CN105671010A - Aldehyde ketone reductase mutant, gene, engineering bacterium and application of mutant - Google Patents

Abstract

The invention discloses an aldehyde ketone reductase mutant, gene, engineering bacterium and an application in preparation of tert-butyl-6-cyano-(3R,5R)-dihydroxyl hexanoate. The aldehyde ketone reductase mutant is obtained by carrying out single mutation or amphimutation on 295th and 296th sites of an amino acid sequence shown in SEQ ID NO.1. Compared with wild type aldehyde ketone reductase, the aldehyde ketone reductase mutant Kluyveromyces lactis provided by the invention has the advantages that specific enzyme activity in catalysis of asymmetric reduction of tert-butyl-6-cyano-(5R)-hydroxyl-3-carbonyl hexanoate is greatly improved and is improved to 7.95U/mg from 0.56U/mg, namely the specific enzyme activity is improved by 14.2 times, and diastereomer selectivity of the tert-butyl-6-cyano-(3R,5R)-dihydroxyl hexanoate maintains to be more than 99.5%; and multiple aldehyde ketone reductase mutants obtained according to the invention are especially applicable to the catalysis of the asymmetric reduction of the tert-butyl-6-cyano-(5R)-hydroxyl-3-carbonyl hexanoate for preparing the tert-butyl-6-cyano-(3R,5R)-dihydroxyl hexanoate, thereby having a relatively good industrial application prospect.

Description

A kind of aldehyde and ketone reductase mutant, gene, engineering bacteria and application thereof

(1) Technical field

The present invention relates to a method for preparing an atorvastatin bichiral intermediate, in particular to a Kluyveromyces lactis aldo-ketone reductase mutant, a coding gene, a carrier, a recombinant engineering bacterium, and the aldo-ketone reductase in different Symmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate to prepare atorvastatin chiral intermediate 6-cyano-(3R,5R)-tert-butyl dihydroxyhexanoate application.

(2) Background technology

6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester is an important chiral intermediate in the synthesis process route of atorvastatin and also a key pharmacophore. Due to the strict restrictions set by the drug regulatory departments of various countries on the chiral purity of drugs (e.e. value>99.5%, d.e. value>99%), the synthesis technology of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate Become the key core technology for the synthesis of atorvastatin. The traditional chemical synthesis process of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate starts from ketoacids (esters) and constructs chiral centers through asymmetric synthesis. The reaction route is complex and requires the use of flammable and explosive Borane, n-butyllithium, etc. and special environments such as cryogenics lead to low product d.e. values, low yields, and high energy consumption. Moreover, the boride waste produced by the reaction is difficult to deal with, and needs to go through tedious repeated methanol quenching and vacuum distillation. Enzymes have excellent selectivity and mild reaction conditions, and are generally carried out under normal temperature, normal pressure and near-neutral conditions, which minimize adverse side reactions such as decomposition, isomerization, racemization, rearrangement, etc., and biocatalysis The technology has the basic conditions to improve the atomic economy of the process and realize the green and environment-friendly process. Therefore, the development of biological asymmetric reduction of 6-cyano-(5R)-hydroxyl-3-oxoylhexanoic acid tert-butyl ester to synthesize 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester technology has huge economic and social benefits.

Aldo-ketoreductase superfamily is a kind of NAD(P)H-dependent oxidoreductase, which is widely distributed in animal, plant and microbial cells, participates in intracellular metabolic reactions, and eliminates the adverse effects of harmful substances in the environment on cells. So far, more than 190 kinds of aldehydes and ketone reductases have been discovered, distributed in 16 families. Aldehyde and ketone reductase is usually a single-subunit protein composed of about 320 amino acids, with a size of 34-37kDa and a (α/β) ₈ cylindrical structure. Its catalytic quadruplex is composed of tyrosine (Tyr), histidine ( His), aspartic acid (Asp) and lysine (Lys), the substrate spectrum is broad, including aliphatic and aromatic aldehydes and ketones, monosaccharides, steroids and prostaglandins, etc. At present, many aldehyde and ketone reductase genes have been cloned and expressed in foreign hosts and are widely used in the asymmetric synthesis of chiral intermediates, some of which are used in the asymmetric reduction synthesis of atorvastatin intermediate 6-cyano-(3R ,5R)-tert-butyl dihydroxyhexanoate.

We have cloned the aldehyde and ketone reductase K1AKR from Kluyveromyceslactis CCTCCM2014380, and achieved heterologous overexpression in Escherichia coli (Enzyme and Microbial Technology 2015, 77:68-77). The enzyme can catalyze the asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate to synthesize tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate, and the de value of the product is greater than 99%. However, the activity of the enzyme to tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate is not high, which limits its industrial application. Through the reported crystal structure of aldehyde and ketone reductase, the spatial structure of the enzyme and the possible amino acid sites related to the activity were determined by means of molecular simulation, and the ability of aldehyde and ketone reductase to 6-cyano-(5R )-Hydroxy-3-oxoylhexanoic acid tert-butyl ester has a strong industrial application value.

(3) Contents of the invention

The object of the present invention is to provide a kind of aldehyde and ketone reductase mutation for the problem that the asymmetric reduction activity of aldehyde and ketone reductase K1AKR to 6-cyano-(5R)-hydroxyl-3-oxoylhexanoic acid tert-butyl ester is lower as reported before. Body protein and its coding gene, recombinant expression vector and recombinant engineering bacteria containing the gene, the crude enzyme liquid after the crushing of recombinant engineering bacteria expressing the aldehyde and ketone reductase mutant is used as a catalyst to catalyze 6-cyano-(5R)- Asymmetric reduction of tert-butyl hydroxy-3-oxohexanoate to prepare optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate. Compared with the wild-type aldehyde and ketone reductase, the aldehyde and ketone reductase mutant provided by the invention has higher catalytic activity.

The technical scheme adopted in the present invention is:

The present invention provides a mutant of aldehyde and ketone reductase, which is obtained by performing a single or double mutation on the 295th and 296th positions of the amino acid sequence shown in SEQ ID NO.1, preferably the aldehyde The ketoreductase mutant is obtained by performing a single mutation at position 295 of the amino acid sequence shown in SEQ ID NO.1 or performing double mutations at positions 295 and 296.

Further, preferably, the single mutation is to mutate the 295th tyrosine in the amino acid sequence shown in SEQ ID NO.1 to tryptophan, the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.5; The above double mutation is to mutate the 295th tyrosine in the amino acid sequence shown in SEQ ID NO.1 to tryptophan, and mutate the 296th tryptophan to leucine. Shown as SEQ ID NO.6.

The invention adopts the fixed-point saturation mutation technique to mutate the gene encoding the aldehyde and ketone reductase K1AKR, connects the expression vector, transforms the host Escherichia coli, induces the expression, and detects positive mutations with improved activity by a high-performance liquid phase detection method. The obtained mutant can catalyze the asymmetric reduction of (1～50g/L) tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate to prepare optically pure 6-cyano-(3R,5R)-di Tert-butyl hydroxyhexanoate, the specific method is as follows: the aldehyde and ketone reductase KlAKR (GenBankaccessionnumber: KU145407) cloned from K.lactisCCTCCM2014380 consists of 309 amino acid residues, and the expression vector pET28b-klakr has been successfully constructed, a functioning enzyme The protein was overexpressed in Escherichia coli BL21(DE3). Then, through homology modeling and molecular docking, potential sites that may affect enzyme activity are selected according to the optimal conformation. Set the site-directed saturation mutation site, then design and synthesize appropriate primers, and use the recombinant expression plasmid containing the parental aldehyde and ketone reductase gene as a template to amplify the plasmid of the full-length mutant gene by PCR. By transforming the plasmid containing the full-length mutation into an appropriate host cell, the positive mutant with high activity is selected by culturing, inducing expression, and screening. Finally, the plasmid DNA was extracted from the positive mutants and subjected to DNA sequencing analysis to determine the mutation of the primers. In the preparation of the aldehyde and ketone reductase mutants of the present invention, any suitable carrier can be used.

The present invention also provides a gene encoding the aldehyde and ketone reductase mutant, a recombinant vector and a recombinant genetically engineered bacterium constructed by the aldehyde and ketone reductase mutant encoding gene, preferably the recombinant plasmid is pET28b; the host cell is Escherichia coli E. coli BL21(DE3).

The present invention also relates to the application of the gene encoding the aldehyde and ketone reductase mutant in the preparation of the recombinant aldehyde and ketone reductase. In the host bacteria (preferably Escherichia coli), the obtained recombinant genetically engineered bacteria are induced and cultured, and the bacterial cells containing the recombinant aldehyde and ketone reductase are isolated from the culture medium, which has higher catalytic activity compared with the wild type aldehyde and ketone reductase .

The present invention also relates to an application of the aldehyde and ketone reductase mutant in the preparation of 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester. The wet thallus obtained by fermenting and cultivating the recombinant genetically engineered bacteria with the body gene is used as the catalyst, tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate is used as the substrate, and the glucose dehydrogenase gene is used as the substrate. The wet cells of glucose dehydrogenase obtained from the fermentation of engineering bacteria are coenzyme regeneration enzymes, glucose is used as an auxiliary substrate, and pH 7.0 and 100mM phosphate buffer are used as reaction media. The reaction medium is suspended, ultrasonically crushed, then the substrate and auxiliary substrate are added, and the reaction is carried out at 30°C and 200r/min. After the reaction is complete, tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is obtained The glucose dehydrogenase is fermented and cultivated to obtain wet thallus with the engineering bacteria (EscherichiaColiBL21(DE3)/pET28b-esgdh) containing the glucose dehydrogenase gene, and the specifically described recombinant genetically engineered bacteria EscherichiaColiBL21(DE3)/pET28b-esgdh is The glucose dehydrogenase gene (GenBankNo.KM817194.1) derived from Exiguobacterium sibiricum was inserted into pET-28b to construct a recombinant expression plasmid, and the recombinant plasmid was introduced into Escherichia coli to produce; the glucose dehydrogenase wet cell dosage was 10 -250g/L buffer solution (preferably 25g/L), the consumption of described catalyst is 10-250g/L buffer solution (preferably 75g/L) by wet thalline weight, and described substrate final concentration is 1-100g/L L buffer solution (preferably 50g/L), the final concentration of the glucose is 5-300g/L buffer solution (preferably 200g/L).

Further, the catalyst is prepared as follows: Inoculate the recombinant engineering bacteria containing the aldehyde and ketone reductase mutant gene into the LB liquid medium containing the final concentration of 50mg/L kanamycin, cultivate at 37°C for 10h, and then Concentration 4% was inoculated into fresh LB liquid medium containing kanamycin at a final concentration of 50mg/L, cultured at 37°C until the cell concentration _OD600 was 0.6-0.8, and then added to the culture medium with a final concentration of 9g/L lactose, cultured at 28°C for 12h, centrifuged at 8000g for 10min at 4°C, and collected bacterial cells. The preparation method of the wet thallus obtained from the fermentation culture of the engineered bacteria containing the glucose dehydrogenase gene is the same as that of the recombinant engineered bacteria containing the aldehyde and ketone reductase mutant gene. Further, the ultrasonic crushing conditions are as follows: place an ice bath in an ultrasonic crusher, crush with a power of 400W for 20 minutes, break for 1 second and stop for 1 second.

The glucose dehydrogenase wet cell preparation method of the present invention is: insert the glucose dehydrogenase gene (GenBank No. KM817194.1) derived from Exiguobacterium sibiricum into pET-28b to construct a recombinant expression plasmid, and introduce the recombinant plasmid into Escherichia coli BL21 (DE3) Obtain the recombinant genetically engineered bacteria containing the glucose dehydrogenase gene; inoculate the recombinant engineered bacteria containing the glucose dehydrogenase gene into LB liquid medium containing a final concentration of 50 mg/L kanamycin, and cultivate at 37° C. for 10 h , then inoculated into fresh LB liquid medium containing kanamycin at a final concentration of 50mg/L at a volume concentration of 4%, cultivated at 37°C until the _OD600 of the bacterial cell concentration was 0.6-0.8, and then added the final concentration of It is 9g/L lactose, cultured at 28°C for 12h, then centrifuged at 8000g for 10min at 4°C to collect bacterial cells.

The aldehyde and ketone reductase mutants of the present invention can be used in the form of whole cells of engineered bacteria, or in the form of unpurified crude enzymes or purified enzymes. If necessary, the aldehyde and ketone reductase mutants of the present invention can also be made into immobilized enzymes or immobilized cells using immobilization techniques known in the art.

Compared with the prior art, the beneficial results of the present invention are mainly reflected in: compared with the wild-type aldehyde and ketone reductase, the Kluyveromyceslactis aldehyde and ketone reductase mutant provided by the invention can asymmetrically reduce 6-cyano-(5R)- Hydroxy-3-oxoylhexanoic acid tert-butyl ester has a significant increase in catalytic specific enzyme activity (from 0.56U/mg to 7.95U/mg, an increase of 14.2 times), and the product 6-cyano-(3R,5R)-di The diastereoselectivity of tert-butyl hydroxyhexanoate was maintained above 99.5%. The multiple aldehyde and ketone reductase mutants obtained in the present invention are particularly suitable for catalyzing the asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate to prepare 6-cyano-(3R,5R) -Dihydroxyhexanoic acid tert-butyl ester has good industrial application prospect.

(4) Description of drawings

Figure 1 is the coupling of aldehyde and ketone reductase and glucose dehydrogenase to catalyze the asymmetric reduction of 6-cyano-(5R)-hydroxy-3-oxohexanoic acid tert-butyl ester to prepare 6-cyano-(3R,5R)-di Schematic diagram of the reaction of tert-butyl hydroxycaproate.

Fig. 2 is the effect of the mass ratio of the aldehyde and ketone reductase mutant Y295W/W296L and the recombinant glucose dehydrogenase cell on the asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate.

Figure 3 is the effect of glucose concentration on the asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate.

Figure 4 is the asymmetric reduction of 50g/L 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl ester catalyzed by the coupling of aldehyde and ketone reductase mutant KlAKR-Y295W/W296K with glucose dehydrogenase to prepare 6-cyanide The reaction process of tert-butyl-(3R,5R)-dihydroxyhexanoate.

(5) Specific implementation methods

The present invention can be better understood from the following examples. Those skilled in the art will easily understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in the claims.

The recombinant aldehyde and ketone reductase gene of the present invention is derived from Kluyveromyceslactis isolated from the environment, and is preserved in the China Center for Type Culture Collection, with a preservation date of August 14, 2014, a preservation number of CCTCCNO: M2014380, and a preservation address of China Wuhan, Wuhan University, postcode 430072, has been disclosed in the patent application (201510004669.2).

Embodiment 1: Preparation of aldehyde and ketone reductase mutants

The aldehyde and ketone reductase K1AKR gene (GenBankaccessionnumber: KU145407) cloned from KluyveromyceslactisCCTCCM2014380 (the nucleotide sequence is shown in SEQ ID NO.4, the amino acid sequence of the encoded protein is shown in SEQ ID NO.1), and the expression vector pET28b-klakr has been successfully constructed.

Aldoketone reductase mutants were prepared by two rounds of site-directed saturation mutagenesis. In the first round, the 295th tyrosine in the amino acid sequence of aldehyde and ketone reductase KlAKR shown in SEQ ID NO.1 was mutated into tryptophan, and the recombinant plasmid pET28b-klakr was used as a template for full plasmid amplification, and the primer pair was designed as Y295-F and For Y295-R (as shown in Table 1), the aldehyde and ketone reductase mutant Y295W (the amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence is shown in SEQ ID NO.5) was obtained. In the second round, the recombinant plasmid containing the aldehyde and ketone reductase mutant gene (that is, the nucleotide sequence shown in SEQ ID NO.5) containing 295 tyrosine mutations to tryptophan was used as a template for full plasmid amplification, and primer pairs were designed. For Y295W/W296-F and Y295W/W296-R (as shown in Table 1), the tyrosine at position 295 of the amino acid sequence shown in SEQ ID NO.1 was mutated into tryptophan, and the tyrosine at position 296 was Aldoketone reductase mutant Y295W/W296L in which tryptophan is mutated to leucine (the amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.6). The PCR system is: 10 μL of 5×PS buffer, 4 μL of dNTP (2.5 mM of each nucleotide), 0.5 μL of each mutation primer, 0.5 μL of template (recombinant plasmid), 0.5 μL of PrimeSTAR DNA polymerase, and replenish water to 50 μL. PCR conditions were: 95°C pre-denaturation for 5 minutes, followed by 27 cycles: 95°C for 15s, 56°C for 15s, 72°C for 7 minutes, and finally 72°C for 10 minutes. After the PCR was positive by 0.9% agarose gel electrophoresis analysis, take 20 μL of the PCR solution, add 1 μL of LpnI, digest at 37°C for 3 hours to remove the template plasmid, inactivate at 65°C for 10 minutes, transform the competent cells E.coliBL21(DE3), and coat LB plates containing kanamycin (50 μg/mL) were cultured overnight at 37°C. Confirmed by sequencing by Shanghai Sunny Biotechnology Co., Ltd., the aldehyde and ketone reductase mutant strains E.coliBL21(DE3)/pET28b-klakr-Y295W(Y295W) and E.coliBL21(DE3)/pET28b-klakr-Y295W/W295L were obtained (Y295W/W295L).

The recombinant wild-type aldehyde and ketone reductase strain E.coliBL21(DE3)/pET28b-klakr and the recombinant glucose dehydrogenase strain E.coliBL21(DE3)/pET28b-esgdh are the aldehyde and ketone reductase KlAKR genes cloned from KluyveromyceslactisCCTCCM2014380 respectively ( GenBankaccessionnumber: KU145407) and the glucose dehydrogenase gene (GenBankNo. KM817194.1) from Exiguobacterium sibiricum were inserted into pET-28b to construct a recombinant expression plasmid, and the recombinant plasmid was introduced into Escherichia coli BL21 (DE3) to obtain.

Table 1 Aldehyde and ketone reductase mutation primers

Example 2: Induced expression of aldehyde and ketone reductase parents and mutants and glucose dehydrogenase

The aldehyde and ketone reductase mutant strain E.coliBL21 (DE3)/pET28b-klakr-Y295W (Y295W) and E.coliBL21 (DE3) in the starting strain E.coliBL21 (DE3)/pET28b-klakr and embodiment 1 of embodiment 1 /pET28b-klakr-Y295W/W295L (Y295W/W295L) and recombinant glucose dehydrogenase strain E.coliBL21 (DE3)/pET28b-esgdh were inoculated into LB liquid medium containing a final concentration of 50 mg/L kanamycin, Cultivate at 37°C for 10 hours, then inoculate it into fresh LB liquid medium containing a final concentration of 50mg/L kanamycin at a volume concentration of 4%, and cultivate at 37°C until the cell concentration _OD600 is 0.6-0.8. Lactose with a final concentration of 9g/L was added to the mixture, cultured at 28°C for 12h, centrifuged at 8000g for 10min at 4°C, and the bacterial cells were collected. It can be used for enzyme purification and biocatalysis to prepare 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester.

Example 3: Purification of aldehyde and ketone reductase parents and mutants thereof

Suspend the aldehyde and ketone reductase bacterial cells described in Example 2 in buffer A (20 mM, pH 8.0 sodium phosphate buffer containing 500 mM NaCl and 20 mM imidazole), and sonicate for 20 min (ice bath, power 400W, break for 1 s and stop for 1 s) , centrifuge at 12000rpm for 20min at 4°C, and take the supernatant. Use a Ni affinity column (1.6cm × 10cm, Bio-Rad Company, USA) to purify the aldehyde and ketone reductase parent and its mutant protein, and the specific operations are as follows:

(1) Equilibrate the Ni column with the above bufferA;

(2) Pass the supernatant obtained by centrifugation after the above-mentioned ultrasonication through the Ni column at a flow rate of 1 mL/min, so that the target enzyme is adsorbed on the Ni column filler;

(3) Wash the Ni column with buffer A of 5 times the column volume above at a flow rate of 1 mL/min to elute unadsorbed protein;

(4) Use buffer B (20 mM, pH 8.0 sodium phosphate buffer containing 500 mM NaCland and 500 mM imidazole) to elute the target protein at a flow rate of 1 mL/min. Combine the collected solution containing the target enzyme activity, dialyze overnight in sodium phosphate buffer (20mM, pH8.0), collect the retentate, and obtain the enzyme solution of wild-type aldehyde and ketone reductase KLAKR and its mutants Y295W and Y295W/W296L respectively . All purification steps were performed at 4°C.

Embodiment 4: the determination of aldehyde and ketone reductase and mutant enzyme specific enzyme activity thereof

Enzyme activity unit (U) is defined as: at 30°C and pH 7.0, the amount of enzyme required to oxidize 1 micromole of NADH per minute is defined as 1U. Specific enzyme activity is defined as the number of units of activity per milligram of protein.

The protein concentration was determined with a biquinolinic acid protein assay kit (Nanjing KGI Biotechnology Development Co., Ltd., Nanjing).

Determination method of specific enzyme activity: use 6-cyano-(5R)-hydroxy-3-oxoylhexanoic acid tert-butyl ester as substrate, continuously monitor the decrease of NADH absorbance at 340nm to measure aldehyde and ketone reductase and its mutants catalytic activity. The enzyme activity detection system consists of potassium phosphate buffer (100mM, pH7.0), 0.5mMNADH, 0.5mM tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate and the aldehydes and ketones prepared in Example 3 Reductase or its mutant enzyme solution 0.5 μg/μL, the total volume is 200 μL.

The specific enzymatic activities of aldehyde and ketone reductase and its mutants are shown in Table 2.

Example 5: Determination of 6-cyano-(5R)-hydroxy-3-oxoylhexanoic acid tert-butyl ester diastereomers by aldehyde and ketone reductase and mutants thereof

The asymmetric reduction reaction was carried out in a 1.5mL centrifuge tube, and the reaction system consisted of 20mM 6-cyano-(5R)-hydroxyl-3-oxoylhexanoic acid tert-butyl ester, 15mMNADH and 5U of the aldehyde and ketone reductase prepared in Example 3 or For its mutant, potassium phosphate buffer (100 mM, pH 7.0) was added to form a total volume of 1 mL. React at 30°C and 200 rpm for 2 hours, take a sample, and detect the de value of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate formed by liquid chromatography.

Liquid phase detection conditions: ODS-2C ₁₈ column (4.6×250mm, 5μm), mobile phase acetonitrile:water=1:3 (v/v), column temperature 40°C, flow rate 1mL/min, UV detection wavelength 210nm, sample injection Volume 20 μL. 6-cyano-(3S,5R)-dihydroxyhexanoic acid tert-butyl ester, 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester, 6-cyano-(5R)-hydroxy-3 - The retention times of tert-butyl carbonylhexanoate were 7.4min, 8.1min, and 12.5min, respectively.

The selectivity of aldehyde and ketone reductase and its mutants are shown in Table 2.

Table 2 The specific activity and diastereoselectivity of wild-type K1AKR and its mutants to 6-cyano-(5R)-hydroxyl-3-oxoylhexanoic acid tert-butyl ester

Example 6: Comparison of asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate by aldehyde and ketone reductase and its mutant Y295W/W296L

Preparation of glucose dehydrogenase cells: the glucose dehydrogenase gene (GenBankNo.KM817194.1) derived from Exiguobacterium sibiricum was inserted into pET-28b to construct a recombinant expression plasmid, and the recombinant plasmid was introduced into Escherichia Coli BL21 (DE3) to obtain glucose-containing Recombinant genetically engineered bacteria with dehydrogenase gene; inoculate recombinant engineered bacteria containing glucose dehydrogenase gene into LB liquid medium containing final concentration of 50mg/L kanamycin, culture at 37°C for 10h, and then inoculate at a volume concentration of 4 % inoculated into fresh LB liquid medium containing kanamycin at a final concentration of 50mg/L, cultivated at 37°C until the cell concentration _OD600 was 0.6-0.8, and then added lactose with a final concentration of 9g/L to the culture medium After culturing at 28°C for 12h, centrifuge at 8000g for 10min at 4°C to collect bacterial cells.

Mix 3.75 g of aldehyde and ketone reductase (starting strain or its mutant Y295W/W296L) wet thallus obtained in Example 2 with 1.25 g of glucose dehydrogenase thalline, and mix with 50 ml of potassium phosphate buffer (100 mM, pH7.0 ) suspension, the total concentration of bacteria is 20gDCW/L. The bacterial suspension was ultrasonically disrupted according to the method in Example 3, and the resulting disrupted solution was used for the asymmetric reduction reaction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate, as shown in FIG. 1 . The reduction reaction was carried out in a 100mL round bottom flask, and the reaction system consisted of different concentrations of 6-cyano-(5R)-hydroxy-3-oxoylhexanoic acid tert-butyl ester (0.5g or 1.5g or 2.5g) and 1.5 times the mass ( To 6-cyano-(5R)-hydroxy-3-carbonylhexanoic acid tert-butyl) glucose, 50 mL of the above crushing solution was added to form. The reaction was carried out at 30° C. with a magnetic stirring speed of 300 rpm, and 1M Na ₂ CO ₃ solution was added to maintain the pH of the reaction solution at 7.0. Sampling is carried out by the liquid phase method shown in Example 5. The substrate conversion rate and product de value are shown in Table 3.

Table 3 Asymmetric reduction of wild-type KlAKR and its mutant Y295W/W296L 6-cyano-(5R)-hydroxy-3-oxoylhexanoic acid tert-butyl ester

Example 7: The effect of the weight ratio of the aldehyde and ketone reductase mutant Y295W/W296L and the recombinant glucose dehydrogenase cell on the asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate

The aldehyde and ketone reductase mutant Y295W/W296L wet thallus obtained in Example 2 and the glucose dehydrogenase thallus (the preparation method is the same as in Example 6) were respectively 2:1, 1:1, and 1.5 according to the weight ratio of the thalline :1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1 and 4.5:1 were mixed, suspended in 50ml of potassium phosphate buffer (100mM, pH7.0), the total concentration of bacteria was measured as wet bacteria Calculated as 100g/L. The bacterial suspension was ultrasonically disrupted according to the method in Example 3, and the resulting disrupted solution was used for the asymmetric reduction reaction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate, as shown in FIG. 1 . The reaction was carried out in a 10 mL shake flask, and the reaction system consisted of adding 0.1 g of tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate and 0.1 g of glucose to 10 mL of the above crushing solution. React at 30°C and 200 rpm for 10 minutes, take a sample, and detect the yield and de value of the product tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate by the liquid phase method shown in Example 5. As shown in Figure 2, when the mass ratio of aldoketone reductase mutant Y295W/W296L to recombinant glucose dehydrogenase cell is 4:1, the product yield reaches the highest value of 56.8%.

Example 8: Effect of glucose concentration on asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate

Mix 3.75 g of the aldehyde and ketone reductase mutant Y295W/W296L wet thallus obtained in Example 2 with 1.25 g of the glucose dehydrogenase thallus prepared by the method in Example 6, and add 50 ml of potassium phosphate buffer (100 mM, pH 7.0) Suspended, the total concentration of bacteria is 20gDCW/L. The bacterial suspension was ultrasonically disrupted according to the method in Example 3, and the resulting disrupted solution was used for the asymmetric reduction reaction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate. The reaction was carried out in a 10mL shake flask, 0.1g of tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate was added, and glucose was respectively 0.05g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3 g, 0.35g, 0.4g and 0.45g, and 10mL of the above broken solution. React at 30° C. and 200 rpm for 10 minutes, take samples, and detect the yield and de value of the product 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester by the liquid phase method shown in Example 5. As shown in Figure 3, when the glucose concentration is 15g/L (that is, 1.5 times of the substrate concentration), the product yield reaches the highest value of 68%, and the de value is greater than 99.5%.

Example 9: Application of aldehyde and ketone reductase mutant Y295W/W296L in asymmetric reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate.

Mix 3.75 g of the aldehyde and ketone reductase mutant Y295W/W296L wet thallus obtained in Example 2 with 1.25 g of the glucose dehydrogenase thalline, suspend in 50 ml of potassium phosphate buffer (100 mM, pH7.0), and the total concentration of the thalline It is 20gDCW/L. The bacterial suspension was ultrasonically disrupted according to the method in Example 3, and the resulting disrupted solution was used for the asymmetric reduction reaction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate. The reduction reaction was carried out in a 100 mL round bottom flask, and the reaction system consisted of adding 2.5 g of tert-butyl 6-cyano-(5R)-hydroxy-3-carbonylhexanoate and 3.75 g of glucose to 50 mL of the above crushing solution. The reaction was carried out at 30° C. with a magnetic stirring speed of 300 rpm, and 1M Na ₂ CO ₃ solution was added to maintain the pH of the reaction solution at 7.0. The liquid phase method shown in Example 5 was used to monitor the formation of the product 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester and the change of de value during the reaction process. The reaction progress curve is shown in Figure 4. This figure shows that the product concentration gradually increases with the passage of time, and the reaction is completed at 80min, and finally 162.7mM of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is obtained, and the de value of the product is always maintained at More than 99.5%.

Claims (10)

1. A mutant of aldehyde and ketone reductase, characterized in that the mutant of aldehyde and ketone reductase is obtained by performing a single or double mutation on the 295th and 296th positions of the amino acid sequence shown in SEQ ID NO.1. 2. The aldehyde and ketone reductase mutant as claimed in claim 1, wherein the aldehyde and ketone reductase mutant is that the 295th tyrosine in the amino acid sequence shown in SEQ ID NO.1 is mutated into tryptophan, and the amino acid sequence Shown as SEQ ID NO2. 3. The aldehyde and ketone reductase mutant as claimed in claim 1 is characterized in that the aldehyde and ketone reductase mutant is that the 295th tyrosine of the amino acid sequence shown in SEQ ID NO.1 is mutated into tryptophan, and The 296th tryptophan is mutated into leucine, and the amino acid sequence is shown in SEQ ID NO3. 4. A gene encoding the aldehyde and ketone reductase mutant described in claim 1. 5. A recombinant vector constructed by the coding gene according to claim 4. 6. A recombinant genetically engineered bacterium constructed by the recombinant vector according to claim 5. 7. The use of the aldehyde and ketone reductase mutant described in claim 1 in the preparation of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate. 8. application as claimed in claim 7, it is characterized in that described application uses the wet thalline that the recombinant genetically engineered bacterium that contains aldehyde and ketone reductase mutant gene obtains through fermenting and cultivating as catalyzer, with 6-cyano group-( 5R)-Hydroxy-3-oxoylhexanoic acid tert-butyl ester is used as the substrate, the glucose dehydrogenase wet cell obtained from the fermentation culture of engineering bacteria containing the glucose dehydrogenase gene is used as the coenzyme regeneration enzyme, and glucose is used as the auxiliary substrate. Using pH 7.0, 100mM phosphate buffer as the reaction medium, suspend the catalyst and glucose dehydrogenase wet bacteria in the reaction medium, ultrasonically disrupt, then add the substrate and auxiliary substrate, at 30°C, 200r/min Reaction, after the reaction is complete, obtain tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate; the amount of wet cells of the glucose dehydrogenase is 10-250g/L buffer solution, and the amount of the catalyst The weight of wet cells is 10-250g/L buffer solution, the final concentration of the substrate is 1-100g/L buffer solution, and the final concentration of glucose is 5-300g/L buffer solution. 9. application as claimed in claim 8, it is characterized in that described catalyzer is prepared as follows: the recombinant engineered bacterium that contains aldehyde and ketone reductase mutant gene is inoculated to the LB liquid that contains final concentration 50mg/L kanamycin culture medium at 37°C for 10 hours, then inoculate it into fresh LB liquid medium containing a final concentration of 50 mg/L kanamycin at a volume concentration of 4%, and cultivate at 37°C until the OD ₆₀₀ of the cell concentration is 0.6-0.8, Then, lactose with a final concentration of 9 g/L was added to the culture medium, cultured at 28° C. for 12 hours, centrifuged at 8000 g at 4° C. for 10 minutes, and bacterial cells were collected. 10. The application according to claim 8, characterized in that the ultrasonic crushing condition is: place an ice bath in an ultrasonic crusher, crush with 400W power for 20 minutes, break for 1 second and stop for 1 second.