CN102618513B - Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound - Google Patents
Carbonyl reductase, gene and mutant and application thereof to asymmetrical reduced carbonyl compound Download PDFInfo
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- CN102618513B CN102618513B CN201210136823.8A CN201210136823A CN102618513B CN 102618513 B CN102618513 B CN 102618513B CN 201210136823 A CN201210136823 A CN 201210136823A CN 102618513 B CN102618513 B CN 102618513B
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Abstract
The invention discloses a novel carbonyl reductase, a gene, a mutant thereof, a recombinant expression vector containing the gene and the mutant, a recombinant expression transformant, a recombinase preparation method, and applications of the carbonyl reductase and recombinase to preparation of active chiral alcohols with a chiral carbonyl compound before asymmetrical reduction. The carbonyl reductase is derived from candida glabrata, is applied to preparation of a plurality of optically-active chiral alcohols such as (R)-chloromandelic acid methyl ester, (R)-2-hydroxy-4-phenyl ethyl butyrate, (R)-4-chlorin-3-phenyl ethyl butyrate and the like. Compared with other preparation methods, a product prepared through the method has high concentration, does not require additionally or slightly adding any expensive coenzyme, has high optical purity, and has the advantages of mild reaction conditions, easiness and convenience for operating, easiness for amplifying and the like, and has a good industrial application prospect in the production of clopidogrel, L-carnitine and perindopril antihypertensive medicinal intermediates.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to carbonyl reductase, a gene and a mutant thereof, a recombinant expression vector and a recombinant expression transformant containing the gene, recombinase of the carbonyl reductase, a preparation method of the recombinase, and application of the carbonyl reductase or the recombinase of the carbonyl reductase as a catalyst in asymmetric reduction of a chiral carbonyl compound to prepare optically active chiral alcohol.
Background
Chiral alcohols are important intermediates for many marketable drugs and chiral chemicals. For example, methyl (R) -o-chloromandelate (formula o-Cl-C)6H4CH(OH)COOCH3Molecular weight 200.62, CAS number: 32345-59-8) is an important chiral intermediate for synthesizing clopidogrel, a platelet aggregation inhibitor. Clopidogrel is an Adenosine Diphosphate (ADP) receptor blocker, can be combined with an ADP receptor on the surface of a platelet membrane to enable fibrinogen to be generatedCan not be combined with glycoprotein GP IIb/IIIa receptor, thereby inhibiting mutual aggregation of platelets, is mainly used for treating acute myocardial infarction and has wide market in China. (R) -2-hydroxy-4-phenylbutyric acid ethyl ester (molecular formula C)6H5(CH2)2CH(OH)COOCH2CH3Molecular weight 208.25, CAS number: 90315-82-5) is an important chiral building block for synthesizing various Angiotensin Converting Enzyme Inhibitor (ACEI) pril drugs, such as benazepril, cilazapril, and the like. The medicine is mainly used for treating diseases such as hypertension, congestive heart failure and the like. (R) -4-chloro-3-hydroxybutyric acid ethyl ester (molecular formula is ClCH)2CH(OH)CH2COOCH2CH3Molecular weight 166.60, CAS number: 90866-33-4) is an important chiral intermediate of L-carnitine. L-carnitine is an amino acid-like substance which promotes fat to be converted into energy, and the administration of the L-carnitine can reduce body fat and body weight without reducing water and muscles, and is considered as the safest weight-losing nutritional supplement without side effects by the international obesity health organization in 2003. Therefore, the research on the chiral synthesis preparation of the optical pure chiral alcohol has wide application prospect.
The synthesis of optically active chiral alcohols is carried out by two methods, chemical and biological. Compared with chemical methods, biological methods have the advantages of mild reaction conditions, high conversion rate, high enantioselectivity and the like, and particularly, the application of the biocatalytic asymmetric carbonyl reduction reaction in asymmetric synthesis of chiral alcohol is more and more emphasized.
The biological asymmetric synthesis method of (R) -o-chloromandelic acid or the derivative ester thereof mainly comprises nitrilase-catalyzed asymmetric hydrolysis of o-chloromandelic nitrile and carbonyl reductase-catalyzed asymmetric reduction of o-chlorobenzoyl formate. Zhang Shi Jun et al prepared (R) -o-chloromandelic acid by dynamic kinetic hydrolytic resolution of racemic o-chloromandelic acid in a water-toluene two-phase system using nitrilase derived from Alcaligenes sp.ECU0401, with a substrate concentration of 200mM, an enantiomeric excess (ee) of the product of 90.4%, and a yield of 76.5% (J.Biotechnol.2011, 152, 24-29). Ema catalyzing o-chlorine with Saccharomyces cerevisiae-derived carbonyl reductase Gre2pThe methyl benzoylformate is asymmetrically reduced to prepare (R) -methyl o-chloromandelate, the concentration of a substrate is 200g/L, the ee value of the product is higher than 99%, and the separation yield is 89% (adv. Synth. Catal.2008, 350, 2039-plus 2044). To achieve high yields, the reaction was supplemented with 1g/L additional NADP+Coenzyme, the price of coenzyme is expensive, making the reaction unsatisfactory. The preparation of (R) -2-hydroxy-4-phenyl ethyl butyrate is generally a method for catalyzing asymmetric reduction of 2-carbonyl-4-phenyl ethyl butyrate by using cells of wild bacteria or recombinant engineering bacteria or free enzymes. In the reported application, the highest level of substrate concentration was 400mM (82g/L), the catalyst used was pre-treated Baker's yeast (Baker' syeast), but only 41.9% of the substrate was converted to ethyl (R) -2-hydroxy-4-phenylbutyrate after 48h of reaction, and the enantiomeric excess (ee) was only 87.5% (biocatal. biotransform. 2009, 27, 211-. In other reports, products with high optical purity can be obtained, but the product concentration is generally low, and the industrialization is not feasible. Zhang et al prepared ethyl (R) -2-hydroxy-4-phenylbutyrate by catalysis of Candida kluyveri (Candida krusei SW2026), the ee value reached 99.7% at a substrate concentration of 2.5g/L, and the isolation yield was 95.1%. However, when the substrate concentration was increased to 20g/L, the ee value was reduced to 97.4% (Process Biochemistry 2009, 44, 1270-. Lavandera et al, catalyzed 5g/L of ethyl 2-carbonyl-4-phenylbutyrate by recombinant alcohol dehydrogenase to prepare ethyl (R) -2-hydroxy-4-phenylbutyrate through reduction, the ee value of the product is higher than 99%, but 1mM NADH needs to be exogenously added in the reaction, and the cost is greatly increased (ChemSusChem 2008, 1, 431-one 436). Linvingqing and the like catalyze asymmetric reduction of 2-carbonyl-4-phenylbutyric acid ethyl ester in an aqueous phase and an aqueous/organic two-phase system by using screened strains such as Candida boidinii and the like, the ee value of a product is 84.9-98.9%, and the highest product concentration reported is only 50g/L (Chinese patent, publication No. CN 101314784A). The aldehyde reductase SSAR from the brown Sporobolomyces salmonicolor can catalyze the asymmetric reduction of 4-chloro-3-carbonyl ethyl butyrate to prepare (R) -4-chloro-3-hydroxy ethyl butyrate, the concentration of a substrate can reach 300g/L, but the ee value is 91.7%, and the preparation is not satisfactory. Thus for the three substrates mentioned aboveThe carbonyl reductase is sought, the asymmetric reduction with high stereoselectivity can be catalyzed under the condition of high substrate concentration, no coenzyme or a small amount of coenzyme is required to be added in the reaction process, and the application prospect is wide undoubtedly.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a carbonyl reductase with high catalytic activity, strong enantioselectivity and good substrate tolerance, a gene of the carbonyl reductase, a recombinant expression vector containing the gene, a recombinant expression transformant and a high-efficiency preparation method thereof, and application of the carbonyl reductase in catalyzing asymmetric reduction of a carbonyl substrate, aiming at the problems that enzymes with different sources are required in the reported reactions for preparing (R) -methyl o-chloromandelate, (R) -ethyl 2-hydroxy-4-phenylbutyrate and (R) -ethyl 4-chloro-3-hydroxybutyrate through biocatalytic asymmetric reduction, the enzyme catalytic activity is low, the product concentration is low, the optical purity is not high, expensive coenzyme is additionally added and the like.
The invention solves the technical problems through the following technical scheme:
one of the technical schemes adopted by the invention is as follows: an isolated protein which is a protein of (a) or (b) below:
(a) a protein consisting of an amino acid sequence shown by SEQ ID No.2 in a sequence table;
(b) a protein derived from (a) and having carbonyl reductase activity, which is at least 62% identical to the amino acid sequence of (a), by substituting, deleting or adding one or several amino acids in the amino acid sequence of (a).
The protein consisting of the amino acid sequence shown in SEQ ID No.2 is derived from Candida glabrata (Candida glabrata) CGMCC 2.234, has the function of carbonyl reductase, and is a novel carbonyl reductase.
The protein consisting of the amino acid sequence shown in SEQ ID No.2 can be obtained by separating from Candida glabrata (CGMCC 2.234), can also be obtained by separating from an expression transformant for recombinant expression of the protein, and can also be obtained by artificial synthesis.
The homology (i.e., amino acid sequence similarity) of the carbonyl reductase represented by SEQ ID No.2 to other known carbonyl reductases is shown in Table 1. The homology of the carbonyl reductase shown in the amino acid sequence of SEQ ID No.2 and the amino acid sequence of the known carbonyl reductase is lower than 62%, and the carbonyl reductase has obvious difference.
TABLE 1 homology of the novel carbonyl reductases of the invention to other known carbonyl reductases
The protein (b) is a protein which is derived from (a) by substituting, deleting or adding one or several amino acids in the amino acid sequence of (a) and has carbonyl reductase activity and which is at least 62% identical to the amino acid sequence of (a). Wherein the number of "several" means two to less than 100, more preferably less than 30. Such as a fusion protein to which an exocrine signal peptide has been added, it has been found by the present invention that such a fusion protein also has carbonyl reductase activity. As another example, according to the present invention, the protein (b) can be obtained by mutating 1 to 5 amino acid residues in the protein (a) molecule having the amino acid sequence shown in SEQ ID No.2, while still maintaining the carbonyl reductase activity. That is, the object of the present invention can be achieved if the protein derived from (a) has carbonyl reductase activity, is derived in the manner described above, and has 62% or more homology with the protein (a). Wherein the homology between the protein and the protein (a) is 62% or more, preferably 90% or more, and most preferably 98.6% or more.
On the basis of screening to obtain high-activity stereoselective enzyme, we have carried out protein modification on wild type enzyme to direct active center and activityAmino acid residues at some sites in the hydrophobic channel of the sexual center are mutated into other amino acid residues so as to further enhance the catalytic performance of the enzyme and improve the expression of the enzyme. The active center is defined as about the vicinity of the substrate binding site
The spherical space of (a).
Preferably, the protein (b) is a protein derived from (a) by substituting one amino acid for one or more of the amino acids at positions 85, 92, 94, 99 and 174 in the amino acid sequence of (a) and having carbonyl reductase activity.
Preferably, the valine at position 85 of the amino acid sequence in (a) is substituted by isoleucine to obtain the mutant protein CgKR1V85I, or the glycine at position 174 is substituted by alanine to obtain the mutant protein CgKR1G174A, and the thermal stability of the mutant protein CgKR1G174A is improved to a certain extent.
Preferably, the phenylalanine at position 92 of the amino acid sequence in (a) is substituted by a nonpolar amino acid selected from alanine, valine, leucine, isoleucine and methionine, more preferably leucine, to obtain the mutein CgKR1F92L, which still has a certain carbonyl reductase activity.
Among them, the amino acid sequence of (a) preferably 94 th phenylalanine substitution is selected from alanine, valine, leucine, isoleucine or methionine nonpolar amino acid, preferably valine, mutant protein CgKR1F94V, its carbonyl reductase activity is improved to 7 times.
Preferably, the isoleucine at position 99 of the amino acid sequence in (a) is substituted by a polar amino acid selected from serine, cysteine, glutamine, asparagine or tyrosine, more preferably tyrosine, to obtain the derivative protein CgKR1I99Y, which has significantly improved soluble expression and thermostability.
Preferably, the amino acid sequence of (a) is substituted by leucine for phenylalanine 92 and valine for phenylalanine 94 to obtain the mutant protein CgKR1F92L/F94V, which has carbonyl reductase activity 12 times higher than that of the original protein and greatly improved thermal stability.
Preferably, the phenylalanine at position 92 of the amino acid sequence in (a) is substituted by leucine, the phenylalanine at position 94 is substituted by valine, the isoleucine at position 99 is substituted by tyrosine, and the glycine at position 174 is substituted by alanine, so that the mutant protein CgKR1F92L/F94V/I99Y/G174A is obtained, the carbonyl reductase activity of the mutant protein is improved to 12 times of the original activity, and the thermal stability of the mutant protein is greatly improved.
The second technical scheme adopted by the invention is as follows: an isolated nucleic acid which is a nucleic acid of the following (1) or (2):
(1) nucleic acid consisting of a nucleotide sequence shown as SEQ ID No.1 in a sequence table;
(2) a nucleic acid encoding the following protein (a) or (b):
(a) a protein consisting of an amino acid sequence shown by SEQ ID No.2 in a sequence table;
(b) a protein derived from (a) and having carbonyl reductase activity, which is at least 62% identical to the amino acid sequence of (a), by substituting, deleting or adding one or several amino acids in the amino acid sequence of (a).
The nucleic acid shown in SEQ ID No.1 is derived from Candida glabrata (Candidaglabrata) CGMCC 2.234. The nucleic acid shown in SEQ ID No.1 can be obtained by separating from Candida glabrata (Candida glabrata) CGMCC 2.234 genome, can be obtained by separating from a recombinant expression vector or a recombinant transformant containing the nucleic acid shown in SEQ ID No.1, and can also be obtained by whole gene artificial synthesis.
In the invention, the nucleotide sequence is as shown in SEQ ID No.1 in the sequence table, which is named as CgKR1 and has a total length of 1059 bp. Wherein the coding sequence (CDS) is from 1 st base to 1056 th base, the initiation codon is ATG, and the termination codon is TAA. The sequence has no intron, and the amino acid sequence of the encoded protein is shown as SEQ ID No.2 in the sequence table.
As known to the person skilled in the art, the nucleic acid sequence encoding the amino acid sequence of SEQ ID No.2 is not limited to SEQ ID No.1 only, due to the degeneracy of the codons. The nucleic acid sequence of the carbonyl reductase gene of the invention can also be any other nucleic acid sequence encoding the amino acid sequence shown by SEQ ID No.2 in the sequence list. In addition, a polynucleotide homologue can also be provided by appropriately introducing substitutions, deletions or insertions. A homologue of a polynucleotide of the present invention may be produced by substituting, deleting or adding one or more bases of the nucleic acid sequence of SEQ ID No.1 within a range that retains the activity of an enzyme.
Homologs of SEQ ID No.1 are also referred to as promoter variants. The promoter or signal sequence preceding the nucleic acid sequence may be altered by one or more nucleic acid substitutions, insertions or deletions without these alterations having a negative effect on the function of the promoter. Furthermore, the expression level of the target protein can be increased by changing the sequence of the promoter or even completely replacing it with a more efficient promoter from a different species of organism.
A homologue of SEQ ID No.1 also refers to a polynucleic acid having a base sequence which is capable of hybridizing under standard conditions with a polynucleic acid of the sequence shown in SEQ ID No. 1. Hybridization under standard conditions can be carried out according to the procedure described in "molecular cloning": cold Spring Harbor laboratory Press, a general protocol in Molecular Biology (Current Protocols in Molecular Biology). Specifically, hybridization can be carried out by hybridizing a membrane carrying the transcribed DNA or RNA molecule to be detected with a labeled probe in a hybridization buffer. The hybridization buffer comprises 0.1 wt% SDS, 5 wt% dextran sulfate, a dilution inhibitor of 1/20, and 2-8 XSSC. 20 XSSC is a solution of 3M sodium chloride and 0.3M citric acid. The hybridization temperature is 50-70 ℃. After incubation for several hours or overnight, the membranes were washed with washing buffer. The washing temperature is room temperature, more preferably the hybridization temperature. The composition of the washing buffer is 6 XSSC +0.1 wt% SDS solution, more preferably 5 XSSC +0.1 wt% SDS. When the membrane is washed with such a washing buffer, the DNA or RNA molecules can be recognized by the label on the probe hybridized within the DNA or RNA molecule.
The third technical scheme adopted by the invention is as follows: a recombinant expression vector comprising a nucleic acid sequence of the invention. The carbonyl reductase gene or the nucleic acid of the mutant thereof can be constructed by connecting the carbonyl reductase gene or the nucleic acid of the mutant to various expression vectors by a conventional method in the field. The expression vector may be any vector conventionally used in the art, such as a commercially available plasmid, cosmid, phage or viral vector, and the like, and preferably is plasmid pET28 a. Preferably, the recombinant expression vector of the present invention can be prepared by the following method: the nucleic acid product obtained by PCR amplification and expression vector pET28a are double-digested by restriction enzymes Nde I and BamH I respectively to form complementary cohesive ends, and the cohesive ends are connected by T4DNA ligase to form recombinant expression plasmid pET28a-CgKR1 or mutant expression plasmid thereof containing the carbonyl reductase gene of the invention.
The fourth technical scheme adopted by the invention is as follows: a recombinant expression transformant comprising the recombinant expression vector of the present invention. Can be produced by transforming the recombinant expression vector of the present invention into a host cell. The host cell may be a host cell which is conventional in the art, as long as it satisfies that the recombinant expression vector can stably self-replicate and that the carried reductase gene of the present invention can be efficiently expressed. Coli (e.coli) BL21(DE3) or e.coli (e.coli) DH5 α are preferred in the present invention. The preferred genetically engineered strain of the invention, i.e., E.coli (E.coli) BL21(DE3)/pET28a-CgKR1 or a mutant thereof, can be obtained by transforming the aforementioned recombinant expression plasmid pET28a-CgKR1 or a mutant thereof into E.coli (E.coli) BL21(DE 3). The transformation method can be selected from conventional methods in the field, such as an electric transformation method, a heat shock method and the like, preferably the heat shock method is selected for transformation, and the heat shock conditions are preferably as follows: the mixture was heat-shocked at 45 ℃ for 90 seconds.
The fifth technical scheme adopted by the invention is as follows: a preparation method of recombinant carbonyl reductase comprises the following steps: culturing the recombinant expression transformant of the present invention, and obtaining the recombinant carbonyl reductase from the culture.
Wherein, the recombinant expression transformant is obtained by transforming the recombinant expression vector of the present invention into a host cell, as described above. The medium used in said culturing of the recombinant expression transformant may be any medium that is conventional in the art and that allows the transformant to grow and produce the carbonyl reductase of the present invention, and for the E.coli strain, LB medium is preferred: 10g/L of peptone, 5g/L of yeast extract, 10g/L of NaCl and 7.0 of pH. The culture method and culture conditions are not particularly limited, and may be appropriately selected according to the ordinary knowledge in the art depending on the type of host, the culture method, and the like, as long as the transformant can grow and produce the carbonyl reductase of the present invention. Other specific procedures for culturing the transformant can be performed according to the routine procedures in the art. For the Escherichia coli strain, the following method is preferably used for producing the enzyme by shake flask culture fermentation: inoculating the recombinant Escherichia coli (preferably E.coli BL21(DE3)/pET28a-CgKR1 or its mutant) to LB culture medium containing kanamycin, and culturing when the optical density OD of the culture solution600When the concentration reaches 0.5 to 0.7 (preferably 0.6), isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.05 to 1.0mmol/L (preferably 0.1mmol/L) is added for induction, and the induction temperature is 10 to 30 ℃ (preferably 16 ℃), so that the recombinant carbonyl reductase can be efficiently expressed.
The catalyst for catalyzing the asymmetric reduction of the prochiral carbonyl compound to form the optically active chiral alcohol in the present invention may be a culture of a transformant of the recombinant carbonyl reductase produced as described above, or a transformant cell obtained by centrifuging a culture medium, or a product processed using the same. The term "processed product" as used herein means an extract obtained from a transformant, an isolated product obtained by isolating and/or purifying carbonyl reductase in the extract, or an immobilized product obtained by immobilizing cells of the transformant or the extract or the isolated product of the transformant.
The sixth technical scheme adopted by the invention is as follows: the invention relates to an application of protein in catalyzing a prochiral carbonyl compound to perform asymmetric reduction reaction to form chiral alcohol.
In the above application, the conditions of the asymmetric reduction reaction can be selected according to the conventional conditions of such reactions in the art, and are preferably as follows:
the protein is preferably the carbonyl reductase or the recombinant carbonyl reductase of the present invention. The prochiral carbonyl compound is preferably an alpha-ketoester, beta-ketoester or aryl ketone compound, and is a compound represented by formula 1, 2 or 3:
wherein,
R1is alkyl, phenyl or phenyl with substituent, and the substituent of the phenyl is halogen or alkyl;
R2is an alkyl group;
R3is alkyl or haloalkyl;
R4is a haloalkyl group.
Preferably, the first and second liquid crystal films are made of a polymer,
R1the alkyl group with a carbon chain length of 1-8, the phenyl group or the phenyl group with a substituent, wherein the substituent of the phenyl group is halogen or the alkyl group with a carbon chain length of 1-2;
R2is an alkyl group with a carbon chain length of 1-2;
R3is alkyl or halo with a carbon chain length of 1-2Alkyl, more preferably, the halogen of said haloalkyl is Cl, Br or F; most preferably, R3Is methyl substituted by one to three Cl, Br or F atoms.
R4Is a haloalkyl group. Preferably, the halogen of the haloalkyl is Cl or F.
More preferably, ,
R1is-CH3、-C6H5、-C6H4-o-Cl or- (CH)2)2C6H5；
R2is-CH3or-CH2CH3；
R3is-CH2CH3、-CH2Cl、-CH2Br or-CF3；
R4is-CH2Cl or-CF3。
Most preferably, R1is-C6H4-o-Cl，R2is-CH3I.e., formula 1 is o-chlorobenzoyl methyl ester.
Most preferably, R1Is- (CH)2)2C6H5，R2is-CH2CH3I.e., formula 1 is ethyl 2-carbonyl-4-phenylbutyrate.
Most preferably, R3is-CH2Cl, i.e., formula 2, is ethyl 4-chloro-3-carbonylbutyrate.
The conditions of the asymmetric reduction reaction of the present invention may be selected according to the conditions conventional in such reactions in the art, and preferably, the application comprises the steps of: in an aqueous solution with pH of 5.5-7.0, under the conditions of glucose dehydrogenase, glucose and NADP+In the presence of the carbonyl reductase or the recombinant carbonyl reductase, the prochiral carbonyl compound is subjected to asymmetric reduction reaction to form the optically active chiral alcohol.
Preferably, the amount of the protein is 5-60 kU/L, the amount of the glucose dehydrogenase is 5-60 kU/L, the amount of the glucose is 5-600 g/L, and the NADP is+The dosage of the prochiral carbonyl compound is 0-0.5 mmol/L, the concentration of the prochiral carbonyl compound is 10-2.0 mol/L, the aqueous solution with the pH value of 5.5-7.0 is phosphate buffer solution, and the reaction temperature is 20-35 ℃.
The preferable concentration of the prochiral carbonyl compound (except methyl o-chlorobenzoyl formate, ethyl 2-carbonyl-4-phenylbutyrate and ethyl 4-chloro-3-carbonylbutanoate) in the reaction solution is 10-100 mmol/L. The dosage of the carbonyl reductase is catalytic effective amount, preferably 5-60 kU/L. The dosage of the glucose dehydrogenase is preferably 5-60 kU/L. The amount of glucose is preferably 5 to 600 g/L. Additional added NADP+The amount of the surfactant is preferably 0 to 1.0 mmol/L. The aqueous solution can be a buffer solution which is conventional in the field as long as the pH range is 5.5-7.0, and a phosphate buffer solution, such as a phosphate-sodium phosphate buffer solution, is preferred. The concentration of the phosphate buffer solution is preferably 0.05-0.1 mol/L, and the concentration refers to the total concentration of conjugate acid and alkali in the buffer solution. The asymmetric reduction reaction is preferably carried out under shaking or stirring conditions. The temperature of the asymmetric reduction reaction is preferably 20-35 ℃. The time of the asymmetric reduction reaction is preferably determined by the time when the concentration of the product is not continuously increased in the reaction process.
After the asymmetric reduction reaction is finished, the chiral alcohol product can be extracted from the reaction solution according to the conventional method in the field. The substrate concentrations of methyl o-chlorobenzoate, ethyl 2-carbonyl-4-phenylbutyrate and ethyl 4-chloro-3-carbonylbutyrate can reach 1.5mol/L, 2.0mol/L and 2.0mol/L respectively at the highest. The dosage of the reductase is catalytic effective amount, and the dosage of the carbonyl reductase and o-chlorobenzoyl methyl formate, 2-carbonyl-4-phenyl ethyl butyrate or 4-chloro-3-carbonyl ethyl butyrate is preferably 10-100 kU carbonyl reductase/mol substrate. The amount of glucose to be used is preferably 200 to 300g of glucose per mol of substrate, in comparison with methyl o-chlorobenzoylformate, ethyl 2-carbonyl-4-phenylbutyrate or ethyl 4-chloro-3-carbonylbutyrate. GrapeThe dosage of the sugar dehydrogenase is preferably 10-100 kU of glucose dehydrogenase/mol of substrate, and additionally added NADP+The amount of the surfactant is preferably 0 to 1.0 mmol/L. The buffer solution can be a buffer solution conventional in the art as long as the pH range is 5.5-7.0, and a phosphate buffer solution with a pH of 6.0, such as a phosphate-sodium phosphate buffer solution, is preferably selected. The concentration of the phosphate buffer solution is preferably 0.05-0.1 mol/L, and the concentration refers to the total concentration of conjugate acid and alkali in the buffer solution. The asymmetric reduction reaction is preferably carried out under stirring. The temperature of the asymmetric reduction reaction is preferably 20 to 35 ℃, more preferably 25 ℃. The time of the asymmetric reduction reaction is preferably determined by the time when the concentration of the product is not increased any more in the reaction process. After the asymmetric reduction reaction is finished, the chiral alcohol product can be extracted from the reaction solution according to the conventional method in the field.
In the present invention, in the case where the carbonyl reductase described above catalyzes the asymmetric reduction reaction of a prochiral carbonyl compound to form a chiral alcohol, if a pure carbonyl reductase is used, it is necessary to add NADP as a coenzyme+When a crude enzyme solution of carbonyl reductase extracted from microbial cells or resting cells expressing carbonyl reductase are used, it is only necessary to use the coenzyme contained in the cells without adding the coenzyme.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the invention provides a novel carbonyl reductase and a method for preparing optically pure (R) -2-hydroxy-4-phenyl ethyl butyrate by regenerating and asymmetrically reducing 2-carbonyl-4-phenyl ethyl butyrate by combining recombinant reductase with coenzyme aiming at the problems that the product concentration is not high and expensive coenzyme needs to be additionally added in the reported research of biocatalytic asymmetric synthesis of (R) -2-hydroxy-4-phenyl ethyl butyrate. At catalytic concentrations of up to 2mol/L (412g/L) of substrate, the optical purity of the product is still up toMore than 99 percent, and does not need to add expensive coenzyme additionally. Compared with other asymmetric reduction preparation methods, the product prepared by the method has high concentration, and expensive coenzyme NADP does not need to be additionally added+And the method has the advantages of high optical purity of the product, mild reaction conditions, environmental friendliness, simplicity and convenience in operation and easiness in industrial amplification, so that the method has a good industrial application prospect.
Drawings
The features and advantages of the present invention are described below with reference to the accompanying drawings.
FIG. 1 is a PCR amplification electropherogram of gene CgKR1, wherein, 1.DNA Marker (MarkerII, Beijing Tiangen Biochemical technology Co., Ltd.); 2-3, PCR amplification products of gene CgKR 1.
FIG. 2 is a PCR amplification electrophoresis chart of a bacterial liquid of Escherichia coli (E.coli) DH5 alpha/pET 28a-CgKR1, wherein, 1.DNA Marker (Marker II, Beijing Tiangen Biochemical technology Co., Ltd.); 2-3, carrying out PCR amplification electrophoresis on a bacterial liquid of Escherichia coli (E.coli) DH5 alpha/pET-CgKR 1.
FIG. 3 is a PCR amplification electrophoresis chart of the bacterial liquid of Escherichia coli (E.coli) BL21(DE3)/pET28a-CgKR1, wherein, 1.DNA Marker (Marker II, Beijing Tiangen Biochemical technology Co., Ltd.); 2-3, a bacterial liquid PCR amplification electrophoresis picture of Escherichia coli (E.coli) BL21(DE3)/pET28a-CgKR 1.
FIG. 4 is a PCR amplification electrophoresis chart of the bacterial liquid of Escherichia coli (E.coli) BL21(DE3)/pET28a-CgKR1F92L/F94V/I99Y/G174A, wherein, 1.DNA Marker (Marker II, Beijing Tiangen Biochemical technology Co., Ltd.); 2-3, bacterial liquid PCR amplification electrophoresis picture of Escherichia coli (E.coli) BL21(DE3)/pET28a-CgKR1F 92L/F94V/I99Y/G174A.
FIG. 5 is a schematic diagram of the construction of recombinant expression plasmid pET28a-CgKR 1.
FIG. 6 is a polypropylene gel electrophoresis of recombinant carbonyl reductase CgKR 1.
FIG. 7 is a polypropylene gel electrophoresis diagram of recombinant carbonyl reductase CgKR1F 92L/F94V/I99Y/G174A.
Detailed Description
The present inventors analyzed some genome sequences by a method of genome database mining, and candidate a lot of sequences predicted to be carbonyl reductase genes in some bioinformatics. The mining method specifically comprises the steps of taking an amino acid sequence of carbonyl reductase Gre2p (or named as YOL151w) from Saccharomyces cerevisiae with good biocatalytic performance as a probe, carrying out pBLAST search in NCBI database, and selecting a batch of predicted carbonyl reductase gene sequences. Then, the candidate genes are cloned and expressed respectively to construct recombinant Escherichia coli cells. The activity and stereoselectivity of the carbonyl reductase on the o-chlorobenzoyl methyl formate, 2-carbonyl-4-phenyl ethyl butyrate or 4-chloro-3-carbonyl ethyl butyrate and the like are measured, and the cloned and expressed enzyme is repeatedly compared and screened to finally obtain the carbonyl reductase CgKR1 with the best catalytic performance, thereby completing the invention.
On the basis of screening to obtain high-activity and stereoselective enzymes, the invention carries out protein modification on wild enzymes, and mutates the active center and amino acid residues at some sites in a hydrophobic channel leading to the active center into other amino acid residues, so as to further strengthen the catalytic performance of the enzyme and improve the expression of the enzyme.
The present invention is further illustrated by the following examples, but is not limited thereto. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
The material sources in the following examples are:
candida glabrata (Candida glabrata) CGMCC 2.234.
Expression plasmid pET28a was purchased from Novagen, Shanghai.
Coli DH5 α and e.coli BL21(DE3) competent cells, 2 × Taq PCR MasterMix, agarose gel DNA recovery kit was purchased from beijing tiangen biochemical science ltd.
EXAMPLE 1 cloning of carbonyl reductase Gene
Based on the Genbank record of the gene sequence predicted to be C.glabrata (Candida glabrata) carbonyl reductase (NCBI accession number: CAG58832), PCR primers were designed as follows:
CgKR1f：5’-CATATGGCTTCTGATAACAGCAAC-3’；
CgKR1r：5’-GGATCCTTAATTAGAGTTCTTCTCGGC-3’。
wherein, the underlined part of the upstream primer is NdeI restriction site, and the underlined part of the downstream primer is BamHI restriction site.
PCR amplification was performed using the genomic DNA of Candida glabrata (Candida glabrata) CGMCC 2.234 as a template. The PCR system is as follows: 2 XTaq PCR MasterMix 10. mu.l, upstream and downstream primers 1. mu.l each (0.3. mu. mol/L), DNA template 1. mu.l (0.1. mu.g) and ddH2O7. mu.l. The PCR amplification step is as follows: (1) pre-denaturation at 95 ℃ for 3 min; (2) denaturation at 94 deg.C for 1 min; (3) annealing at 55 ℃ for 30 s; (4) extending for 1min at 72 ℃; repeating the steps (2) to (4) for 30 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. The PCR product was purified by agarose gel electrophoresis, and the target band of 900-1200 bp was recovered using an agarose gel DNA recovery kit (FIG. 1). Obtaining a complete carbonyl reductase full-length gene sequence of Candida glabrata (Candida glabrata) CGMCC 2.234, carrying out DNA sequencing to obtain a full-length 1059bp named as CgKR1, wherein the base sequence is shown as SEQ ID No.1 in a sequence table.
Example 2 site-directed mutagenesis of the CgKR1 Gene
The full-length gene sequence (SEQ ID No.1) of carbonyl reductase of Candida glabrata CGMCC 2.234 obtained in example 1 was subjected to base mutation.
PCR primers were designed as follows:
mutation V85I:
an upstream primer: 5'-CAAGGTTATCTTACACACCGCCTCTCC-3'
A downstream primer: 5'-CGGTGTGTAAGATAACCTTGATATCCTTGCCATG-3'
Mutation F92L:
an upstream primer: 5'-CCGCCTCTCCACTGCACTTCAACACCACTGACATT-3'
A downstream primer: 5'-GTGCAGTGGAGAGGCGGTGTGTAAG-3'
Mutation F94V:
an upstream primer: 5' -CACACCGCCTCTCCATTCCACGTTAACACCACTGA
CATTGAA-3’
A downstream primer: 5'-TGGAGAGGCGGTGTG-3'
Mutation I99Y:
an upstream primer: 5'-CCACTGACTATGAAAAGGATCTATTGATCCC-3'
A downstream primer: 5'-GATCCTTTTCATAGTCAGTGGTGTTGAAGTGG-3'
Mutation G174A:
an upstream primer: 5'-CCAATCAGAGCTTACTGTGGTTCAAAGAAGTTTG-3'
A downstream primer: 5'-CCACAGTAAGCTCTGATTGGATCGGATTGAC-3'
Combination mutation F92L/F94V:
an upstream primer: 5'-CTCCACTGCACGTGAACACCACTGACATTGAAAAG-3'
A downstream primer: 5'-GTGTTCACGTGCAGTGGAGAGGCGGTGTGTAAG-3'
Carbonyl reduction of Candida glabrata CGMCC 2.234 obtained in example 1The full-length gene sequence of the enzyme (SEQ ID No.1) is taken as a template to carry out PCR amplification. The PCR system is as follows: 10 XKOD-Plus PCRbuffer 2. mu.l, 25mM MgSO41.2. mu.l, 2mM dNTP 2. mu.l, KOD-Plus PCR Hi-Fi enzyme 0.3. mu.l, DNA template 0.5. mu.l (0.1. mu.g), ddH2Mu.l of O13, the first DNA fragment was amplified by PCR with CgKR1f (see example 1) and 0.5. mu.l (10mmol/L) of each downstream primer, and the second DNA fragment was amplified by PCR with CgKR1r and 0.5. mu.l (10mmol/L) of each upstream primer. The PCR amplification step is as follows: (1) pre-denaturation at 95 ℃ for 3 min; (2) denaturation at 98 ℃ for 15 s; (3) annealing at 55 ℃ for 30 s; (4) extending for 1min at 72 ℃; repeating the steps (2) to (4) for 30 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. The PCR product was purified by agarose gel electrophoresis, and the target band was recovered using an agarose gel DNA recovery kit.
Then, PCR amplification is performed by using the first DNA fragment and the second DNA fragment obtained above as templates. The PCR system is as follows: 10 XKOD-Plus PCR buffer 5. mu.l, 25mM MgSO43. mu.l, 5. mu.l of 2mM dNTP, 1. mu.l of KOD-Plus PCR Hi-Fi enzyme, 1. mu.l (0.1. mu.g) of each of the first DNA fragment and the second DNA fragment as a DNA template, ddH2O31. mu.l, CgKR1f and CgKR1r each 1.5. mu.l (10 mmol/L). The PCR amplification step is as follows: (1) pre-denaturation at 95 ℃ for 3 min; (2) denaturation at 98 ℃ for 15 s; (3) annealing at 55 ℃ for 30 s; (4) extending for 1min at 72 ℃; repeating the steps (2) to (4) for 30 times; (5) extension was continued for 10min at 72 ℃ and cooled to 4 ℃. The PCR product was purified by agarose gel electrophoresis, and a target band of about 1.0 to 1.2kbp was recovered using an agarose gel DNA recovery kit.
The F92L/F94V/I99Y/G174A mutation construction process comprises the following steps: firstly, using SEQ ID No.1 as a template, using a PCR primer combining mutation F92L/F94V to construct F92L/F94V, then using the mutation as the template, using the PCR primer combining mutation I99Y to construct F92L/F94V/I99Y, finally using the mutation as the template, and using the PCR primer combining mutation G174A to construct F92L/F94V/I99Y/G174A.
The DNA sequencing is correct, and the obtained target band is the gene of the mutant protein, and the names of the target band are respectively as follows:
CgKR1V85I, the amino acid sequence SEQ ID No.2 with valine at position 85 substituted by isoleucine;
CgKR1F92L, amino acid sequence SEQ ID No.2 with a substitution of phenylalanine at position 92 to leucine;
CgKR1F94V, amino acid sequence SEQ ID No.2 with the 94 th phenylalanine substituted into valine;
CgKR1F92L/F94V, the amino acid sequence SEQ ID No.2 with the substitution of phenylalanine at position 92 to leucine and phenylalanine at position 94 to valine;
CgKR1I99Y, amino acid sequence SEQ ID No.2 with the 99 th isoleucine substituted into tyrosine;
CgKR1G174A, glycine at position 174 of amino acid sequence SEQ ID No.2 is substituted into alanine;
CgKR1F92L/F94V/I99Y/G174A, the amino acid sequence SEQ ID No.2 with phenylalanine at position 92 substituted with leucine, phenylalanine at position 94 substituted with valine, isoleucine at position 99 substituted with tyrosine, and glycine at position 174 substituted with alanine.
EXAMPLE 3 construction of recombinant expression vector (plasmid) and preparation of recombinant expression transformant
The DNA fragment of carbonyl reductase gene obtained in example 1 or 2 was digested with restriction enzymes NdeI and BamHI at 37 ℃ for 12 hours, purified by agarose gel electrophoresis, and the target fragment was recovered by agarose gel DNA recovery kit. The target fragment was ligated with plasmid pET28a, which was also digested with NdeI and BamHI, at 4 ℃ overnight under the action of T4DNA ligase to give recombinant expression plasmid pET28a-CgKR1 and its mutants, and the plasmid construction map is shown in FIG. 5.
The recombinant expression plasmid was transformed into escherichia coli (e.coli) DH5 α competent cells, and positive recombinants were selected on kanamycin-containing resistance plates by heat shock for 90 seconds under transformation conditions at 45 ℃, and single clones were picked up and colony PCR verified for positive clones (fig. 2). Culturing the recombinant bacteria, extracting plasmids after the plasmids are amplified, retransforming the plasmids into competent cells of Escherichia coli (E.coli) BL21(DE3), coating a transformation solution on an LB plate containing kanamycin, and carrying out inversion culture at 37 ℃ overnight to obtain a bacterial solution PCR amplification electrophoretogram (figure 4) of a positive recombinant transformant Escherichia coli (E.coli) BL21(DE3)/pET28a-CgKR1 and mutants thereof, wherein the bacterial solution PCR amplification electrophoretogram (figure 3) verifies positive clones, and BL21(DE3)/pET28a-CgKR1F L/F94V/I99Y/G174A.
Example 4 expression and enzyme Activity and stability assay of recombinant carbonyl reductase
The recombinant Escherichia coli obtained in example 3 was inoculated into LB medium (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH 7.0) containing kanamycin, cultured overnight with shaking at 37 ℃ and 1% (v/v) of the inoculum size in a 500ml Erlenmeyer flask containing 100ml of LB medium, cultured with shaking at 37 ℃ and 180rpm, and when OD of the culture solution is determined600When the concentration reaches 0.6, IPTG with the final concentration of 0.5mmol/L is added as an inducer, after induction is carried out for 12h at 25 ℃, the culture solution is centrifuged, cells are collected and washed twice by normal saline, and the resting cells are obtained. Suspending the obtained resting cells in a buffer solution with the pH value of 7.0, carrying out ultrasonic disruption in an ice bath, centrifuging and collecting supernatant fluid, namely the crude enzyme solution of the recombinant carbonyl reductase. The crude enzyme solution and the precipitate are analyzed by polyacrylamide gel electrophoresis chart, wherein the polypropylene gel electrophoresis chart of CgKR1 is shown in figure 6, and the polypropylene gel electrophoresis chart of recombinant carbonyl reductase CgKR1F92L/F94V/I99Y/G174A is shown in figure 7.
The recombinant protein is present in a partially soluble form. The soluble protein content calculation method comprises the following steps: and integrating the recombinant carbonyl reductase in the supernatant and the precipitate in the polyacrylamide gel electrophoresis image, wherein the percentage of the recombinant carbonyl reductase in the supernatant to the sum of the two is the content of the soluble protein. The solubility of the recombinant carbonyl reductases CgKR1 and CgKR1F92L/F94V/I99Y/G174A are shown in Table 2. And (3) freeze-drying the crude enzyme solution by using a freeze-drying machine to obtain freeze-dried crude enzyme powder.
Determination of the Activity of carbonyl reductase: measured by detecting the change in absorbance at 340 nm. The specific method comprises the following steps: in 1mlAdding substrate o-chlorobenzoyl methyl formate with the final concentration of 2mmol/L and NADPH with the final concentration of 0.1mmol/L into a reaction system (100mmol/L sodium phosphate buffer solution, pH 6.0), keeping the temperature at 30 ℃ for 2 minutes, adding a proper amount of crude enzyme solution after cell disruption, quickly mixing uniformly, and detecting the change of the light absorption value at 340 nm. The calculation formula of the enzyme activity is as follows: enzyme activity (U) ═ EW × V × 103V (6220X l); wherein EW is the change value of absorbance at 340nm within 1 min; v is the volume of the reaction solution, unit mL; 6220 molar extinction coefficient of NADPH, unit L/(mol. cm); l is the path length in cm. Each unit (U) of carbonyl reductase is defined as the amount of enzyme required to catalyze the oxidation of 1. mu. mol NADPH per minute under the above-mentioned conditions. The enzyme activities of the recombinant carbonyl reductase CgKR1 and its mutant are shown in Table 2.
Carbonyl reductase stability determination method: determination of the T of the carbonyl reductase enzyme according to the document Angew. chem. int. Ed., 2006, 27, 7745-50The value is obtained. T of recombinant carbonyl reductase CgKR1 and mutant strain thereof50The values are shown in Table 2.
The glucose dehydrogenase activity determination method comprises the following steps: to a 1ml reaction system (100mmol/L sodium phosphate buffer, pH 7.0), 10mmol/L glucose, 1mmol/L NADP was added+Keeping the temperature at 30 ℃ for 2 minutes, adding a proper amount of enzyme solution, quickly and uniformly mixing, and detecting the change of the light absorption value at 340 nm. Per unit (U) of glucose dehydrogenase is defined as catalyzing 1. mu. mol NADP per minute under the above conditions+The amount of enzyme required for reduction.
TABLE 2 CgKR1 and mutant Properties thereof
Examples 5-17 recombinant reductase CgKR1 catalyzing asymmetric reduction of carbonyl Compounds
2U of the crude enzyme solution CgKR1 prepared in example 4 and 2U of the crude enzyme solution of glucose dehydrogenase (preparation method see below) were added to 0.4ml of sodium phosphate buffer (100mmol/L, pH 7.0)See: journal of Industrial Microbiology and Biotechnology 2011, 38, 633-641), ketone ester or aryl ketone (examples 5-17) was added to a final concentration of 10mmol/L, and NADP was added to a final concentration of 0.5mmol/L+And 5g/L glucose. The reaction was shaken at 1100rpm at 30 ℃ for 24 hours, at which time the concentration of the reaction product did not increase further. After the reaction is finished, the mixture is extracted twice by using equal volume of ethyl acetate, the extracts are combined, and after the mixture is dried overnight by adding anhydrous sodium sulfate, the conversion rate of the substrate and the ee value of the reduction product are analyzed and determined. The results are shown in Table 3.
The specific analysis conditions for the conversion and ee value of the product are as follows:
in examples 5 and 10 to 17, the conversion rate and ee value were analyzed by using a gas chromatograph, the chromatographic column was a chiral capillary column CP-chiralil-DEX CB, nitrogen was used as a carrier gas, the injection port temperature was 280 ℃, the detector temperature was 280 ℃, and other conditions were as follows:
example 5: the column temperature is 80 ℃;
example 10: maintaining the initial column temperature at 90 deg.C for 2min, heating to 120 deg.C at 1 deg.C/min, and maintaining for 5 min;
examples 11 and 12: after acetylation of the reaction product, analyzing, wherein the initial column temperature is 110 ℃, after maintaining for 2min, the temperature is raised to 126 ℃ at the speed of 2 ℃/min, and the temperature is maintained for 2 min;
example 13: the column temperature is 120 ℃;
example 14: the column temperature is 130 ℃;
examples 15 and 16: the column temperature is 140 ℃;
example 17: the column temperature is 160 ℃;
examples 6 to 9 were carried out for the analysis of the conversion rate using a gas chromatograph with a chiral capillary column CP-chiralil-DEX CB, nitrogen as carrier gas, a sample inlet temperature of 280 ℃, a detector temperature of 280 ℃, and a column temperature of 180 ℃;
examples 6 and 7 analysis of ee values using liquid chromatography, chiral OD-H column, mobile phase: n-hexane/isopropanol/trifluoroacetic acid 94/6/0.2, flow rate 1ml/min, detector wavelength 228 nm.
Examples 8 and 9 analysis of ee values using liquid chromatography, chiral OD-H column, mobile phase: 97/3 (normal hexane/isopropanol), flow rate of 1ml/min and detector wavelength of 254 nm.
TABLE 3 results of asymmetric reduction of carbonyl compounds catalyzed by CgKR1
Example 18 CgKR1 catalysis of asymmetric reduction of methyl o-chlorobenzoate
To 100ml of a sodium phosphate buffer solution (100mmol/L, pH 5.5), 4000U of the crude CgKR1 enzyme solution prepared in example 4 and 4000U of the crude glucose dehydrogenase enzyme solution were added, and methyl o-chlorobenzoate and 220g/L of glucose were added to a final concentration of 0.75 mol/L. The reaction was carried out at 20 ℃ with the pH of the reaction solution controlled to 5.5 until the reaction was complete, i.e., the concentration of the reaction product did not increase further, at which time the reaction time was 12 hours. After the reaction is finished, the mixture is extracted by equal volume of ethyl acetate twice, the extract liquor is combined and dried by adding anhydrous sodium sulfate overnight, the solvent is removed by rotary evaporation, and the reduced pressure distillation is carried out to obtain 1.16g of (R) -methyl o-chloromandelate, the yield is 76 percent, and the ee value of the product is 98.0 percent.
Example 19 asymmetric reduction of methyl o-chlorobenzoate catalyzed by CgKR1
6000U of the crude enzyme solution of CgKR1 prepared in example 4 and 6000U of glucose dehydrogenase were added to 100ml of a sodium phosphate buffer solution (100mmol/L, pH 6.0), and methyl o-chlorobenzoate and 450g/L of glucose were added to the mixture to a final concentration of 1.5 mol/L. The reaction was carried out at 25 ℃ with the pH of the reaction solution controlled to 6.0 and the reaction was completed until the concentration of the reaction product did not increase any more, at which time the reaction time was 6 hours. After the reaction is finished, the mixture is extracted by equal volume of ethyl acetate twice, the extract liquor is combined and dried by adding anhydrous sodium sulfate overnight, the solvent is removed by rotary evaporation, and the reduced pressure distillation is carried out to obtain 2.61g of (R) -methyl o-chloromandelate, the yield is 87%, and the ee value of the product is 98.5%.
Example 20 asymmetric reduction of ethyl 2-carbonyl-4-phenylbutyrate catalyzed by CgKR1
6000U of the crude enzyme solution of CgKR1 prepared in example 4 and 6000U of the crude enzyme solution of glucose dehydrogenase were added to 100ml of a sodium phosphate buffer solution (100mmol/L, pH 6.0), and ethyl 2-carbonyl-4-phenylbutyrate and 600g/L of glucose were added to a final concentration of 2.0 mol/L. The reaction was carried out at 25 ℃ with the pH of the reaction solution controlled to 6.0 and the reaction was completed until the concentration of the reaction product did not increase any more, at which time the reaction time was 12 hours. After the reaction, the mixture is extracted twice by using equal volume of ethyl acetate, the extracts are combined and dried overnight by adding anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and the ethyl (R) -2-hydroxy-4-phenylbutyrate is obtained by reduced pressure distillation, wherein the yield is 89%, and the ee value of the product is 98%.
Example 21 asymmetric reduction of 4-chloro-3-carbonyl ethyl butyrate catalyzed by CgKR1
6000U of the crude enzyme solution of CgKR1 prepared in example 4 and 6000U of the crude enzyme solution of glucose dehydrogenase were added to 100ml of a sodium phosphate buffer solution (100mmol/L, pH 6.0), and 4-chloro-3-carbonyl ethyl butyrate and 600g/L of glucose were added to a final concentration of 2.0 mol/L. The reaction was carried out at 25 ℃ with the pH of the reaction solution controlled to 6.0 and the reaction was completed until the concentration of the reaction product did not increase any more, at which time the reaction time was 4 hours. After the reaction is finished, the ethyl acetate with the same volume is used for extraction, extraction is carried out twice, extraction liquid is combined, anhydrous sodium sulfate is added for drying overnight, the solvent is removed by rotary evaporation, and reduced pressure distillation is carried out to obtain 3.0g of (R) -4-chloro-3-hydroxy ethyl butyrate, the yield is 91 percent, and the ee value of the product is 97 percent.
Examples 22-31 CgKR1 and its mutants catalyze asymmetric reduction of methyl o-chlorobenzoate
A mutant strain of CgKR1 was constructed according to example 2, recombinant expression vectors (plasmids) and recombinant expression transformants were prepared according to example 3, and crude enzyme powders were prepared according to example 4.
4000U of crude enzyme powder of CgKR1 and its mutant strain prepared in example 4 and 4000U of crude enzyme powder of glucose dehydrogenase were added to 100ml of sodium phosphate buffer (100mmol/L, pH 6.0), and methyl o-chlorobenzoate, 150g/L of glucose and 0.1mmol/L of NADP were added to the mixture to give a final concentration of 100g/L+. The reaction is carried out at 25-35 ℃, the pH of the reaction solution is controlled to be 6.0, and the reaction lasts for 24 hours, so that the concentration of the reaction products in the embodiments 21-31 is not increased any more. The conversion of the reaction and the ee value were measured in accordance with example 8, and the results are shown in Table 4.
TABLE 4 asymmetric reduction results of CgKR1 and its mutants catalyzing methyl o-chlorobenzoate
It should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.
Claims (8)
1. An isolated protein characterized by: it is a protein as follows:
in the amino acid sequence of the protein consisting of the amino acid sequence shown in SEQ ID No.2 in the sequence table, phenylalanine at position 92 is substituted by leucine, phenylalanine at position 94 is substituted by valine, isoleucine at position 99 is substituted by tyrosine, and glycine at position 174 is substituted by alanine.
2. An isolated nucleic acid, characterized in that: it is a gene encoding the following proteins: in the protein consisting of the amino acid sequence shown in SEQ ID No.2 in the sequence table, phenylalanine at position 92 is substituted by leucine, phenylalanine at position 94 is substituted by valine, isoleucine at position 99 is substituted by tyrosine, and glycine at position 174 is substituted by alanine.
3. A recombinant expression vector comprising the nucleic acid of claim 2.
4. A recombinant expression transformant comprising the recombinant expression vector of claim 3.
5. A preparation method of recombinant carbonyl reductase is characterized by comprising the following steps: culturing the recombinant expression transformant according to claim 4 to obtain the recombinant carbonyl reductase from the culture.
6. Use of a protein according to claim 1 for catalyzing the asymmetric reduction of a prochiral carbonyl compound to form a chiral alcohol, wherein the prochiral carbonyl compound is a compound of formula 1, 2 or 3:
wherein R is1is-CH3Phenyl or phenyl with a substituent group, wherein the substituent group of the phenyl is halogen or alkyl with a carbon chain length of 1-2;
R2is an alkyl group with a carbon chain length of 1-2;
R3the halogen of the halogenated alkyl is Cl, Br or F;
R4is-CH2Cl or-CF3。
7. The application of claim 6, wherein said application packageThe method comprises the following steps: in an aqueous solution at pH 5.5-7.0, in the presence of glucose dehydrogenase, glucose and NADP+In the presence of the protein of claim 1, the prochiral carbonyl compound undergoes an asymmetric reduction reaction to form an optically active chiral alcohol.
8. The use of claim 7, wherein the amount of protein is 5 to 60kU/L, the amount of glucose dehydrogenase is 5 to 60kU/L, the amount of glucose is 5 to 600g/L, and NADP+The using amount of the prochiral carbonyl compound is 0-0.5 mmol/L, the concentration of the prochiral carbonyl compound is 10-2.0 mol/L, the aqueous solution with the pH value of 5.5-7.0 is phosphate buffer solution, and the reaction temperature is 20-35 ℃.
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| CN102876734B (en) * | 2012-10-30 | 2014-01-01 | 华东理工大学 | Carbonyl reductase, gene and applications of carbonyl reductase in asymmetric reduction of prochiral carbonyl compound |
| CN102925500B (en) * | 2012-11-05 | 2014-07-30 | 遵义医学院 | Preparation method of chiral 4-hydroxy-4 phenyl butyric acid ester |
| SG11201508053WA (en) * | 2013-03-28 | 2015-10-29 | Kaneka Corp | Modified carbonyl reducing enzyme and gene |
| CN103320403B (en) * | 2013-06-18 | 2016-02-10 | 杭州师范大学 | Ketoreductase LEK mutant and application |
| CN104099305A (en) * | 2014-07-18 | 2014-10-15 | 华东理工大学 | Carbonyl reductase mutant as well as gene and application thereof |
| WO2016138641A1 (en) | 2015-03-04 | 2016-09-09 | 华东理工大学 | Generation and use of candida and carbonyl reductase thereof |
| CN105062985B (en) * | 2015-08-11 | 2018-04-27 | 中国科学院成都生物研究所 | A kind of carbonyl reduction enzyme mutant and application thereof |
| CN105821013B (en) * | 2016-04-05 | 2019-05-03 | 华东理工大学 | Carbonyl reductase and its application in the chiral N-protected-hydroxyl azacyclo- of preparation |
| CN106701698B (en) * | 2016-11-15 | 2019-11-15 | 华东理工大学 | Carbonyl reductase, mutant and its preparing the application in antimycotic pharmaceutical intermediate |
| CN106947772B (en) * | 2016-12-28 | 2020-08-21 | 江苏阿尔法药业有限公司 | Carbonyl reductase mutant and application thereof in preparation of chiral alcohol |
| CN106929490B (en) * | 2017-01-25 | 2019-10-01 | 华东理工大学 | A kind of carbonyl reductase, mutant and its application in preparation statin synthetic intermediate |
| CN106929491B (en) * | 2017-03-13 | 2019-09-17 | 江南大学 | (S)-carbonyl reductase heteromer and its application in the more benzene ring compounds of catalysis |
| CN109112166B (en) * | 2017-06-26 | 2023-08-15 | 弈柯莱生物科技(上海)股份有限公司 | Enzymatic preparation of ticagrelor intermediate |
| CN107574158A (en) * | 2017-08-31 | 2018-01-12 | 沈阳药科大学 | The enzyme process of chiral indenes alcohol compound prepares and its draws more applications in lucky synthesis in chiral drug |
| CN109852592B (en) * | 2019-01-14 | 2022-05-31 | 中国科学院成都生物研究所 | Carbonyl reductase mutant with improved heat resistance |
| CN110592035B (en) * | 2019-08-29 | 2022-07-15 | 浙江大学 | Carbonyl reductase mutant, recombinant expression vector and application of carbonyl reductase mutant in production of chiral alcohol |
| CN113174377B (en) * | 2021-04-28 | 2022-11-25 | 华东理工大学 | Carbonyl reductase, mutant and application of carbonyl reductase in preparation of diltiazem intermediate |
| CN113249348B (en) * | 2021-05-19 | 2023-06-23 | 华东理工大学 | Carbonyl reductase, gene thereof, recombinant expression transformant containing the gene and use thereof |
| CN113528588A (en) * | 2021-06-15 | 2021-10-22 | 海南卓科制药有限公司 | Preparation method of levocarnitine |
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