CN108486075B - Recombinant carbonyl reductase mutant, gene, engineering bacterium and application thereof - Google Patents

Abstract

The invention discloses a recombinant carbonyl reductase mutant, a gene, a vector, an engineering bacterium and application thereof, wherein the mutant is obtained by carrying out combined mutation on the 145 th site and the 152 th site of an amino acid sequence shown in SEQ ID NO.2, and the mutation points are as follows: the 145 th phenylalanine is mutated into methionine and the 152 th threonine is mutated into serine; the 145 th phenylalanine is mutated into tyrosine, and the 152 th threonine is mutated into serine. The invention provides a recombinant carbonyl reductase mutant for reducing (S) -6-chloro-5-hydroxy-3-oxohexanoate tert-butyl ester, which has greatly improved catalytic activity and substrate tolerance compared with wild enzyme in the conversion reaction, and obviously shortens the reaction process time.

Description

Recombinant carbonyl reductase mutant, gene, engineering bacterium and application thereof

(I) technical field

The invention belongs to the fields of biological pharmacy and biological transformation, and particularly relates to a recombinant carbonyl reductase mutant, a gene, a vector, an engineering bacterium and application thereof, as well as a method for preparing tert-butyl (3R,5S) -6-chloro-3,5-dihydroxyhexanoate (tert-butyl (3R,5S) -6-chloro-3,5-dihydroxyhexanoate, (3R,5S) -CDHH) by biocatalysis of (S) -6-chloro-5-hydroxy-3-carbonylhexanoate and (S) -CHOH) of recombinant escherichia coli expressing carbonyl reductase.

(II) background of the invention

Stereoselective Carbonyl reductases (E.C. 1.1.1.x) belong to the class of redox enzymes, which are enzymes capable of catalyzing reversible redox reactions between alcohols and aldehydes/ketones in both directions, and require the coenzymes NAD (H) (nicotinamide adenine dinucleotide) or NADP (H) (nicotinamide adenine dinucleotide phosphate) as hydrogen transporters, capable of reducing large amounts of endogenous or exogenous Carbonyl compounds. NADH and NADPH as electron donors involved in their reductionReaction, NAD⁺And NADP⁺It participates in the oxidation reaction as an electron acceptor. Carbonyl reductases are currently reported in the literature to be distributed predominantly in three superfamilies: the first type is Short-chain Dehydrogenase/reductase (SDRs), each molecule has about 250-350 amino acids in length, the catalytic triad is serine (Ser), tyrosine (Tyr) and lysine (Lys), and the SDRs family is one of the largest enzyme families at present; the second type is Medium-chain Dehydrogenases/Reductases (MDRs), each molecule of which is about 350-400 amino acids in length, and cysteine (Cys), histidine (His), and aspartic acid (Asp) are catalytic triads thereof; the third type is Aldo-Keto Reductases (AKRs), each molecule has a length of about 300-350 amino acids, and histidine (His), tyrosine (Tyr) and lysine (Lys) are catalytic triads thereof. The three have similar catalytic functions but have larger structural difference. Carbonyl reductase is a very ancient family, widely distributed in various animals, microorganisms and plants in the nature, and is a main source of carbonyl reductase due to various microorganisms and wide distribution, such as: pichia finlandica, Clostridium ljungdahlii, Vibrio vulgaris, Candida glabrata, Serratia quinvorans, Polygonum minus, Arabidopsis thaliana, Ocnococcus oeni, Serratia marcocens, Chryseobacterium sp, Rhodococcus erythropolis, Candida magnoae, Lactobacillus jensenii, and Lactobacillus coyniferis, etc. Furthermore, extremophilic carbonyl reductases, such as carbonyl reductases derived from extremophiles such as Thermococcus sibiricus, Thermococcus guaymansensis, Halofax volcanii, Thermus thermophilus, Sulfolobus acidocaldarius, Carboxydothermus hydrogenofamomans, Thermococcus kodakarensis, Thermotoga maritime, Koliella Antarctica, Pyrobaculum californica and Halobacterium sp.

(3R,5S) -CDHH is chiral side chain intermediate of HMG-CoA enzyme inhibitor atorvastatin and rosuvastatin medicine, and can be synthesized by a chemical method and a biological catalysis method. Chemical synthesis of (3R,5S) -CDHH often starts from a simple compound, such as (S) -ringThe method comprises the steps of synthesizing a target intermediate product by using oxychloropropane through a series of chemical reactions, wherein the introduction of a C3 chiral center requires the use of flammable and explosive NaBH₄As a reducing agent, the synthetic reaction needs to be carried out at the low temperature of minus 65 ℃, and the environmental pollution and the energy consumption are large. In addition, the chemical synthesis of (3R,5S) -CDHH has insufficient diastereoinduction, the optical purity of the product is low, the production requirements cannot be met, and the final yield is not high. In recent years, the enzyme method is used for replacing the chemical method, so that the method has high chemical, regional and stereoselectivity, is mild in reaction condition, avoids introduction of highly toxic substances, is environment-friendly, and makes up for the defects of the chemical method. Therefore, the synthesis of (3R,5S) -CDHH by the biocatalytic method has been the focus of attention in recent years. The carbonyl reductase LbADH derived from Lactobacillus brevis has good catalytic activity on the precursor compound of (3R,5S) -CDHH, namely 6-chloro-3, 5-dioxohexanoic acid tert-butyl ester, and can asymmetrically reduce the carbonyl at C5, e.e.>99.5 percent. In addition, a double-carbonyl reductase catalytic system consisting of carbonyl reductases LkADH1 and LkADH2 obtained from Lactobacillus kefir catalyzes asymmetric synthesis of (3R,5S) -CDHH from 6-chloro-3, 5-dioxohexanoic acid tert-butyl ester, and the yield reaches 47.5% and e.e.>99.5 percent. Subsequently, the Japan scholars cloned the carbonyl reductase gene from the Magnolia canadensis (Canadian magnoliae IFO 0705) and co-expressed with glucose dehydrogenase in Escherichia coli, and the co-expressed strain can catalyze 200g/L of (S) -CHOH with the yield of 97.2%, d.e.>98.6% (US 6645746B 1, US 6472544B 1). Recently, researchers have catalyzed the substrate of (3R,5S) -CDHH by immobilized Saccharomyces cerevisiae CGMCC No.2233, with a substrate concentration of 50g/L and a conversion rate of 100%, d.e.>99 percent. In addition, the scholars added 10g/L of recombinant carbonyl reductase cell dry weight, 0.1mM NAD using isopropanol as co-substrate⁺Catalyzes 100g/L of (S) -CHOH, and the yield reaches 96 percent, d.e.>97.2% (CN 104630125A). Shangke biological medicine (Shanghai) Co., Ltd., a whole cell co-expressed by carbonyl reductase (KRED) and Glucose Dehydrogenase (GDH) was used as a catalyst, the concentration of substrate (S) -CHOH was 250g/L, the concentration of whole cell was 180g/L, NADPH was 0.12g/L, triethanolamine was used as a buffer,reaction for 24h, conversion>95% optical purity>99.9% (CN 104328148A). After glucose and glucose dehydrogenase are used as coenzyme for circulation, a double-enzyme coupling system is constructed, the (S) -CHOH concentration is between 250-300g/L, the carbonyl reductase thallus concentration is about 75g/L, the GDH concentration is about 25g/L, the NADP is 0.6g/L, the reaction lasts for 24 hours, the yield is 96.7 percent and the d.e are added into the reaction system.>99.9% and 99% purity (CN 102965403A, CN 104726506A).

Therefore, the research on the (3R,5S) -CDHH biocatalytic process is of great significance, and not only can a new route be provided for the synthesis of (3R,5S) -CDHH, but also an enzyme source with high stereoselectivity and high catalytic activity can be provided for the (3R,5S) -CDHH.

Disclosure of the invention

The invention aims to provide a recombinant carbonyl reductase mutant, a coding gene, a recombinant vector containing the mutant gene, a recombinant genetic engineering bacterium obtained by transforming the recombinant vector and application in preparing (3R,5S) -CDHH. The recombinant carbonyl reductase mutant provided by the invention has higher substrate tolerance and catalytic activity, so that the reaction condition is mild, the catalytic efficiency is improved, the production cost is reduced, and the mutant is environment-friendly.

The technical scheme adopted by the invention is as follows:

the present invention provides a recombinant carbonyl reductase mutant which can be reduced to (3R,5S) -CDHH under suitable conditions using (S) -CHOH as a substrate, wherein the recombinant carbonyl reductase mutant is obtained by single-or double-mutation at position 43, 46, 90, 92, 94, 141, 145, 152, 157, 189, 190, 206, 211, 245, 249, or 190 of the amino acid sequence shown in SEQ ID NO. 2.

Further, preferably, the mutant is obtained by mutating phenylalanine at position 145 to methionine or threonine at position 152 to serine in the amino acid sequence shown in SEQ ID NO. 2.

Further, preferably, the mutant is obtained by mutating phenylalanine at position 145 to tyrosine and threonine at position 152 to serine of an amino acid sequence shown in SEQ ID NO. 2.

Due to the specificity of the amino acid sequence, any fragment of the peptide protein or its variant, such as conservative variant, bioactive fragment or derivative thereof, containing the amino acid sequence shown in the present invention, as long as the homology of the fragment of the peptide protein or the peptide protein variant with the aforementioned amino acid sequence is above 90%, is included in the protection scope of the present invention. In particular, the alteration comprises a deletion, insertion or substitution of an amino acid in the amino acid sequence; where conservative changes to a variant are made, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, and the variant may also have non-conservative changes, e.g., replacement of glycine with tryptophan.

The invention provides a coding gene of the recombinant carbonyl reductase mutant.

Due to the specificity of the nucleotide sequence, any variant of the polynucleotide of the present invention, as long as it has more than 90% homology with the aforementioned polynucleotide, is included in the scope of the present invention. A variant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Variants of the polynucleotide may be naturally occurring mutator variants or non-naturally occurring variants, including substitution, deletion and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, without substantially altering the function of the peptide protein encoded thereby.

The invention relates to a recombinant vector containing the coding gene of the recombinant carbonyl reductase mutant. The recombinant vector comprises a polynucleotide operably linked to control sequences suitable for directing expression in a host cell. Preferably, the expression vector is pET28 b.

The invention relates to a recombinant gene engineering bacterium obtained by utilizing the recombinant vector for transformation, which specifically comprises the following steps: the coding gene of the carbonyl reductase mutant is connected with an expression vector pET28b, and heterologous expression recombinant plasmids containing the coding gene of the carbonyl reductase mutant are constructed. And transforming the expression recombinant plasmid into host bacteria to obtain recombinant gene engineering bacteria containing the recombinant plasmid.

The invention relates to an application of a carbonyl reductase mutant encoding gene in preparing a recombinant carbonyl reductase mutant, which specifically comprises the following steps: constructing a recombinant vector containing the coding gene of the carbonyl reductase mutant, transforming the recombinant vector into host bacteria (preferably Escherichia coli E.coli BL21(DE3)), carrying out induced culture on the obtained recombinant gene engineering bacteria, separating culture solution to obtain thallus cells containing the recombinant carbonyl reductase mutant, and purifying carbonyl reductase crude enzyme liquid obtained after crushing to obtain carbonyl reductase pure enzyme.

The invention relates to application of a recombinant carbonyl reductase mutant in preparation of (3R,5S) -CDHH, which is specifically characterized in that: using wet thalli obtained by fermenting and culturing engineering bacteria containing recombinant carbonyl reductase coding gene mutants or pure enzyme extracted after the wet thalli is subjected to ultrasonic disruption as a catalyst, using (S) -CHOH as a substrate, using an organic solvent as an auxiliary substrate, using a buffer solution with the pH value of 6-8 (preferably 6.5-7.5) as a reaction medium to form a reaction system, reacting at the temperature of 25-35 ℃ (preferably 30 ℃) and at the speed of 150-600rpm, completely reacting, and separating and purifying the reaction solution to obtain (3R,5S) -CDHH. The dosage of the catalyst is 3-50g/L (preferably 50g/L) of buffer solution based on the weight of wet bacteria, the initial concentration of the substrate is 2-700g/L (preferably 500g/L) of buffer solution, and the dosage of the auxiliary substrate is 5-70% (v/v) (preferably 40%) of the volume of the buffer solution.

The wet thallus obtained by fermentation culture of the engineering bacteria containing the coding gene of the recombinant carbonyl reductase mutant is prepared by the following method: inoculating the engineering bacteria containing the coding gene of the recombinant carbonyl reductase mutant into LB liquid culture medium containing 50 mug/mL kanamycin resistance at the final concentration, culturing for 8h at 37 ℃ and 180rpm, then inoculating into fresh LB liquid culture medium containing 50 mug/mL kanamycin resistance at the final concentration by 1% of inoculation amount by volume, and culturing at 37 ℃ and 180rpm until the thallus OD₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, performing induction culture at 28 deg.C for 12h, centrifuging at 4 deg.C and 8000rpm for 10min, discarding supernatant, and collecting wet thallus.

The organic solvent is a water-soluble organic solvent or a non-water-soluble organic solvent, the volume usage of the water-soluble organic solvent is 5-50% (preferably 40%) by volume of the buffer solution, the volume usage of the non-water-soluble organic solvent is 10-70% by volume of the buffer solution, and the water-soluble organic solvent is one of the following: dimethyl sulfoxide, N-dimethylformamide, isopropanol, acetone, ethanol, methanol, Tween-20 or Tween-80 (preferably isopropanol); the water-insoluble organic solvent is one of the following: tetrahydrofuran, dichloromethane, tert-amyl alcohol, toluene, xylene, n-octanol, n-hexane, ethyl acetate, butyl acetate, isobutyl acetate, isopropyl acetate or methyl tert-butyl ether.

The preparation of the carbonyl reductase pure enzyme comprises the following steps: the wet recombinant carbonyl reductase genetic engineering bacteria are washed for three times by physiological saline, 1g of the wet bacteria is added with 20mL of 100mM potassium phosphate buffer solution with pH of 7.0 to resuspend the wet bacteria, and ultrasonic disruption is carried out under ice bath conditions (60W, continuous 1s and intermittent 1s and continuous 30min) to obtain cell disruption solution. And centrifuging the cell disruption solution obtained after ultrasonic disruption at 8000rpm and 4 ℃ for 25min to obtain supernatant, namely the required crude enzyme solution, which is designated as crude enzyme solution I. Fractional salting-out of ammonium sulfate: after the cells are subjected to ultrasonic disruption, the crude enzyme solution I is continuously stirred in an ice-water bath, ammonium sulfate is slowly added until the final concentration is 50 percent of saturation, and the mixture is kept stand for 4 hours in a refrigerator at 4 ℃. Centrifuging at 8000rpm for 25min, discarding precipitate, adding ammonium sulfate to the supernatant until the final concentration is 70% saturation, and standing in refrigerator at 4 deg.C for 4 hr. The mixture was centrifuged again at 8000rpm for 25min, the supernatant was discarded, and the precipitate was reconstituted with an appropriate amount of 100mM potassium phosphate buffer, pH 7.0. The resulting solution was dialyzed against 20mM, pH7.0 potassium phosphate buffer at 4 ℃ overnight, and concentrated to give a crude enzyme solution II. DEAE Sepharose Fast Flow anion exchange chromatography: a DEAE Sepharose Fast Flow anion exchange column (1.6 cm. times.20 cm) was first equilibrated with 20mM, pH7.0 potassium phosphate buffer solution at an equilibrium Flow rate of 1.5mL/min, and after the base line was leveled, the dialyzed crude enzyme solution II was applied to the column at a loading Flow rate of 1.0 mL/min. After loading, the sample was first eluted with 20mM, pH7.0 pB potassium phosphate buffer at a flow rate of 1.5mL/min, and after the baseline was leveled, the sample was eluted with a 0-0.8M NaCl gradient. Collecting active part, and concentrating to obtain pure enzyme. The whole purification process was carried out at 4 ℃.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a recombinant carbonyl reductase mutant with reduced (S) -CHOH, compared with a wild enzyme, the mutant improves the substrate tolerance to 500g/L from 400g/L when the reaction is converted, shortens the reaction time by 2 times when catalyzing 400g/L of substrate (S) -CHOH, and improves the catalysis efficiency by 1.89-2.34 times respectively. Compared with a chemical method for preparing (3R,5S) -CDHH, the technology provided by the invention has the advantages that the stereoselectivity of the obtained product is high, the complicated chemical catalysis steps are simplified, the reaction conditions are mild, the requirement on equipment is low, the reaction cost is reduced, and the method is environment-friendly.

(IV) description of the drawings

FIG. 1 is an agarose gel electrophoresis of error-prone PCR products of carbonyl reductase genes; wherein lane 1 is Marker; lanes 2, 3, 4, 5, 6, 7Mn²⁺The resulting carbonyl reductase gene fragments were at concentrations of 0mM, 5mM, 10mM, 15mM, 20mM, and 25mM, respectively.

FIG. 2 is a diagram of agarose gel electrophoresis of a large primer PCR product of carbonyl reductase gene; wherein lane 1 is Marker; lanes 2, 3, 4, 5, 6, 7Mn²⁺The resulting carbonyl reductase gene fragments were at concentrations of 0mM, 5mM, 10mM, 15mM, 20mM, and 25mM, respectively.

FIG. 3 is a SDS-PAGE pattern of carbonyl reductase purification: lane M is the protein molecular weight Marker, lane 1 is the supernatant of the SCR disruption of the original strain of recombinant carbonyl reductase, lane 2 is the supernatant of the mut-Phe145Tyr/Thr152Ser disruption, lane 3 is the supernatant of the mut-Phe145Met/Thr152Ser disruption, lane 4 is the sample of the SCR purification of the original strain of recombinant carbonyl reductase, lane 5 is the sample of the mut-Phe145Tyr/Thr152Ser purification, and lane 6 is the sample of the mut-Phe145Met/Thr152Ser purification.

FIG. 4 is a schematic diagram of asymmetric synthesis of (3R,5S) -CDHH.

FIG. 5 is a diagram showing the result of optimizing the concentration of isopropanol in the coenzyme circulation system.

FIG. 6 is a graph showing the effect of organic solvent on the catalytic reaction.

FIG. 7 is a schematic representation of the detection of substrate (S) -CHOH.

FIG. 8 is a schematic representation of the detection of the product (3R,5S) -CDHHHPLC.

(V) detailed description of the preferred embodiments

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not indicated are those in routine experiments.

Example 1: acquisition of recombinant carbonyl reductase mutant

A recombinant strain (E.coli BL21(DE3)/pET28b-SCR) containing an expression vector pET28b-SCR is used as an original strain, and the catalytic activity and the substrate tolerance of carbonyl reductase to a substrate (S) -CHOH are further improved through random mutation and site-directed saturated mutation technologies.

(1) Error-prone PCR and Large primer PCR

Error-prone PCR upstream primer 1: 5'-TATGTCTTCGCCTACTCCCAAC-3'

Error-prone PCR downstream primer 2: 5'-TCTACCATGGCAAGAACGTCC-3'

Error-prone PCR by changing Mn in PCR system²⁺，Mg²⁺So that the erroneous bases are randomly incorporated into the amplified gene at a certain frequency, thereby obtaining a randomly mutated DNA population. The invention takes the plasmid DNA of the SCR gene (the nucleotide sequence is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO. 2) as a template to carry out PCR amplification, and the amplification conditions are as follows: 3min at 98 ℃; 30 cycles of 98 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1 min; extension was continued for 10min at 72 ℃. The PCR product was detected by 0.9% agarose gel electrophoresis, and the detection results are shown in FIG. 1. And (3) taking the PCR product with the mutation site recovered after amplification as a primer for amplifying plasmid DNA (large primer PCR) to obtain the recombinant vector with the mutation site. The large primer PCR program was as follows: 3min at 98 ℃; 10s at 98 ℃,5s at 55 ℃ and 6min at 72 ℃ for 30s, repeating 26 cycles; extension was continued for 10min at 72 ℃. The PCR product was detected by 0.9% agarose gel electrophoresis, and the detection results are shown in FIG. 2. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing kanamycin resistance at a final concentration of 50mg/L, and cultured at 37 ℃ for 12 hours. Randomly picking single bacteriumAfter enzyme activity screening, the colony obtains an activity-improved mutant.

(2) Site-directed saturation mutagenesis

Phe(F)145：

An upstream primer 3: 5' -ATGTCCTCTATTNNKGGCATGGTAGGC-3’

A downstream primer 4: 5' -GCCTACCATGCMNNAATAGAGGACAT-3’

Thr(T)152：

An upstream primer 5: 5' -GTAGGCGATCCGNNKGTAGGCGCTTAT-3’

A downstream primer 6: 5' -ATAAGCGCCTACMNNCGGATCGCCTAC-3’

Gly(G)92：

An upstream primer 7: 5' -GTGAACAACGCCNNKTATCGGTGTGGT-3’

A downstream primer 8: 5' -ACCACACCGATAMNNGGCGTTGTTCAC-3’

Trp(W)206：

An upstream primer 9: 5' -GCGGAAGAAATGNNKTCCCAGCGTACT-3’

A downstream primer 10: 5' -AGTACGCTGGGAMNNCATTTCTTCCGC-3’

Met(M)141：

An upstream primer 11: 5' -AGCATCATCAACNNKTCCTCTATTTTC-3’

A downstream primer 12: 5' -GAAAATAGAGGAMNNGTTGATGATGCT-3’

Gly(G)94：

An upstream primer 13: 5' -AACGCCGGTATCNNKGTGGTCAAATCT-3’

A downstream primer 14: 5' -AGATTTGACCACMNNGATACCGGCGTT-3’

Val(V)96：

An upstream primer 15: 5' -GGTATCGGTGTGNNKAAATCTGTTGAA-3’

A downstream primer 16: 5' -TTCAACAGATTTMNNCACACCGATACC-3’

Met(M)195：

An upstream primer 17: 5' -ATTAAAACCCCTNNKCTGGACGACGTT-3’

A downstream primer 18: 5' -AACGTCGTCCAGMNNAGGGGTTTTAAT-3’

Lys(K)211：

An upstream primer 19: 5' -TCCCAGCGTACTNNKACCCCGATGGGC-3’

A downstream primer 20: 5' -GCCCATCGGGGTMNNAGTACGCTGGGA-3’

Trp(W)249：

An upstream primer 21: 5' -ATCGATGGTGGCNNKACCGCACAGTAA-3’

A downstream primer 22: 5' -TTACTGTGCGGTMNNGCCACCATCGAT-3’

Val(V)153：

An upstream primer 23: 5' -GGCGATCCGACTNNKGGCGCTTATAAC-3’

A downstream primer 24: 5' -GTTATAAGCGCCMNNAGTCGGATCGCC-3’

Val(V)199：

An upstream primer 25: 5' -ATGCTGGACGACNNKGAGGGCGCGGAA-3’

The downstream primer 26: 5' -TTCCGCGCCCTCMNNGTCGTCCAGCAT-3’

Ile(I)249：

An upstream primer 27: 5' -GCTGAATTCGTANNKGATGGTGGCTGG-3’

The downstream primer 28: 5' -CCAGCCACCATCMNNTACGAATTCAGC-3’

Saturated mutant primers are as described above, and the mutation sites are underlined. Similarly, using plasmid DNA containing the SCR gene as a template, mutations were introduced by PCR, and the PCR reaction procedure was as follows: 3min at 98 ℃; 10s at 98 ℃,5s at 55 ℃ and 6min at 72 ℃ for 30s, repeating 26 cycles; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing kanamycin resistance at a final concentration of 50mg/L, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony for sequencing analysis to obtain different mutant amino acids of different mutation points, and then carrying out enzyme activity determination analysis to determine the optimal mutation point.

(4) Site-directed mutagenesis

Asn(N)90Phe(F)：

An upstream primer 3: 5' -ACCGTCGTGAACTTCGCCGGTATCGGT-3’

A downstream primer 4: 5' -ACCGATACCGGCGAAGTTCACGACGGT-3’

Pro(P)190Phe(F)：

An upstream primer 5: 5' -GTACATCCGGGTTTCATTAAAACCCCT-3’

A downstream primer 6: 5' -AGGGGTTTTAATGAAACCCGGATGTAC-3’

Asn(N)157Thr(T)：

An upstream primer 7: 5' -GTAGGCGCTTATACAGCGTCCAAAGGC-3’

A downstream primer 8: 5' -GCCTTTGGACGCTGTATAAGCGCCTAC-3’

Gly(G)117Ser(S)：

An upstream primer 9: 5' -GTGAACCTGGACTCAGTTTTCTTCGGT-3’

A downstream primer 10: 5' -ACCGAAGAAAACTGAGTCCAGGTTCAC-3’

Ile(I)223Val(V)：

An upstream primer 11: 5' -GAGCCGAACGACGTCGCATGGGTATGT-3’

A downstream primer 12: 5' -ACATACCCATGCGACGTCGTTCGGCTC-3’

Gly(G)7Ser(S)：

An upstream primer 11: 5' -GATCGTCTGAAATCCAAGGTAGCTATT-3’

A downstream primer 12: 5' -AATAGCTACCTTGGATTTCAGACGATC-3’

Site-directed mutagenesis primers are as described above, and the mutation sites are underlined. Similarly, using plasmid DNA containing the SCR gene as a template, mutations were introduced by PCR, and the PCR reaction procedure was as follows: 3min at 98 ℃; 10s at 98 ℃,5s at 55 ℃ and 6min at 72 ℃ for 30s, repeating 26 cycles; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing kanamycin resistance at a final concentration of 50mg/L, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony for sequencing analysis to obtain different mutant amino acids of different mutation points, and then carrying out enzyme activity determination analysis to determine the optimal mutation point.

(5) Screening for Positive clones

Positive clones on plates were randomly selected for 96 wellsTo the plate, 800. mu.L of LB medium (50. mu.g/mL, Kan) was added, and the plate was incubated overnight at 37 ℃ and 180 rpm. 50 μ L of the seed solution was transferred to another new 96-well plate, cultured at 37 ℃ for 4 hours at 150rpm, then IPTG (0.1mM) was added, and the plate was transferred to 28 ℃ and cultured for 16 hours. The obtained cells were centrifuged at 1000rpm for 30min in a 96-well plate centrifuge, and 180. mu.L of phosphate buffer solution of pH7.0 was added to each well to resuspend the cells, 120. mu.L of isopropanol containing 200mM (S) -CHOH was added thereto, and the reaction was carried out at 30 ℃ and 180rpm for 30 min. 30. mu.L of the reaction solution was pipetted into each well and 30. mu.L of 20mM 2, 4-dinitrobenzene (1ml of 3% concentrated sulfuric acid in ethanol dissolved in 3.9628mg of 2, 4-dinitrobenzonitrile) was added to each well. Keeping the temperature at 37 ℃ for 15min, respectively adding 150 mu L of 6M NaOH, and determining the OD of the reaction system₄₈₀The variation value of (c). Correspondingly, the higher the enzyme activity of the mutant, the lighter the color of the reaction solution, and the OD₄₈₀The smaller the absorbance value is, the more active mutants can be selected, and the primary screening schematic diagram is shown in FIG. 3.

We obtained a series of different amino acid mutants by analysis of saturation mutations at positions Phe (F)145, Thr (T)152, Gly (G)92, Trp (W)206, Met (M)141, Gly (G)94, Val (V)96, Met (M)195, Lys (K)211, Trp (W)249, Val (V)153, Val (V)199, Ile (I) 249. The optimal mutant is determined by comparing the conversion rate of different mutants in catalyzing a substrate. The transformation reaction was carried out in a 10mL transformation flask, the substrate ((S) -CHOH) concentration was 100g/L, isopropanol was 4mL, the mutant wet cells were 15g/L, the reaction was magnetically stirred at 0 ℃ and 600rpm for 20min, and the reaction solution was analyzed by HPLC to determine the conversion rate of the reaction and to determine the optimum mutant. A schematic diagram of activity detection of each mutant by site-directed saturation mutagenesis is shown in FIG. 4. Similarly, site-directed mutagenesis can be used to determine the optimal mutant by comparing the conversion rates of different mutants in catalyzing a substrate. The transformation reaction was carried out in a 10mL transformation flask, the concentration of substrate (S) -CHOH) was 100g/L, isopropanol was 4mL, the mutant wet cells were 15g/L, the reaction was carried out at 30 ℃ and 600rpm with magnetic stirring for 20min, and the optimum mutant was determined by determining the conversion rate of the reaction by HPLC analysis of the reaction solution.

The results show that the optimal mutants obtained at two different mutation sites by random mutation, site-directed saturation mutation and site-directed mutation methods are mut-Phe145Met, mut-Phe145Tyr and mut-Thr152 Ser.

(6) Multiple point combinatorial mutation

Phe(F)145Met(M)/Thr(T)152Ser(S)：

An upstream primer 3: 5' -ATGTCCTCTATTATGGGCATGGTAGGC-3’

A downstream primer 4: 5' -GCCTACCATGCCCATAATAGAGGACAT-3’

Phe(F)145Tyr(Y)/Thr(T)152Ser(S)：

An upstream primer 3: 5' -ATGTCCTCTATTTACGGCATGGTAGGC-3’

A downstream primer 4: 5' -GCCTACCATGCCGTAAATAGAGGACAT-3’

The multi-point combination mutation primers are as described above, and the mutation sites are underlined. Mutations were introduced by PCR using plasmid DNA containing the mut-Thr (152) Ser (S) gene as a template, and the PCR reaction procedure was as follows: 3min at 98 ℃; 10s at 98 ℃,5s at 55 ℃ and 6min at 72 ℃ for 30s, repeating 26 cycles; extension was continued for 10min at 72 ℃. The PCR product was treated with DpnI at 37 ℃ for 3 hours, inactivated, transformed into E.coli BL21(DE3) recipient bacteria, spread on LB solid plates containing kanamycin resistance at a final concentration of 50mg/L, and cultured at 37 ℃ for 12 hours. Randomly picking a single colony for sequencing analysis to obtain different mutant amino acids of different mutation points, and then carrying out enzyme activity determination analysis to determine the optimal mutation point. The results show that the optimal mutants obtained by the multi-point combined mutation method after combined mutation at two different mutation sites are mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152 Ser. Transferred into Escherichia coli (BL21(DE3) to obtain recombinant Escherichia coli, namely recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Met/Thr152Ser and recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Tyr/Thr152 Ser.

Example 2: preparation of recombinant carbonyl reductase mutant wet thallus

The recombinant Escherichia coli (recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Met/Thr152 Ser), recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Tyr/Thr152Ser) containing the gene expressing the recombinant carbonyl reductase mutant obtained in example 1 was inoculated into LB broth containing kanamycin resistance at a final concentration of 50. mu.g/mL, cultured at 37 ℃ and 180rpmCulturing for 8h, inoculating with 1% inoculum size (v/v) to fresh LB liquid medium containing kanamycin resistance with final concentration of 50 μ g/mL, culturing at 37 deg.C and 180rpm until the thallus OD₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, inducing and culturing at 28 deg.C for 12h, centrifuging at 4 deg.C and 8000rpm for 10min, discarding supernatant, and collecting precipitate to obtain recombinant Escherichia coli wet thallus containing gene for expressing recombinant carbonyl reductase mutant. The wet thallus can be directly used as a biocatalyst or used for protein purification. Recombinant E.coli (BL21(DE3)/pET28b-SCR) wet cells containing the gene for expressing the recombinant carbonyl reductase were prepared in the same manner.

Example 3: isolation and purification of carbonyl reductase mutant

The wet cells obtained in example 3 were washed three times with physiological saline, and 1g of the wet cells were resuspended in 20mL of 20mM pH7.0 potassium phosphate buffer, and disrupted by sonication in ice bath (60W, 1s continuous and 1s intermittent, and 30min continuous). And centrifuging the cell disruption solution obtained after ultrasonic disruption at 8000rpm and 4 ℃ for 25min to obtain supernatant, namely the required crude enzyme solution, which is designated as crude enzyme solution I.

Fractional salting-out of ammonium sulfate: determination of the optimum precipitation concentration, the most common salt used in salting out is (NH)₄)₂SO₄、Na₂SO₄、MgSO₄。(NH₄)₂SO₄Because of its high solubility and small temperature coefficient, the separation effect is good, generally will not denature protein, and cheap and available, become the most commonly used salt in the salting-out step. Because the protein can be removed from the hydration layer under the influence of high-concentration neutral salt, the carried charges are neutralized, the colloidal stability of the protein is damaged and separated out, and meanwhile, the salting-out function of the protein is a reversible process and can be redissolved when diluted by water or buffer solution. Thus, as the saturation of ammonium sulfate increases, the protein content in the supernatant gradually decreases; on the contrary, the amount of protein precipitated from the precipitate gradually increased. The method is to add 5mL of crude enzyme solution into 8 small beakers numbered 1-8. 0.57g, 0.88g, 1.215g, 1.565g, 1.95g, 2.36g, 2.805g, 3.31g of ground ammonium sulfate were added to the crude enzyme solutionThe saturation degrees of the ammonium sulfate respectively reach 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%. Slowly adding ammonium sulfate, stirring on ice to dissolve, and standing in a refrigerator at 4 deg.C for 4 hr. And respectively taking out the liquid in the beaker, centrifuging (8000rpm, 25min), taking the supernatant, weighing, calculating the volume of the supernatant, and measuring the enzyme activity and the protein content. The ammonium sulfate fractional salting-out specific activity diagram and the ammonium sulfate fractional salting-out protein precipitation rate diagram are obtained through calculation, and two saturations with the highest specific activity and the highest protein precipitation rate can be found, namely the optimal precipitation saturation of the hybrid protein and the optimal precipitation saturation of the target protein, so that a large amount of hybrid protein can be removed; salting out ammonium sulfate in stages, carrying out ultrasonic cell disruption, continuously stirring the crude enzyme solution I in an ice water bath, slowly adding ammonium sulfate until the final concentration is 50% saturation, and standing for 4h in a refrigerator at 4 ℃. Centrifuging at 8000rpm for 25min, discarding precipitate, adding ammonium sulfate to the supernatant until the final concentration is 70% saturation, and standing in refrigerator at 4 deg.C for 4 hr. The mixture was centrifuged again at 8000rpm for 25min, the supernatant was discarded, and the precipitate was reconstituted with an appropriate amount of 20mM potassium phosphate buffer, pH 7.0. The resulting solution was dialyzed against 20mM, pH7.0 potassium phosphate buffer at 4 ℃ overnight, and concentrated to give a crude enzyme solution II.

DEAE Sepharose Fast Flow anion exchange chromatography: ion exchange chromatography is a method of separation by reversible ion exchange between ion exchange groups coupled to a stationary phase and ionic compounds dissociated from a mobile phase. Most of the preliminary chromatographic purification processes for oxidoreductases employ anion exchange chromatography. A DEAE Sepharose Fast Flow anion exchange column (1.6 cm. times.20 cm) was first equilibrated with 20mM, pH7.0, pB potassium phosphate buffer solution at an equilibrium Flow rate of 1.5mL/min, and after the base line was leveled, the dialyzed crude enzyme solution II was applied to the column at a loading Flow rate of 1.0 mL/min. After the sample is loaded, the sample is eluted by 20mM potassium phosphate buffer solution with pH7.0 at the elution flow rate of 1.5mL/min, and after the base line is leveled, the sample is eluted by a 0-0.8M NaCl gradient. Collecting active part, and concentrating to obtain pure enzyme. The whole purification process was carried out at 4 ℃. And (3) taking trapped fluid, determining the protein content by adopting a BCA kit method, and freezing and storing the trapped fluid in a refrigerator at the temperature of-80 ℃ (the protein electrophoresis diagram of the carbonyl reductase mutant is shown in figure 3) to obtain carbonyl reductase and mutants SCR, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser pure enzymes thereof.

Example 4: determination of carbonyl reductase activity and apparent kinetic parameters

The carbonyl reductase SCR separated and purified in the example 3 and the mutants mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser thereof are used for catalyzing the substrate (S) -CHOH.

The enzyme activity catalytic system comprises the following components under the catalytic conditions: 5mL, 100mM, pH7.0 phosphate buffer was added carbonyl reductase mutant pure enzyme (final concentration SCR 2.5. mu.g/mL, mut-Phe145 Met/Thr152Ser1.8. mu.g/mL and mut-Phe145 Tyr/Thr152Ser1.80. mu.g/mL) diluted with the same buffer solution, and initial rates at final concentrations of NAD (P) H of 0.5, 1.0, 1.5, 2.0 and 2.5mM were determined when the final concentration of (S) -CHOH was 0 mM; initial rates at final concentrations of NAD (P) H of 0.5, 1.0, 1.5, 2.0 and 2.5mM were determined at final (S) -CHOH concentrations of 0.5 mM; initial rates at final concentrations of NAD (P) H of 0.5, 1.0, 1.5, 2.0 and 2.5mM were determined at a final (S) -CHOH concentration of 0.75 mM; initial rates were determined for NAD (P) H final concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5mM when the final (S) -CHOH concentration was 1.0 mM; initial rates at final concentrations of NAD (P) H of 0.5, 1.0, 1.5, 2.0 and 2.5mM were determined at a final (S) -CHOH concentration of 1.25 mM; when the final concentration of (S) -CHOH was 1.5mM, the initial rates of NAD (P) H at final concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5mM were determined, and the reaction was carried out at 30 ℃ and 600rpm for 3min by preheating for 3 min. 5mL of 30% acetonitrile aqueous solution with volume concentration is added to terminate the reaction, and sampling is carried out after uniform mixing to detect the enzyme activity. Under the same conditions, the control solutions were obtained by dialyzing disrupted supernatants of E.coli BL21(DE3) and E.coli BL21(DE3)/pET28 b.

TABLE 1 results of apparent kinetic parameters of SCR and mutants

The enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. mu. mol of the product (3R,5S) -CDHH within 1min at 30 ℃ and pH7.0 was defined as 1U. The amount of product formed was determined by HPLC detection. The results are shown in Table 1.

Example 5: establishment of recombinant carbonyl reductase coenzyme regeneration system

The recombinant Escherichia coli BL21(DE3)/pET28b-SCR wet bacteria obtained by the method of example 2 were used as a biocatalyst, and (S) -CHOH was used as a substrate.

(1) And a single-enzyme double-substrate coupling system is selected for a coenzyme regeneration system of the carbonyl reductase SCR, and the production cost is saved when isopropanol is selected as the coenzyme regeneration system.

(2) Optimization of isopropanol concentration in coenzyme cyclic regeneration system

The recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Tyr/Thr152Ser containing the expression recombinant plasmid obtained by the method of example 2 has the optimal catalytic activity, so the recombinant bacterium is used as a catalyst in the catalytic reaction condition optimization process, and (S) -CHOH is used as a substrate.

Conversion system composition and catalytic conditions: 10mL of potassium phosphate buffer solution (100mM, pH7.0), wet recombinant carbonyl reductase mutant mut-Phe145Tyr/Thr152Ser bacteria (15 g/L buffer solution), 100g/L of buffer solution as substrate, and 10%, 20%, 30%, 40%, 50%, 60% and 70% of isopropanol in volume. Reacting in a magnetic stirring water bath kettle at the speed of 600rpm for 20min at the temperature of 30 ℃. After the reaction is finished, adding 1mL of acetonitrile to terminate the reaction, fully and uniformly mixing the reaction solution, and taking a proper amount of the reaction solution to detect the conversion rate by HPLC. The optimization results are shown in fig. 5.

The results show that the coenzyme cycle is substantially able to meet the requirement of carbonyl reductase for NADPH when the volume concentration of isopropanol is 40%, the above catalytic reaction product e.e. > 99%.

Example 6: effect of organic solvents on recombinant carbonyl reductase in preparation of (3R,5S) -CDHH applications

The recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Tyr/Thr152Ser wet cell containing the expression recombinant plasmid obtained by the method of example 2 was used as a biocatalyst, and (S) -CHOH was used as a substrate.

(1) Effect of organic solvents on the conversion reaction

Conversion system composition and catalytic conditions: 10mL of potassium phosphate buffer solution (100mM, pH7.0), recombinant carbonyl reductase mutant mut-Phe145Tyr/Thr152Ser wet bacteria (the dosage is 15g/L buffer solution), the volume concentration of isopropanol is 40%, the final concentration of substrate is 100g/L buffer solution, and a certain amount of organic solvent is added. Reacting in a magnetic stirring water bath kettle at the speed of 600rpm for 20min at the temperature of 30 ℃. After the reaction is finished, 1mL of acetonitrile is added to terminate the reaction, the reaction solution is fully and uniformly mixed, a proper amount of reaction solution is taken, the conversion rate is detected by HPLC, and the screening result is shown in figure 6.

The water-soluble organic solvent is: dimethyl sulfoxide (DMSO), N-Dimethylformamide (DMF), Isopropanol (IPA), Acetone (Acetone), Ethanol (Ethanol), Methanol (Methanol), Tween-20 (Tween-20), Tween-60 (Tween-60) and Tween-80 (Tween-80), wherein the addition amount in the reaction system is 5% (v/v).

The water-insoluble organic solvent is: tetrahydrofuran (THF), methylene Chloride (CH)₂Cl₂) T-amyl alcohol (tAmyl-OH), Toluene (Toluene), Xylene (Xylene), n-Octanol (Octanol), n-Hexane (c-Hexane), ethyl acetate (EtOAc), butyl acetate (BuOAc), isobutyl acetate (iBuOAc), isopropyl acetate (iPrOAc), and methyl t-butyl ether (MTBE) were added in an amount of 10% (v/v) to the reaction system.

The results show that when p-xylene, isopropyl acetate, isobutyl acetate and cyclohexane are used, the conversion rate of the catalytic reaction is obviously improved.

Example 7: application of recombinant carbonyl reductase SCR in preparation of (3R,5S) -CDHH

(1) (3R,5S) -CDHH was prepared by biotransformation reaction using (S) -CHOH as substrate and the recombinant Escherichia coli BL21(DE3)/pET28b-SCR wet cell containing the recombinant plasmid obtained in example 2 as biocatalyst. Examples 9-12 are comparative.

The composition of the catalytic system and the catalytic conditions are as follows: to 10mL of the reaction system, 6mL of potassium phosphate buffer solution (pH7.0) was added, wet cells of recombinant carbonyl reductase SCR (used in an amount of 50g/L buffer solution) were added, 4mL of isopropanol was added, the final concentration of the initial substrate was 400g/L, a 30 ℃ water bath was used, a magnetic stirrer was used at 600rpm, the reaction was periodically sampled, the sampling volume was 100. mu.L, the reaction mixture was diluted 50-fold with 30% by volume of acetonitrile aqueous solution, and the conversion rate was measured by HPLC analysis. The result shows that the yield of the product (3R,5S)) -6-chloro-3, 5-dihydroxy-tert-butyl hexanoate in the presence of the catalyst for 8 hours reaches 94.1%, and the yield of the product is e.e. > 99%.

(2) Liquid phase detection method of (S) -CHOH and (3R,5S) -CDHH

High performance liquid chromatography instrument: shimadzu LC-20AD system-SPD-20A ultraviolet detector

And (3) detecting the conversion rate by adopting a chromatographic column: agilent Zorbax SB-C8 column (150X 4.6mm,5 μm), mobile phase: acetonitrile: water 30: 70, flow rate of 1mL/min, detection wavelength 210 nm. The retention times of tert-butyl (S) -CHOH and (3R,5S)) -6-chloro-3,5-dihydroxyhexanoate were 10.104min (FIG. 7) and 6.588min (FIG. 8), respectively.

The e.e. was detected using a chiral chromatography OD-H column (250 × 4.6mm,5 μm), mobile phase: n-hexane: isopropanol 85: 15, flow rate 1mL/min, detection wavelength 215 nm. The retention times of (S) -CHOH with (3R,5S) -CDHH and tert-butyl (3S,5S) -6-chlorodihydroxyhexanoate were 5.99min, 5.09min and 4.93min, respectively.

Example 8: application of recombinant carbonyl reductase SCR in preparation of (3R,5S) -CDHH

(1) (3R,5S) -CDHH was prepared by biotransformation reaction using (S) -CHOH as substrate and the recombinant Escherichia coli BL21(DE3)/pET28b-SCR wet cell containing the recombinant plasmid obtained in example 2 as biocatalyst. Examples 9-12 are comparative.

The composition of the catalytic system and the catalytic conditions are as follows: to 10mL of the reaction system, 6mL of potassium phosphate buffer solution (pH7.0) was added, wet cells of recombinant carbonyl reductase SCR (used in an amount of 50g/L buffer solution) were added, 4mL of isopropanol was added, the final concentration of the initial substrate was 500g/L, a 30 ℃ water bath was used, a magnetic stirrer was used at 600rpm, the reaction was periodically sampled, the sampling volume was 100. mu.L, the reaction mixture was diluted 50-fold with 30% by volume of acetonitrile aqueous solution, and the conversion rate was measured by HPLC analysis. The result shows that the yield of the product (3R,5S) 6-chloro-3,5-dihydroxy tert-butyl hexanoate in the catalysis of 8h reaches 68.4%, and the yield of e.e. > 99%.

Example 9: application of recombinant carbonyl reductase mutant mut-Phe145Tyr in preparation of (3R,5S) -CDHH

(3R,5S) -CDHH was prepared by biotransformation reaction using the recombinant E.coli BL21(DE3)/pET28b-mut-Phe145Tyr wet cell containing the recombinant plasmid obtained in example 2 as a biocatalyst and (S) -CHOH as a substrate.

The composition of the catalytic system and the catalytic conditions are as follows: to 10mL of the reaction system, 6mL of potassium phosphate buffer solution (pH7.0) was added, wet cells of the recombinant carbonyl reductase mutant mut-Phe145Tyr (in an amount of 50g/L buffer solution) and 4mL of isopropyl alcohol were added, the final concentration of the initial substrate was 500g/L, a 30 ℃ water bath was used, the reaction was carried out at 600rpm, samples were taken at regular time, the sample volume was 100. mu.L, the sample was diluted 50-fold with 30% by volume of acetonitrile aqueous solution, and the conversion rate was measured by HPLC analysis. The results show that the yield of the product (3R,5S) -CDHH after 8h of catalysis reached 76.8%, e.e. > 99% at a substrate concentration of 500 g/L.

Example 10: application of recombinant carbonyl reductase mutant mut-Phe145Met in preparation of (3R,5S) -CDHH

(3R,5S) -CDHH was prepared by biotransformation reaction using wet cells of recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Met containing the recombinant plasmid obtained in example 2 as biocatalyst and (S) -CHOH as substrate.

The composition of the catalytic system and the catalytic conditions are as follows: to 10mL of the reaction system, 6mL of potassium phosphate buffer solution (pH7.0) was added, wet cells of recombinant carbonyl reductase mutant mut-Phe145Met (in an amount of 50g/L buffer solution) were added, 4mL of isopropanol was added, the final concentration of the initial substrate was 500g/L, a 30 ℃ water bath was used, the reaction was carried out at 600rpm, sampling was carried out at regular time, the sampling volume was 100. mu.L, the reaction solution was diluted 50-fold with 30% by volume of acetonitrile aqueous solution, and the conversion rate was determined by HPLC analysis. The results show that the yield of the product (3R,5S) -CDHH after 8h of catalysis reached 70.5%, e.e. > 99% at a substrate concentration of 500 g/L.

Example 11: application of recombinant carbonyl reductase mutant mut-Thr152Ser in preparation of (3R,5S) -CDHH

(3R,5S) -CDHH was prepared by biotransformation reaction using (S) -CHOH as substrate, using the recombinant Escherichia coli BL21(DE3)/pET28b-mut-Thr152Ser wet cell containing the recombinant plasmid obtained in example 2 as biocatalyst.

The composition of the catalytic system and the catalytic conditions are as follows: to 10mL of the reaction system, 6mL of potassium phosphate buffer solution (pH7.0) was added, wet recombinant carbonyl reductase mutant mut-Thr152Ser (50 g/L buffer solution) was added, 4mL of isopropanol was added, the final initial substrate concentration was 500g/L, a 30 ℃ water bath was used, the reaction was carried out at 600rpm, the sample was taken at regular time, the sample volume was 100. mu.L, the sample was diluted 50-fold with 30% acetonitrile aqueous solution, and the conversion rate was measured by HPLC analysis. The results show that the yield of the product (3R,5S) -CDHH after 8h of catalysis reached 78.7%, e.e. > 99% at a substrate concentration of 500 g/L.

Example 12: application of recombinant carbonyl reductase mutant mut-Phe145Met/Thr152Ser in preparation of (3R,5S) -CDHH

(3R,5S) -CDHH was prepared by biotransformation reaction using (S) -CHOH as substrate and wet cell of recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Met/Thr152Ser containing the recombinant plasmid obtained in example 2 as biocatalyst.

The composition of the catalytic system and the catalytic conditions are as follows: to 10mL of the reaction system, 6mL of potassium phosphate buffer solution (pH7.0) was added, wet cells of recombinant carbonyl reductase mutant mut-Phe145Met/Thr152Ser (in an amount of 50g/L buffer solution) and 4mL of isopropanol were added, the final initial substrate concentration was 500g/L, a 30 ℃ water bath was carried out, a magnetic stirrer was used at 600rpm, samples were taken at regular time of the reaction, the sample volume was 100. mu.L, the sample was diluted 50-fold with 30% acetonitrile aqueous solution, and the conversion rate was determined by HPLC analysis. The results show that the yield of the product (3R,5S) -CDHH after 8h of catalysis reached 80.7%, e.e. > 99% at a substrate concentration of 500 g/L.

Example 13: application of recombinant carbonyl reductase mutant mut-Phe145Tyr/Thr152Ser in preparation of (3R,5S) -CDHH

(3R,5S) -CDHH was prepared by biotransformation reaction using (S) -CHOH as a substrate, using the recombinant Escherichia coli BL21(DE3)/pET28b-mut-Phe145Tyr/Thr152Ser wet cells containing the recombinant plasmid obtained in example 2 as a biocatalyst.

The composition of the catalytic system and the catalytic conditions are as follows: to 10mL of the reaction system, 6mL of potassium phosphate buffer (pH7.0) was added, wet cells of recombinant carbonyl reductase mutant mut-Phe145Tyr/Thr152Ser (in an amount of 50g/L buffer) and 4mL of isopropanol were added, the final initial substrate concentrations were 400g/L, 500g/L, 600g/L and 700g/L, respectively, a 30 ℃ water bath, a magnetic stirrer at 600rpm, sampling at regular time, the sampling volume was 100. mu.L, the samples were diluted 50-fold with 30% by volume aqueous acetonitrile, and the conversion rate was determined by HPLC analysis. The results show that the yield of the product (3R,5S) -CDHH after 4h of catalysis reaches 98.7% at a substrate concentration of 400g/L, e.e. > 99%; the yield of the product (3R,5S) -CDHH after 8h of catalysis reaches 98.7% at a substrate concentration of 500g/L, e.e. > 99%; the yield of the product (3R,5S) -CDHH after 12h of catalysis at a substrate concentration of 600g/L reaches 90.73%, e.e. > 99%; the e.e. value was always greater than 99% at a substrate concentration of 700g/L, with a product yield of 52.45%.

Sequence listing

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Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg Arg Ala Asp Val Gly Glu Arg Ala Ala

35 40 45

Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Gly Val Val

85 90 95

Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Phe Gly Met Val Gly Asp Pro Thr Val Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys

180 185 190

Thr Pro Met Leu Asp Asp Val Glu Gly Ala Glu Glu Met Trp Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Val Cys Val Tyr Leu Ala Ser Gly Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Ile Asp Gly Gly Trp Thr Ala Gln

245 250

Claims (8)

1. A recombinant carbonyl reductase mutant, characterized in that the mutant is one of the following: (1) the 145 th phenylalanine of the amino acid sequence shown in SEQ ID NO.2 is mutated into methionine; (2) mutating threonine 152 th of the amino acid sequence shown in SEQ ID NO.2 into serine; (3) the 145 th phenylalanine of the amino acid sequence shown in SEQ ID NO.2 is mutated into tyrosine, and the 152 th threonine is mutated into serine.

2. A gene encoding the recombinant carbonyl reductase mutant of claim 1.

3. A recombinant genetically engineered bacterium constructed from the coding gene of claim 2.

4. Preparation of a recombinant carbonyl reductase mutant as claimed in claim 1(3R,5S) -6-chloro-3, 5-dihydroxyhexanoic acid tert-butyl ester.

5. The use according to claim 4, characterized in that the use is: wet thallus obtained by fermentation and culture of engineering bacteria containing recombinant carbonyl reductase mutant coding gene or pure enzyme extracted after ultrasonic crushing of wet thallus is used as catalyst, and (B) isS) Taking tert-butyl (6-chloro-5-hydroxy-3-oxohexanoate) as a substrate, taking an organic solvent as an auxiliary substrate, forming a reaction system in a buffer solution with the pH value of 6-8, reacting at the temperature of 25-35 ℃ and the rpm of 150-(3R,5S) -6-chloro-3, 5-dihydroxyhexanoic acid tert-butyl ester.

6. The use according to claim 5, wherein the amount of catalyst is 3-50g/L buffer solution based on the weight of wet cells, the initial concentration of substrate is 2-700g/L buffer solution, and the volume of co-substrate is 5-70% of the buffer solution.

7. The use according to claim 5, wherein the wet biomass is prepared by: inoculating the engineering bacteria containing the coding gene of the recombinant carbonyl reductase mutant into LB liquid culture medium containing 50 mug/mL kanamycin resistance at the final concentration, culturing for 8h at 37 ℃ and 180rpm, then inoculating into fresh LB liquid culture medium containing 50 mug/mL kanamycin resistance at the final concentration by 1% of inoculation amount by volume concentration, and culturing at 37 ℃ and 180rpm until the thallus OD₆₀₀The content of the active carbon is 0.6-0.8,adding IPTG with final concentration of 0.1mM, inducing culture at 28 deg.C for 12h, centrifuging at 4 deg.C and 8000rpm for 10min, discarding supernatant, and collecting wet thallus.

8. The use according to claim 5, wherein the organic solvent is a water-soluble organic solvent or a water-insoluble organic solvent, the volume of the water-soluble organic solvent is 5 to 50% by volume of the buffer solution, the volume of the water-insoluble organic solvent is 10 to 70% by volume of the buffer solution, and the water-soluble organic solvent is one of: dimethyl sulfoxide, N-dimethylformamide, isopropanol, acetone, ethanol, methanol, Tween-20 or Tween-80; the water-insoluble organic solvent is one of the following: tetrahydrofuran, dichloromethane, tert-amyl alcohol, toluene, xylene, n-octanol, n-hexane, ethyl acetate, butyl acetate, isobutyl acetate, isopropyl acetate or methyl tert-butyl ether.

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