CN116496997A - Carbonyl reductase mutant and application thereof - Google Patents

Carbonyl reductase mutant and application thereof Download PDF

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CN116496997A
CN116496997A CN202210062673.4A CN202210062673A CN116496997A CN 116496997 A CN116496997 A CN 116496997A CN 202210062673 A CN202210062673 A CN 202210062673A CN 116496997 A CN116496997 A CN 116496997A
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carbonyl reductase
amino acid
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徐尤勇
范乃贵
杜平
王苑先
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Pharmablock Sciences Nanjing Inc
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Abstract

The invention discloses a carbonyl reductase mutant and application thereof. The amino acid sequence of the carbonyl reductase mutant is SEQ ID NO:1, and the mutated amino acid site comprises: s65; or the amino acid sequence of the carbonyl reductase mutant has a mutated amino acid site and is identical to the amino acid sequence of SEQ ID NO:1 has more than 90 percent of homology and has the catalytic activity of carbonyl reductase. The invention obtains carbonyl reductase mutant with high catalytic efficiency and high selectivity. The partial carbonyl reductase mutant obtained by the method has the advantages that the enzyme dosage is reduced to 0.01-0.03 wt% and the reaction volume is reduced to 10-15V in the synthesis, so that the industrial production cost of the compound is greatly reduced, and the enzyme has better application value in industrial production.

Description

Carbonyl reductase mutant and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a carbonyl reductase mutant and application thereof.
Background
Biocatalysis has become an important method for expanding traditional chemical synthesis in the sustainable development process due to the characteristics of high stereoselectivity, mild reaction conditions, environmental friendliness and the like, and particularly the rapid development of enzyme molecular modification technologies (bioinformatics, genetic engineering and protein engineering) in recent years.
Enzymes that catalyze the reduction of ketones (or aldehydes) to the corresponding alcohols are known as carbonyl reductases (KREDs). Since KRED can highly selectively construct chiral alcohols, its use in organic synthesis is widely growing.
For racemic chiral carbonyl compounds, the action of kinetic resolution of racemic chiral carbonyl compounds can be obtained when the speed of the catalytic reaction of KRED on one absolute configuration is significantly faster than that of the other absolute configuration. Owen W.good et al selectively reduced the enantiomer of the (R) configuration in racemic 2-methylpentanal to the desired product (Development of a Practical Biocatalytic Process for (R) -2-Methylpentanol) by evolution from the Lactobacillus kefir enzyme. However, the research is limited to aldehyde compounds, and the substrate range is narrow, so that the application value is low. The reaction is as follows:
however, few reports have been made on the use of KRED for the resolution of racemic chiral ketones, in particular on the use of ketones having an ee value of not less than 99% and at the same time alcohols having an ee value of not less than 99%.
Disclosure of Invention
The invention aims to provide a carbonyl reductase mutant and application thereof, so as to improve the catalytic activity and resolution selectivity of carbonyl reductase.
In order to achieve the above object, according to one aspect of the present invention, there is provided a carbonyl reductase mutant. The amino acid sequence of the carbonyl reductase mutant is SEQ ID NO:1, and the mutated amino acid site comprises: s65; or the amino acid sequence of the carbonyl reductase mutant has a mutated amino acid site and is identical to the amino acid sequence of SEQ id no:1 has more than 90 percent of homology and has the catalytic activity of carbonyl reductase.
Further, S65 is mutated to S65A, S G or S65D.
Further, the mutation is one of the following combinations of mutation sites: s65A+I V, S Gv+I78Q, S D+I78V, S65A+A201Q, S65A+I78V+M154I, S A+I78V+A201D, S A+I78V+A201Q, S A+P153 S+V36I, S A+I78V+M154I+A201Q, S A+I78V+P153 S+V+ I, S A+I78V+M438I+A202 427A+I78V+D182 A+I Q, S A+I78V+D194A+V I, S5A+P153 S+V360I+A202 5265A+I78V+M431I+A200A200Q+A200L, S65 A+I. 78v+p153s+v198i+a201D, S a+i78v+s145e+p153s+d194A, S a+i78v+m168i+a155 d+a65D, S a+i78v+m168i+v198 i+a1200q+a202L, S a+i78v+m168i+v360i+a201 D+A202L, S65 A+I78V+M1200I+A155 D+V198 I+A1209D+A202L, S A+I78V+M1200I+A155 D+V198 I+A120Q+A217L or S65A+I78V+G94T+M120I+A397D+V198 I+A120Q+A202L.
According to another aspect of the present invention, there is provided a DNA molecule. The DNA molecule encodes any of the carbonyl reductase mutants described above.
According to yet another aspect of the present invention, there is provided a recombinant plasmid. The recombinant plasmid is linked to any of the DNA molecules described above.
Further, the method comprises the steps of, the recombinant plasmids are pET-21b (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b, pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a, pET-29a (+), and the recombinant plasmids are recombinant plasmids obtained by the recombinant plasmids pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-3835-C, pGEX-5X-1, pGEX-6p-1, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pPIC9k, pGAPZalpha A, pUC-18 or pUC-19.
According to another aspect of the invention, a host cell is provided. The host cell contains any of the recombinant plasmids described above.
Further, the host cell is a prokaryotic cell or a eukaryotic cell.
Further, the prokaryotic cells are E.coli DH5 alpha, top10, BL21-DE3 or E.coli Rosetta-DE3 cells; eukaryotic cells are yeast cells.
According to yet another aspect of the present invention, a method of producing chiral ketones is provided. The method comprises the step of adopting carbonyl reductase to reduce and split ketone raceme compounds, wherein the carbonyl reductase is any one of the carbonyl reductase mutants.
Further, the ketone racemate isWherein R is 1 And R is 2 Each independently represents C1-C8 alkyl, C5-C10 cycloalkyl, C6-C10 aryl or C5-C10 heteroaryl, or R 1 And R is 2 And each heteroatom in the C5-C10 heterocyclyl or C5-C10 carbocyclyl, C5-C10 heterocyclyl and C5-C10 heteroaryl is independently selected from at least one of nitrogen, oxygen and sulfur, aryl in the C6-C10 aryl, heteroaryl in the C5-C10 heteroaryl, carbocyclyl in the C5-C10 carbocyclyl or heterocyclyl in the C5-C10 heterocyclyl is independently unsubstituted or substituted with at least one of halogen, alkoxy or alkyl, and has at least one chiral center.
Further, the ketone racemate is
Further, the reaction system for reducing and resolving ketone racemate by carbonyl reductase also comprises a coenzyme or a coenzyme regeneration system, wherein the coenzyme is one or more selected from the group consisting of NADP, NAD, NADPH or NADH.
The invention utilizes a method of combining saturation mutation to improve carbonyl reductase from lactobacillus brevis (Lactobacillus brevis, lbCR) to obtain carbonyl reductase mutant with high catalytic efficiency and high selectivity. In the synthesis of the partial carbonyl reductase mutant, the consumption of the enzyme is reduced to 0.01-0.03 wt%, and the reaction volume is reduced to 10-15V (the volume multiple of the reaction solvent (1 kg of the specified material corresponds to 1L of the solvent, namely 1V), so that the industrial production cost of the compound is greatly reduced, and the enzyme has better application value in industrial production.
Detailed Description
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
According to the invention, carbonyl reductase derived from lactobacillus brevis (Lactobacillus brevis), candida glabra (Candida glabra), arthrobacter (Arthrobacter), saccharomyces cerevisiae (Saccharomyces cerevisiae), anaerobic bacillus (Thermoanaerobacter brockii) and lactobacillus (Lentilactobacillus kefiri) is screened, so that the carbonyl reductase derived from lactobacillus brevis (Lactobacillus brevis) has relatively good resolution effect, but the catalytic efficiency and selectivity of the carbonyl reductase are still required to be further improved. Therefore, the invention uses lactobacillus brevis (Lactobacillus brevis, with the amino acid sequence shown in SEQ ID NO: 1) as the starting gene of the invention to carry out further genetic engineering, thus obtaining a series of carbonyl reductase mutants with higher catalytic efficiency and selectivity.
According to an exemplary embodiment of the present invention, a carbonyl reductase mutant is provided. The amino acid sequence of the carbonyl reductase mutant is SEQ ID NO:1, and the mutated amino acid site comprises: s65; or the amino acid sequence of the carbonyl reductase mutant has a mutated amino acid site and is identical to the amino acid sequence of SEQ ID NO:1 has 90%, 95% or more than 99% homology, and has carbonyl reductase catalytic activity. The carbonyl reductase mutant provided by the invention can be used for well resolving raceme ketone, has high selectivity and yield for obtaining ketone with specific configuration, and is suitable for industrial production.
The term "homology" as used herein has a meaning generally known in the art, and the rules and standards for determining homology between different sequences are also well known to those skilled in the art. The sequences defined according to the invention by different degrees of homology must also have an improved tolerance of the transaminases to organic solvents. In the above embodiments, it is preferred that the amino acid sequence of the transaminase mutant has the above homology and has or encodes an amino acid sequence with improved tolerance to organic solvents. Such variant sequences may be obtained by those skilled in the art in light of the present disclosure.
Preferably, S65 is mutated to S65A, S G or S65D. More preferably, the mutation is one of the following combinations of mutation sites: s65A+I V, S Gv+I78Q, S D+I78V, S65A+A201Q, S65A+I78V+M154I, S A+I78V+A201D, S A+I78V+A201Q, S A+P153 S+V36I, S A+I78V+M154I+A201Q, S A+I78V+P153 S+V+ I, S A+I78V+M438I+A202 427A+I78V+D182 A+I Q, S A+I78V+D194A+V I, S5A+P153 S+V360I+A202 5265A+I78V+M431I+A200A200Q+A200L, S65 A+I. 78v+p153s+v198i+a201D, S a+i78v+s145e+p153s+d194A, S a+i78v+m168i+a155 d+a65D, S a+i78v+m168i+v198 i+a1200q+a202L, S a+i78v+m168i+v360i+a201 D+A202L, S65 A+I78V+M1200I+A155 D+V198 I+A1209D+A202L, S A+I78V+M1200I+A155 D+V198 I+A120Q+A217L or S65A+I78V+G94T+M120I+A397D+V198 I+A120Q+A202L.
According to an exemplary embodiment of the present invention, a DNA molecule is provided. The DNA molecule encodes any of the carbonyl reductase mutants described above. The carbonyl reductase mutant coded by the DNA molecule has higher catalytic activity and selectivity.
The above-described DNA molecules of the invention may also be present in the form of "expression cassettes". "expression cassette" refers to a linear or circular nucleic acid molecule that encompasses DNA and RNA sequences capable of directing expression of a particular nucleotide sequence in an appropriate host cell. Generally, a promoter operably linked to a nucleotide of interest is included, optionally operably linked to a termination signal and/or other regulatory elements. The expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region typically encodes a protein of interest, but also encodes a functional RNA of interest, e.g., antisense RNA or nontranslated RNA, in sense or antisense orientation. The expression cassette comprising the polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component thereof. The expression cassette may also be naturally occurring, but obtained in an efficient recombinant formation for heterologous expression.
According to an exemplary embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the DNA molecules described above. The DNA molecule in the recombinant plasmid is placed at a proper position of the recombinant plasmid, so that the DNA molecule can be correctly and smoothly copied, transcribed or expressed.
Although the term "comprising" is used herein to define the DNA molecule, it is not intended that other sequences not functionally related thereto may be added at any one of the two ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombinant manipulation, it is necessary to add appropriate restriction sites for restriction enzymes at both ends of the DNA sequence, or additionally to add start codons, stop codons, etc., and thus these cases will not be truly covered if defined by a closed expression.
As used herein, the term "plasmid" includes any plasmid, cosmid, phage, or Agrobacterium binary nucleic acid molecule, preferably a recombinant expression plasmid, either prokaryotic or eukaryotic, in certain embodiments, pET-21b (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b, pET-15b (+), pET-16b (+), pET-17b, pET-19b (+), pET-20b (+), pET-21a, pET-23a (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a, pET-29a, pET-30a, pET-31b, pET-32 b, pET-41b, pET-40b, pET-41b pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pPIC9k, pGAPZalpha A, pUC-18 or pUC-19.
According to an exemplary embodiment of the present invention, a host cell is provided, which comprises any of the recombinant plasmids described above. Host cells suitable for use in the present invention include, but are not limited to, prokaryotic cells or eukaryotic cells. Preferably, the prokaryotic cell is E.coli DH5 alpha, top10, BL21-DE3 or E.coli Rosetta-DE3 cell; eukaryotic cells are yeast cells.
According to an exemplary embodiment of the present invention, a method for producing chiral ketones is provided. The method comprises the step of adopting carbonyl reductase to reduce and split ketone racemate, wherein the carbonyl reductase is any one of carbonyl reductase mutants.
Further, the ketone racemate isWherein R1 and R2 each independently represent a C1-C8 alkyl group, a C5-C10 cycloalkyl group, a C6-C10 aryl group, or a C5-C10 heteroaryl group, or R1 and R2 together with the carbon on the carbonyl group form a C5-C10 heterocyclic group or a C5-C10 carbocyclic group, the heteroatoms of the C5-C10 heterocyclic group are each independently selected from at least one of nitrogen, oxygen, and sulfur, the aryl group of the C6-C10 aryl group, the heteroaryl group of the C5-C10 heteroaryl group, the carbocyclyl group of the C5-C10 carbocyclyl group, or the heterocyclic group of the C5-C10 heterocyclic group are each independently unsubstituted or substituted with at least one of halogen, alkoxy, or alkyl, and have at least one chiral center. Preferably, the ketone racemate is
According to an exemplary embodiment of the present invention, the reaction system for reducing and resolving the ketone racemate using carbonyl reductase further includes a coenzyme, and the coenzyme or the coenzyme regeneration system is one or more selected from the group consisting of NADP, NAD, NADPH and NADH.
The advantageous effects of the present invention will be further described below with reference to examples.
Example 1
Carbonyl reductase screening
The screening method provided by the invention is used for screening carbonyl reductase from lactobacillus brevis (Lactobacillus brevis), candida glabra, arthrobacter (Arthrobacter), saccharomyces cerevisiae (Saccharomyces cerevisiae), anaerobic bacillus (Thermoanaerobacter brockii) and lactobacillus (Lentilactobacillus kefiri), the screening steps are as follows, and the screening data are shown in table 1.
Screening substrates and procedures:
to a 4mL centrifuge tube, water (2 mL), disodium hydrogen phosphate (40 mg), sodium dihydrogen phosphate (12 mg), substrate (0.1 g) were added sequentially, glucose (0.2 g), NADP (1 mg), GDH (2 mg) were added sequentially after stirring uniformly, and finally dry cell (2 mg) was added. The reaction was controlled at 35℃and the ee and conversion of the ketone substrate were monitored during the reaction.
Table 1: screening results of carbonyl reductase from different strains
As can be seen from Table 1, strains derived from Lactobacillus brevis (Lactobacillus brevis), candida glabra and Arthrobacter have certain resolution effects on substrate ketones, lactobacillus brevis (Lactobacillus brevis) has relatively good resolution effects, but E is only 10.9, and the activity and resolution selectivity of the strain are greatly improved, so that Lactobacillus brevis (Lactobacillus brevis) (having the amino acid sequence shown in SEQ ID NO: 1) is used as a starting gene of the invention.
Example 2
Construction of Lactobacillus brevis (Lactobacillus brevis) mutant library
Selecting 17 points of non-conserved residues near a substrate binding pocket of lactobacillus brevis (Lactobacillus brevis) to carry out combination saturation mutation, adopting degenerate codon NNK to design mutation primers, and carrying out semi-rational design by adopting a single-point saturation mutation binding iterative combination mutation technology to improve the catalytic activity and selectivity of enzyme. The complete linear fragment is obtained by full plasmid PCR, the PCR product is digested by DpnI to remove the mother template of the original gene, then is transformed into escherichia coli BL21 (DE 3), is coated in an LB culture dish containing 50 mug/mL kanamycin, is cultured overnight at 37 ℃, is subjected to high-throughput screening by a microplate reader after 96-well plate induced expression, and is selected to have higher activity than the mother parent, and the mutation site is determined by sequencing of the re-gene.
The specific operation flow is as follows: PCR was performed using pET28a-LbCR as template and the high fidelity polymerase PrimeSTAR. The PCR conditions were as follows: to a PCR reaction system having a total volume of 20. Mu.L, 0.5 to 20ng of a template, 10. Mu.L of 2X PrimeSTAR (Premix), and 0.4. Mu.L (10. Mu.M) of each of a pair of mutation primers were added, and sterile distilled water was added to 20. Mu.L. PCR reaction procedure: (1) denaturation at 98℃for 10sec, (2) annealing at 55℃for 30sec, (3) elongation at 72℃for 6min, and 30 cycles of steps (1) to (3) were carried out in total, and the product was stored at 4 ℃. The PCR product was verified by agarose gel electrophoresis analysis and digested with DpnI at 37℃for 1 hour. The digestions were transferred to E.coli BL21 (DE 3) competent cells and plated onto plates containing kana antibiotics and placed in a 37℃incubator for approximately 12h of stationary culture. The obtained monoclonal colony is picked into a 96-well deep pore plate for culture, and is subjected to shaking culture at 37 ℃ until OD 600 At=0.6, IPTG was added to a final concentration of 0.2mM and induced expression was performed at 25 ℃ overnight. The supernatant medium was removed by centrifugation in 96-well plates, 200. Mu.L of enzymatic solution (lysozyme 2mg/mL, polymyxin 1mg/mL, pH=7.0) was added to each well, and the mixture was broken for 2 hours at 37 ℃. Centrifuging the cell disruption solution after enzymolysis at 4000rpm for 10min, and collectingThe supernatant yielded a crude enzyme solution. The expressed proteins were subjected to a high throughput viability screen of the microplate reader according to the system shown in table 2.
Table 2: high-flux activity screening reaction system of enzyme-labeled instrument
System of Addition amount of
Ketone substrate (5 mg/mL) 20μL
NADP(1mg/mL) 50μL
0.1M PBS buffer at pH7.0 80μL
Enzyme solution 50μL
Mixing the other components except enzyme solution in 96 shallow holes according to the system shown in the table 3, adding 50 mu L of the prepared mutant enzyme solution into each hole rapidly by a gun, detecting at 340nm of an enzyme marker, recording the light absorption value every 10s, monitoring for 2min, observing the slope change, screening out mutant strains with higher activity by comparing with the enzyme activity of wild carbonyl reductase, carrying out rescreening and gene sequencing, and then carrying out next-wheel combination mutation.
Table 3 provides a list of carbonyl reductase LbCR mutants of the specific sequences disclosed herein having related activities. In the following table, the sequence numbers refer to a series of sequences at the back of Table 3 respectively, and in the activity list, a plus sign "+" indicates that the specific activity of the mutant protein is improved by 0.1-1 times compared with that of the protein consisting of the amino acid sequence shown by SEQ ID NO.1 in the sequence table; two plus signs "++" indicate that the specific activity of the mutant protein is improved by 1 to 2 times compared with the protein composed of the amino acid sequence shown in SEQ ID NO.1, three plus signs "++" indicate that the specific activity of the mutant protein is improved by 2 to 3 times compared with the protein composed of the amino acid sequence shown in SEQ ID NO.1, and four plus signs "++ + ++" indicate that the specific activity of the mutant protein is improved by 3 to 4 times compared with the protein composed of the amino acid sequence shown in SEQ ID NO. 1.
Table 3: carbonyl reductase LbCR mutant sequences and corresponding lists of activity improvements
Example 3
Selective comparison of LbCR mutants
8 carbonyl reductases with mutant numbers M1, M12, M13, M18, M19, M21, M22 and M23 induced to express were used for screening of N- (3-oxoalkoxyxyl) carbamate reactions using the following reaction system: 0.02g of ketone substrate is dissolved in 0.1mL of DMSO and evenly mixed, 40mg of glucose and 0.9mL 0.1M pH7.0PBS,0.4mg GDH,0.2mg NADP are reacted for 1 hour at 35 ℃ at 200rpm, the conversion rate and the ee value are shown in Table 4, and the M23 mutant shows good activity and selectivity, and E reaches 46.9.
Table 4: results of asymmetric reduction reactions of different mutants of LbCR
Mutant numbering Feeding amount Enzyme amount Reaction system Reaction time Conversion rate Ketone ee E
M1 0.02g 0.2wt 50V 1h 15％ 10％ /
M12 0.02g 0.2wt 50V 1h 28％ 31％ 11.9
M13 0.02g 0.2wt 50V 1h 26％ 31％ 21.6
M18 0.02g 0.2wt 50V 1h 34％ 45％ 23
M19 0.02g 0.2wt 50V 1h 36％ 48％ 20
M21 0.02g 0.2wt 50V 1h 53％ 94％ 38.6
M22 0.02g 0.2wt 50V 1h 54％ 95％ 34.7
M23 0.02g 0.2wt 50V 1h 56％ 99.5％ 46.9
Example 4
Carbonyl reductase LbCR M23 Induction of expression of mutants
The mutant E.coli BL21 (DE 3)/pET 28a-LbCR obtained in example 2 was used M23 Inoculating to LB medium containing 50 μg/ml kanamycin, shaking culture at 37deg.C for 12 hr, inoculating to 5L triangular flask containing 2L LB medium according to 1% (V/V) inoculum size, shaking culture at 37deg.C and 180rpm, adding IPTG to final concentration of 0.5mmol/L when OD600 of the culture solution reaches 0.6-0.8, inducing, centrifuging at 8000rpm after overnight induction at 16deg.C, collecting 10g wet cells, and performing downstream enzyme catalytic reaction using the wet cells.
Example 5
Resolution of racemate tert-butyl (3-oxo-cyclohexylamine, compound 1) preparation of tert-butyl (R) - (3-oxo-cyclohexylamine, compound 2) and tert-butyl (1S, 3S) -3-hydroxy-3-hydroxycyclohexylamine, compound 3).
To a four-necked flask, water (450 mL), disodium hydrogen phosphate (9.6 g), sodium dihydrogen phosphate (2.8 g), butyl N- (3-oxoworkbench) carbamate (30 g), and diluted hydrochloric acid or diluted sodium hydroxide were added in this order, followed by stirring, and ph=7.0 was adjusted. After stirring well, glucose (34 g), NADP (150 mg), GDH (300 mg) and LbCR were added sequentially M27 Wet cell (1.5 g). The reaction was carried out at 35℃and pH=7.0, the ee of the ketone substrate was monitored during the reaction, the conversion rate of 3h was 56%, the ee was 100%, and the reaction was completed. The reaction mixture was filtered through 100mL pad celite, the filtrates combined and separated, the aqueous phase extracted with EA (50 mL x 2), the organic phases combined, 50mL of water added, 15g of sodium bisulphite added, stirred at 25 ℃ for 3h at room temperature, a large amount of white solid precipitated, filtered, the filter cake slurried with EA (50 mL x 2), filtered and the filtrate (containing compound 3) and filter cake collected separately. Wherein 50mL of EA and 50mL of water are added into the filter cake, 15g of sodium carbonate is added, and the mixture is stirred for 3 hours at the room temperature of 25 ℃; after the solution was separated, the aqueous phase was extracted with EA (50 ml x 2), the organic phases were combined and concentrated to dryness to give 12g of a pale yellow solid (compound 2) in 40% yield, 100% ee, 97% gas phase purity, and ESI mass spectrum molecular weight 213.14.
The filtrate (containing the compound 3) is concentrated to dryness to obtain 18g of crude compound 3, the content of which is 90 percent and the gas phase purity of which is 97 percent. Further purification gave 11g of pure compound 3, 99% in gas phase purity, 99% ee100% and ESI mass spectrum molecular weight 215.15.
Example 6
Resolution of racemate tert-butyl (3-oxycycloxyl) carbamate (compound 1) preparation tert-butyl (R) - (3-oxycycloxyl) carbamate (compound 2) and tert-butyl ((1 s,3 s) -3-hydroxycycloxyl) carbamate (compound 3) kg-scale production.
High-density fermentation in 5L fermenter to prepare mutant LbCR M23 500g of wet cells were prepared for later use.
2130g of a ketone substrate (compound 1) is added into a 50L cold and hot integrated glass kettle, the rotation speed is 150rpm, 32L of 0.1MPBS (688.0 g of disodium hydrogen phosphate dodecahydrate and 200.0g of sodium dihydrogen phosphate dihydrate are added into water to prepare 32L of aqueous solution), 2378g of glucose, 10.65g of NADP and 21.30g of GDH are added, wet thalli are added, a titrator is added dropwise into 3M potassium carbonate aqueous solution to control pH=7.0, the reaction is carried out at 35 ℃ for 3 hours, sampling GC detects conversion of 56.20%, normal phase monitoring ee value is 100%, diatomite 400.0g is added into the reaction solution, ethyl acetate 10L is added, the temperature is raised to 70 ℃ and stirred for 1 hour to inactivate enzymes, diatomite is filled for filtration after the temperature is lowered to 30 ℃, filtrate is combined and separated, aqueous phase is extracted by EA (5L) and organic phase is combined; adding water 5L, adding sodium bisulphite 1040g, stirring at room temperature 25 ℃ for 3 hours, precipitating a large amount of white solid, filtering, pulping a filter cake with EA (10L x 2), filtering, and respectively collecting filtrate (containing a compound 3) and a filter cake; adding 10L of EA and 10L of water into the filter cake, adding 1060g of sodium carbonate, and stirring for 3h at the room temperature of 25 ℃; separating the solution after the solution is cleared, extracting the aqueous phase with EA (5 L.times.2), and combining the organic phases; the organic phase was concentrated to dryness to give 850.0g of a pale yellow solid (Compound 2), yield 40%, ee100% and gas phase purity 97%.
The filtrate (containing the compound 3) is concentrated to dryness to obtain 1300g of crude compound 3, the content is 90%, and the gas phase purity is 97%. 780g of pure compound 3, 99% of which was obtained as a gas phase purity and 100% of ee, was obtained by further purification treatment.
Examples 7 to 10
LbCR M23 Resolution of series of carbonyl compounds
To a four-necked flask, 15mL of a 0.1M buffer solution (pH 7.0) was sequentially added, and after stirring uniformly a series of carbonyl compounds (1 g), glucose (2 g), NADP (10 mg), GDH (10 mg) were sequentially added, and LbCR was finally added M23 Wet cell, control temperature 35 ℃, ph=7.0 reaction, monitoring conversion and ee of ketone substrate during process, data are shown in table 5, and the compounds of examples 8 and 9 can complete resolution well with 10% wet cell.
Table 5: lbCR (LbCr) M23 Resolution of series of carbonyl Compound results
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the carbonyl reductase mutant provided by the invention can be used for well resolving raceme ketone, has high selectivity and yield for obtaining ketone with specific configuration, and is suitable for industrial production.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Nanjing medical stone technology Co., ltd
<120> carbonyl reductase mutant and use thereof
<130> PN171276YSKJGF
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 249
<212> PRT
<213> Lactobacillus brevis
<400> 1
Met Thr Asp Arg Leu Lys Asp Lys Val Ala Ile Ile Thr Gly Gly Val
1 5 10 15
Ala Gly Ile Gly Leu Gly Ile Ala Glu Cys Tyr Val Arg Glu Gly Ala
20 25 30
Lys Val Val Val Thr Ala Asn His Asn Val Asp Gly Gly Arg Ala Ala
35 40 45
Val Ala Lys Phe Gly Asp Asp Val Ser Leu Phe Val Gln Gln Asp Val
50 55 60
Ser Lys Glu Ala Asp Trp Gln Lys Val Ile Asp Ala Thr Ile Ala Lys
65 70 75 80
Phe Gly Arg Val Asp Ile Leu Val Asn Asn Ala Gly Ile Gly Gly Val
85 90 95
Asn Thr Ala Ile Glu Asp Leu Asp Leu Ala Asp Trp Gln Lys Val Ile
100 105 110
Asp Val Asn Leu Thr Ala Asn Phe Leu Gly Glu Lys Ala Ala Ile Lys
115 120 125
Ala Met Lys Gln Thr Ala Asp Ala Lys Gly Ser Ile Ile Asn Val Ser
130 135 140
Ser Val Ala Gly Leu Val Gly Leu Pro Met Ala Pro Ala Tyr Ser Ala
145 150 155 160
Ser Lys Gly Gly Ser Arg Leu Leu Thr His Ala Thr Ala Leu Asn Leu
165 170 175
Ala Gln Arg Gly Ile Asp Ile Arg Val Asn Ser Val His Pro Gly Trp
180 185 190
Ile Asp Thr Ser Ile Val Pro Glu Ala Ala Arg Lys Gln Ile Ile Ala
195 200 205
Thr Ile Pro Val Gly His Met Gly Gln Pro Gln Asp Ile Gly Glu Val
210 215 220
Cys Val Tyr Leu Gly Ser Asp Glu Ser Arg Phe Ala Asn Gly Ala Glu
225 230 235 240
Phe Thr Val Asp Gly Gly Gln Arg Ala
245
<210> 2
<211> 750
<212> DNA
<213> Lactobacillus brevis
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atgaccgatc gcctgaaaga taaagtggcc attattaccg gtggtgtggc aggtattggt 60
ctgggcattg ccgaatgcta tgtgcgtgaa ggcgccaaag ttgttgtgac cgccaatcat 120
aatgttgatg gcggccgcgc cgccgttgca aaattcggtg atgatgttag tctgttcgtt 180
cagcaggatg ttagtaaaga agcagattgg cagaaagtga ttgatgccac cattgccaaa 240
ttcggtcgtg tggatattct ggttaataat gccggcattg gtggtgtgaa taccgcaatt 300
gaagatctgg atctggccga ttggcagaag gtgattgatg ttaatctgac cgcaaacttc 360
ctgggcgaaa aagcagccat taaagcaatg aaacagaccg ccgatgccaa aggtagtatt 420
attaatgtta gcagcgtggc cggtctggtg ggtctgccga tggcaccggc atatagcgcc 480
agtaaaggtg gtagtcgcct gctgacccat gcaaccgcac tgaatctggc ccagcgtggt 540
attgatattc gcgttaatag tgttcatccg ggctggattg ataccagtat tgtaccggaa 600
gcagctcgca aacagattat tgccaccatt ccggttggtc acatgggtca gccgcaggat 660
attggtgaag tgtgtgtgta tctgggcagc gatgaaagcc gcttcgccaa tggtgcagag 720
ttcaccgtgg atggcggcca gcgcgcataa 750

Claims (13)

1. A carbonyl reductase mutant, wherein the amino acid sequence of the carbonyl reductase mutant is SEQ ID NO:1, the mutated amino acid position comprising: s65; or the amino acid sequence of the carbonyl reductase mutant has the mutated amino acid site and is identical to the amino acid sequence of SEQ ID NO:1 has more than 90 percent of homology and has the catalytic activity of carbonyl reductase.
2. The carbonyl reductase mutant of claim 1, wherein the S65 mutation is S65A, S G or S65D.
3. The carbonyl reductase mutant of claim 1, wherein the mutation is one of the following combinations of mutation sites: s65A+I V, S Gv+I78Q, S D+I78V, S65A+A201Q, S65A+I78V+M154I, S A+I78V+A201D, S A+I78V+A201Q, S A+P153 S+V36I, S A+I78V+M154I+A201Q, S A+I78V+P153 S+V+ I, S A+I78V+M438I+A202 427A+I78V+D182 A+I Q, S A+I78V+D194A+V I, S5A+P153 S+V360I+A202 5265A+I78V+M431I+A200A200Q+A200L, S65 A+I. 78v+p153s+v198i+a201D, S a+i78v+s145e+p153s+d194A, S a+i78v+m168i+a155 d+a65D, S a+i78v+m168i+v198 i+a1200q+a202L, S a+i78v+m168i+v360i+a201 D+A202L, S65 A+I78V+M1200I+A155 D+V198 I+A1209D+A202L, S A+I78V+M1200I+A155 D+V198 I+A120Q+A217L or S65A+I78V+G94T+M120I+A397D+V198 I+A120Q+A202L.
4. A DNA molecule encoding the carbonyl reductase mutant of any one of claims 1 to 3.
5. A recombinant plasmid, wherein the recombinant plasmid is linked to the DNA molecule of claim 4.
6. The recombinant plasmid according to claim 5, wherein, the recombinant plasmid is pET-21b (+), pET-22b (+), pET-3a (+), pET-3d (+), pET-11a (+), pET-12a (+), pET-14b, pET-15b (+), pET-16b (+), pET-17b (+), pET-19b (+), pET-20b (+), pET-21a (+), pET-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), and the recombinant plasmid is a recombinant plasmid pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-3835-C, pGEX-5X-1, pGEX-6p-1, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pPIC9k, pGAPZ alpha A, pUC-18 or pUC-19.
7. A host cell comprising the recombinant plasmid of claim 5 or 6.
8. The host cell of claim 7, wherein the host cell is a prokaryotic cell or a eukaryotic cell.
9. The host cell of claim 8, wherein the prokaryotic cell is an e.coli DH 5a, top10, BL21-DE3 or e.coli Rosetta-DE3 cell; the eukaryotic cell is a yeast cell.
10. A method for producing chiral ketones comprising the step of reductive resolution of ketone racemates using a carbonyl reductase enzyme, characterized in that the carbonyl reductase enzyme is a carbonyl reductase mutant according to any one of claims 1 to 3.
11. The method of claim 10, wherein the ketone racemate isWherein R is 1 And R is 2 Each independently represents C1-C8 alkyl, C5-C10 cycloalkyl, C6-C10 aryl or C5-C10 heteroaryl, or R 1 And R is 2 And carbon on the carbonyl group forms a C5-C10 heterocyclyl or a C5-C10 carbocyclyl, the heteroatoms in the C5-C10 heterocyclyl and the C5-C10 heteroaryl are each independently selected from at least one of nitrogen, oxygen and sulfur, the aryl in the C6-C10 aryl, the heteroaryl in the C5-C10 heteroaryl, the carbocyclyl in the C5-C10 carbocyclyl or the heterocyclyl in the C5-C10 heterocyclyl are each independently unsubstituted or substituted with at least one of halogen, alkoxy or alkyl, and have an outer ring with at least one chiral centerAnd (3) racemes.
12. The method of claim 11, wherein the ketone racemate is
13. The method according to claim 10, wherein the reaction system for the reduction and resolution of the ketone racemate by carbonyl reductase further comprises a coenzyme or a coenzyme regeneration system, wherein the coenzyme is one or more selected from the group consisting of NADP, NAD, NADPH and NADH.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117126823A (en) * 2023-09-01 2023-11-28 华南理工大学 Ketone reductase mutant and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117126823A (en) * 2023-09-01 2023-11-28 华南理工大学 Ketone reductase mutant and application thereof
CN117126823B (en) * 2023-09-01 2024-03-29 华南理工大学 Ketone reductase mutant and application thereof

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