic acid methyl ester as a substrate, adding the Baeyer-Villiger monooxygenase or a mutant thereof according to any one of claims 1-2 or the biological product according to claim 3, then adding formic acid dehydrogenase or a biological material with the function of expressing, secreting or generating the formic acid dehydrogenase, ammonium formate and coenzyme, and reacting for 2-24 hours at a certain catalytic temperature to obtain indoxacarb intermediate;

preferably, the catalytic temperature is in the range of 20-45 ℃;

7. The method of claim 6, wherein the substrate further comprises a solvent and/or a co-solvent comprising at least one of ethyl acetate, isooctane, petroleum ether, methyl t-butyl ether, methanol, n-butanol, and cyclohexane.

8. The method of claim 6, wherein the coenzyme comprises at least one of reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), nicotinamide adenine dinucleotide phosphate (NADP ⁺) or a salt thereof, reduced Nicotinamide Adenine Dinucleotide (NADH), and nicotinamide adenine dinucleotide (NAD ⁺) or a salt thereof;

Preferably, the salts of nicotinamide adenine dinucleotide phosphate (NADP ⁺) include the sodium salt of NADP ⁺ and the potassium salt of NADP ⁺.

9. The method of claim 6, wherein a buffer is further added;

Preferably, the buffer comprises at least one of PB buffer, tris-HCl buffer and TEA buffer;

Preferably, the pH of the buffer is between 6 and 10.

10. A process for the preparation of indoxacarb comprising preparing indoxacarb intermediate using the process of any one of claims 6 to 9 and then preparing indoxacarb.