A carbonyl reductase mutant and its application

Abstract

The present invention discloses a carbonyl reductase mutant and its application. The mutation sites of the carbonyl reductase mutant of the present invention include the 88th, 142nd, 190th and 193rd positions of the amino acid sequence shown in SEQ ID NO:1. The carbonyl reductase mutant of the present invention has higher enzyme activity than the wild-type carbonyl reductase. The enzyme activity of some carbonyl reductase mutants is 50 times that of the wild-type carbonyl. The carbonyl reductase mutant of the present invention can make the compound shown in formula I undergo a reduction reaction as shown in the following formula in the presence of a coenzyme in a liquid reaction system, and prepare the compound shown in formula ⅠⅠ with a conversion rate of >99%, a chiral ee value of >99%, and a chiral de value of >99%. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the field of biocatalytic synthesis and relates to a carbonyl reductase mutant and application thereof. Background Art

[0002] Compounds containing chiral amino alcohol structures have diverse biological activities and are widely used in the field of pharmaceutical chemicals, such as droxidopa for the treatment of Parkinson's disease and hypotension, chloramphenicol with broad-spectrum antibacterial activity, veterinary drugs florfenicol and thiamphenicol, oral Gaucher disease treatment drug iliglustat and other clinical candidate drugs with anti-inflammatory, anti-infective and anti-tumor activities. Among them, chloramphenicol, which was listed in 1949 as a broad-spectrum antibiotic, has a global production mainly concentrated in China, with a domestic production scale of about 3,000 tons. However, with the continuous improvement of environmental protection standards and the increasing cost of treating three wastes (wastewater, waste gas, and waste solids), the existing production process has been difficult to meet the requirements of development. Therefore, there is an urgent need for a more green, economical and environmentally friendly process route to solve the environmental problems existing in the existing production process. The biocatalytic method of dynamic kinetic resolution reduction mediated by carbonyl reductase in the Chinese patent application publication CN111808893A of this laboratory can effectively solve the problems of high pollution, high energy consumption, low efficiency and low quality existing in the existing production process of chloramphenicol. Therefore, the industry urgently needs a more efficient and stable carbonyl reductase for the synthesis of chloramphenicol and other chiral amino alcohol drugs.

[0003] Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defect of low activity of carbonyl reductase mutants in the prior art, and to provide a carbonyl reductase mutant and its application. The carbonyl reductase mutant of the present invention has higher enzyme activity than the wild-type carbonyl reductase, and can be used to catalyze the carbonyl reduction reaction of the compound shown in formula I

[0005] The present invention provides a carbonyl reductase mutant, wherein the mutation sites of the carbonyl reductase mutant include the 88th, 142nd, 190th and 193rd positions of the amino acid sequence shown in SEQ ID NO:1.

[0006] In the present invention, preferably, the mutation site of the carbonyl reductase mutant also includes one or more selected from positions 82, 121, 138, 192, 201, 204, 206 and 207 of the amino acid sequence shown in SEQ ID NO:1.

[0007] In the present invention, more preferably, the mutation sites of the carbonyl reductase mutant also include at least 3 positions selected from positions 82, 121, 138, 192, 201, 204, 206 and 207 of the amino acid sequence as shown in SEQ ID NO: 1.

[0008] In the present invention, preferably, the mutation site of the carbonyl reductase mutant is selected from any one of the following groups:

[0009] (1) The mutation sites of the carbonyl reductase mutant are positions 82, 88, 121, 138, 142, 190 and 193 of the amino acid sequence shown in SEQ ID NO: 1;

[0010] (2) The mutation sites of the carbonyl reductase mutant are positions 82, 88, 121, 138, 142, 190, 193 and 201 of the amino acid sequence shown in SEQ ID NO: 1;

[0011] (3) The mutation sites of the carbonyl reductase mutant are positions 82, 88, 121, 138, 142, 190, 193, 204 and 206 of the amino acid sequence shown in SEQ ID NO: 1;

[0012] (4) The mutation sites of the carbonyl reductase mutant are positions 82, 88, 121, 138, 142, 190, 193, 206 and 207 of the amino acid sequence shown in SEQ ID NO: 1;

[0013] (5) The mutation sites of the carbonyl reductase mutant are positions 82, 88, 121, 138, 142, 190, 193, 201, 206 and 207 of the amino acid sequence shown in SEQ ID NO: 1;

[0014] (6) The mutation sites of the carbonyl reductase mutant are positions 82, 88, 121, 138, 142, 190, 192, 193, 204 and 206 of the amino acid sequence shown in SEQ ID NO: 1;

[0015] (7) The mutation sites of the carbonyl reductase mutant are positions 82, 88, 121, 138, 142, 190, 193, 201 and 204 of the amino acid sequence shown in SEQ ID NO: 1.

[0016] In the present invention, preferably, the amino acid residue at position 82 is mutated from W to L.

[0017] In the present invention, preferably, the amino acid residue at position 88 is mutated from F to V, I or S, such as I or V.

[0018] In the present invention, preferably, the amino acid residue at position 121 is mutated from V to A.

[0019] In the present invention, preferably, the amino acid residue at position 138 is mutated from A to V or L, for example, L.

[0020] In the present invention, preferably, the amino acid residue at position 142 is mutated from R to M, F, H or L, for example, M.

[0021] In the present invention, preferably, the amino acid residue at position 190 is mutated from A to V.

[0022] In the present invention, preferably, the amino acid residue at position 192 is mutated from R to M.

[0023] In the present invention, preferably, the amino acid residue at position 193 is mutated from S to A.

[0024] In the present invention, preferably, the amino acid residue at position 201 is mutated from Y to F.

[0025] In the present invention, preferably, the amino acid residue at position 204 is mutated from N to A or G, such as A. In the present invention, preferably, the amino acid residue at position 206 is mutated from K to H.

[0026] In the present invention, preferably, the amino acid residue at position 207 is mutated from K to N.

[0027] In the present invention, the mutation sites and types of the carbonyl reductase mutants are shown in Table 1 below:

[0028] Table 1

[0029]

[0030]

[0031] The present invention also provides a method for preparing a compound as shown in formula II, which comprises the following steps: in a liquid reaction system, the compound as shown in formula I is subjected to a reduction reaction as shown in the following formula in the presence of a coenzyme and the aforementioned carbonyl reductase mutant;

[0032]

[0033] R1 is H, or benzyl;

[0034] R 1-1 is C1-C6 alkyl or benzyl;

[0035] R2 is H, or benzyl;

[0036] R 2-1 is C1-C6 alkyl or benzyl;

[0037] R3 is Where R 3-1 is a C1-C6 alkyl group;

[0038] R4 is H, NO2, halogen, C1-C6 alkyl, C1-C6 alkoxy or sulfonyl substituted by C1-C6 alkyl.

[0039] In the present invention, preferably, the halogen is F, Cl, Br or I.

[0040] In the present invention, preferably, the C1-C6 alkyl group is a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

[0041] In the present invention, preferably, R1 is H,

[0042] In the present invention, preferably, R2 is H,

[0043] In the present invention, preferably, R4 is H, NO2, F, Cl, Br, I, methyl, methoxy or

[0044] In the present invention, preferably, R3 is

[0045] In the present invention, more preferably, R1 is H, R2 is R3 is R4 is F, Cl or Br.

[0046] In the present invention, preferably, the compound as shown in formula I is

[0047] In the present invention, preferably, the compound as shown in formula II is

[0048] In the present invention, the coenzyme may be a conventional coenzyme in the art, preferably, the coenzyme is a reducing coenzyme and / or an oxidizing coenzyme. The oxidizing coenzyme is preferably NAD + and / or NADP + ; The reducing coenzyme is preferably NADH and / or NADPH.

[0049] In the present invention, the amount of the coenzyme can be the conventional amount of the coenzyme in the art. Preferably, the mass ratio of the coenzyme to the compound as shown in Formula I is 1:(1-100); preferably 1:(50-100); for example 1:100 or 1:75.

[0050] In the present invention, the liquid reaction system can be a conventional liquid reaction system suitable for carbonyl reductase reaction in the art. Preferably, the liquid reaction system includes an enzyme for coenzyme regeneration and a cosubstrate for coenzyme regeneration. The enzyme for coenzyme regeneration is preferably one or more of alcohol dehydrogenase, formate dehydrogenase and glucose dehydrogenase; for example, glucose dehydrogenase. The cosubstrate is preferably one or more of isopropanol, glucose and ammonium formate, for example, glucose.

[0051] In the present invention, in the liquid reaction system, the amount of the co-substrate can be the conventional amount in the art. Preferably, the mass concentration of the co-substrate in the liquid reaction system is 5-30%, more preferably 5%-20%; for example, 16%, 8% or 12%.

[0052] In the present invention, in the liquid reaction system, the amount of the enzyme for coenzyme regeneration can be the conventional amount in the art. Preferably, the mass concentration of the enzyme for coenzyme regeneration in the liquid reaction system is 1-10%; more preferably, 1%-5%; for example, 2.5%, 1.6% or 2.3%.

[0053] In the present invention, the reaction temperature of the reduction reaction can be a conventional reaction temperature in the art, preferably 10°C-50°C, more preferably 25°C-35°C, for example 30°C.

[0054] In the present invention, the reaction time of the reduction reaction is related to the reaction temperature and the reaction scale, and is preferably 0.1-72 hours, and more preferably 3-24 hours.

[0055] In the present invention, the pH of the reduction reaction may be a conventional pH in the art, preferably 6-10, more preferably 7.0-9.0, such as 7.5-8.0.

[0056] In the present invention, the carbonyl reductase mutant is added to the reduction reaction in a conventional form in the art, preferably a free enzyme, an immobilized enzyme, an enzyme in bacterial powder or bacterial form, more preferably an enzyme in bacterial form.

[0057] In the present invention, the liquid reaction system further comprises a buffer such as a phosphate buffer. The phosphate buffer is preferably a 0.1 M phosphate buffer. The phosphate buffer is used to control the pH of the liquid reaction system.

[0058] In the present invention, the liquid reaction system further comprises a co-solvent. The co-solvent may be a conventional co-solvent in the art; preferably, the co-solvent is selected from one or more of dimethyl sulfoxide, isopropanol and toluene, and more preferably, dimethyl sulfoxide.

[0059] In the present invention, the amount of the co-solvent can be the conventional amount in the art. Preferably, the mass concentration of the co-solvent in the liquid reaction system is 10%-50%; more preferably, 20%-30%; for example, 30%, 29% or 28%.

[0060] In the present invention, after the reduction reaction is completed, a post-treatment step is also included.

[0061] In the present invention, the post-treatment step is a conventional post-treatment step in the art. Preferably, the post-treatment step includes: adding an organic solvent to the aforementioned liquid reaction system, heating, filtering the bacteria, extracting, washing the organic phase with water, drying and filtering and concentrating the organic layer to obtain a compound as shown in Formula II.

[0062] In the present invention, the organic solvent may be a conventional organic solvent in the art, preferably an ester solvent, an ether solvent, an alcohol solvent, an aromatic hydrocarbon solvent or a chlorinated alkane solvent. The ester solvent is preferably ethyl acetate or isopropyl acetate. The ether solvent is preferably methyl tert-butyl ether or 2-methyltetrahydrofuran. The alcohol solvent is preferably n-butanol. The aromatic hydrocarbon solvent is preferably toluene. The chlorinated alkane solvent is preferably dichloromethane.

[0063] In the present invention, the heating temperature is based on the denaturation of the protein, preferably 60°C.

[0064] In the present invention, the heating time is based on the denaturation of the protein, preferably 1 hour.

[0065] In the present invention, the water used in the water washing includes pure water and water containing inorganic salts; for example, pure water and / or 5% saline solution.

[0066] In the present invention, the drying method can be a conventional drying method in the art, preferably drying with a desiccant; the desiccant is preferably anhydrous sodium sulfate.

[0067] More preferably, the post-treatment step comprises: adding methyl tert-ether or ethyl acetate to the reaction solution of the aforementioned reduction reaction, heating, filtering the bacteria and extracting, washing the organic phase with pure water, washing with 5% brine, drying with anhydrous sodium sulfate and filtering and concentrating the organic layer to obtain the compound shown in Formula II.

[0068] The present invention also provides a use of the carbonyl reductase mutant as described above in reducing carbonyl groups.

[0069] In the present invention, preferably, the reaction substrate and reaction conditions used are the reaction substrate and reaction conditions in the preparation method of the compound represented by formula II as described above.

[0070] the term

[0071] Enantiomeric excess (ee): usually used to characterize the excess of one enantiomer relative to the other enantiomer in a chiral molecule.

[0072] Diastereomeric excess (de): usually used to characterize the excess of one diastereomer relative to another diastereomer in a molecule with two or more chiral centers.

[0073] Isomer content (ic): usually used to characterize the percentage of one isomer in the total amount of all isomers in a molecule with two or more chiral centers.

[0074] (R,S)-Carbonyl Reductase

[0075] In the present invention, "stereoselective carbonyl reductase" refers to an enzyme that can stereoselectively asymmetric catalyze the reduction of a prochiral ketone to a chiral alcohol.

[0076] Typically, in the present invention, the stereoselective carbonyl reductase is preferably (R, S)-carbonyl reductase, and the stereoselectivity is defined as enantiomeric excess (ee) ≥ 80%, and diastereomeric excess (de) ≥ 80%.

[0077] Similarly, when (R, R)-carbonyl reductase is used, stereoselectivity is defined as enantiomeric excess (ee) ≥ 80%, diastereomeric excess (de) ≥ 80%, and so on.

[0078] Coenzyme

[0079] In the present invention, "coenzyme" refers to a coenzyme that can achieve electron transfer in a redox reaction.

[0080] Solvent

[0081] In the present invention, a co-solvent may or may not be added to the reaction system.

[0082] As used herein, the term "cosolvent" refers to a complex, an association compound or a double salt formed between a poorly soluble substance and a third substance added in a solvent to increase the solubility of the poorly soluble substance in the solvent. This third substance is called a cosolvent.

[0083] Dynamic reduction kinetic resolution reaction principle

[0084] While a single-configuration prochiral ketone (such as the R-configuration) is stereoselectively reduced by carbonyl reductase, a prochiral ketone of another configuration (such as the S-configuration) is racemized at the α-position through enol interconversion of the carbonyl group. The reduction and racemization are carried out under the same reaction conditions, thereby achieving the purpose of efficiently constructing a secondary alcohol containing two chiral centers.

[0085]

[0086] Typically, in the present invention, a carbonyl reductase that only recognizes Ⅰ-S (S-configuration compound Ⅰ) is obtained by screening, and the carbonyl is stereoselectively reduced to obtain a chiral hydroxyl group, while the unrecognized Ⅰ-R (R-configuration compound Ⅰ) is converted to Ⅰ-S by racemization. The racemization and reduction processes are connected and promoted. In theory, the desired chiral product can be obtained 100% by this conversion. The potential chiral carbonyl substrate is reduced by the carbonyl reductase, and the enol interconversion racemization is carried out at the same time. The two are cleverly combined, and two chiral centers are constructed efficiently and economically in one-step reaction, showing good application development prospects.

[0087] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0088] The reagents and raw materials used in the present invention are commercially available.

[0089] The positive and progressive effects of the present invention are:

[0090] The carbonyl reductase mutant of the present invention has higher enzyme activity than the wild-type carbonyl reductase. The enzyme activity of some carbonyl reductase mutants is 50 times that of the wild-type carbonyl. The carbonyl reductase mutant of the present invention can be used to catalyze the carbonyl reduction reaction of the compound shown in formula I.

[0091] The preparation method provided by the present invention can prepare the compound shown in formula ⅠⅠ with a conversion rate of >99%, a chiral ee value of >99%, and a chiral de value of >99%. The preparation method of the present invention has the characteristics of being green, environmentally friendly and economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 Liquid phase comparison diagram of the chiral purity of compound 1b prepared by the four isomers of racemic compound 1b and WTEA enzyme conversion reaction in Comparative Example 1.

[0093] Figure 2 Liquid phase comparison diagram of the chiral purity of compound 1b prepared by the conversion reaction of the four isomers of racemic compound 1b and mutant carbonyl reductase (mutant 65) in Comparative Example 1.

[0094] Figure 3 This is the liquid phase diagram of the four isomers of the racemic compound 2b in Comparative Example 1.

[0095] Figure 4 Liquid phase diagram of the chiral purity of compound 2b prepared by WTEA enzymatic conversion reaction in Comparative Example 1.

[0096] Figure 5 Liquid phase diagram of the chiral purity of compound 2b prepared by the conversion reaction of mutant carbonyl reductase (mutant 65) in Comparative Example 1.

[0097] Figure 6 This is the liquid phase diagram of the four isomers of the racemic compound 5b in Comparative Example 2.

[0098] Figure 7 Liquid phase diagram of the chiral purity of compound 5b prepared by WTEA enzymatic conversion reaction in Comparative Example 2.

[0099] Figure 8 Liquid phase diagram of the chiral purity of compound 5b prepared by the conversion reaction of mutant carbonyl reductase (mutant 65) in Comparative Example 2. DETAILED DESCRIPTION

[0100] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples where specific conditions are not specified are carried out according to conventional methods and conditions, or are selected according to the product specifications. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0101] Specifically, the biological preparation method of the present invention uses compound I (such as compound 1) as a raw material and carbonyl reductase as a biocatalyst to efficiently prepare compound II with a stereo conformation (reduction yield>99%, chiral ee value>99%, chiral de value>99%) in the presence of a coenzyme, and constructs two chiral centers in a one-step reaction, thereby greatly improving production efficiency and reducing production costs.

[0102] Material

[0103] The total gene synthesis was completed by Nanjing GenScript.

[0104] The coding gene is obtained through commercial whole gene synthesis, and then the coding gene is constructed into an expression vector, introduced into a host bacterium, and induced to express to obtain carbonyl reductase.

[0105] The preparation of the enzyme-reduced substrate compound Ⅰ can be found in Tetrahedron.2016,72:1787-1793 The method.

[0106] method

[0107] 1. Enzyme preparation method

[0108] By conventional techniques in the art, the glucose dehydrogenase and formate dehydrogenase for achieving coenzyme regeneration are constructed on the pET28a(+) vector, respectively, and then introduced into the expression host Escherichia coli, and the bacterial cells containing glucose dehydrogenase, formate dehydrogenase and carbonyl reductase are obtained by inducing expression. The bacterial cells can be directly obtained by centrifugation, or the cell wall can be broken to obtain crude enzyme liquid and crude enzyme powder for subsequent bioconversion reactions.

[0109] 2. Method for preparing compound II by biocatalytic reduction of compound I

[0110] The present invention provides a method for preparing compound II by catalyzing the reduction of compound I by carbonyl reductase. The reaction formula is as follows:

[0111]

[0112] R1 is H, or benzyl; R 1-1 is C1-C6 alkyl or benzyl; R2 is H, or benzyl; R 2-1 is C1-C6 alkyl or benzyl; R3 is Where R 3-1 is a C1-C6 alkyl group;

[0113] R4 is H, NO2, halogen, C1-C6 alkyl, C1-C6 alkoxy or sulfonyl substituted by C1-C6 alkyl.

[0114] Wherein, the biocatalytic system comprises carbonyl reductase and coenzyme. The nucleotide sequence of the carbonyl reductase encoding gene in the present invention is SEQ ID NO: 2, and the amino acid sequence of the carbonyl reductase is SEQ ID NO: 1. According to common knowledge in the art, the carbonyl reductase gene can be obtained by commercial whole gene synthesis.

[0115] According to the above preferred system, the implementation process of the preparation method is as follows: the substrate is fully dissolved in a cosolvent, such as dimethyl sulfoxide or isopropanol, and then added to a phosphate buffer, stirred evenly, and then the bacterial cells, crude enzyme solution, crude enzyme powder or pure enzyme are added, and the coenzyme NADP is added. + With co-substrate glucose, maintain at 20℃~40℃, monitor by TLC or HPLC, and terminate the reaction until the remaining raw material is less than 2%. The reaction solution is extracted with an organic solvent, which can be methyl tert-butyl ether, toluene, ethyl acetate, isopropyl acetate, dichloromethane, 2-methyltetrahydrofuran or n-butanol. Extract the aqueous layer 2-3 times, combine the organic phases; wash with saturated brine 2-3 times, and concentrate to obtain a light yellow solid, which is compound II (1b-7b, respectively, depending on the substrate, as shown in Table 3).

[0116] The final concentration of substrate compound I in the system is 10-200 g / L, the reaction temperature is 20-40° C., the rotation speed is 200 rpm / min, and the reaction time is about 3-24 h. The reaction time varies according to the substrate concentration or the raw material conversion is monitored by HPLC. Generally, the reaction is terminated when the remaining raw material is <2%.

[0117] 3. Chiral normal phase monitoring method for compound II:

[0118] The sample was dissolved in methanol at a concentration of 10 mg / mL; the injection volume was 2 μL. The specific detection method is shown in Table 2.

[0119] Table 2 Chiral normal phase monitoring method a .

[0120]

[0121]

[0122] a General detection conditions: temperature: 30℃; flow rate: 1mL / min

[0123] b IB-3:CHIRALPAK IB-3column (3μm, 4.6mm×250mm, DAICEL, Shanghai)

[0124] c OJ-H: CHIRALPAK IB-3 column (5 μm, 4.6 mm × 250 mm, DAICEL, Shanghai) Table 3.1b-7b Compound structure

[0125]

[0126]

[0127] 4. Reverse phase monitoring method of compound II:

[0128] HPLC conditions: phenomenex Gemini 5u C18 110A, 250×4.6mm, 5μm; flow rate: 1mL / min; mobile phase: acetonitrile: water = 55:45; UV detection wavelength: 210, 220, 245nm; column temperature: 30℃; sample concentration: 10mg / mL; injection volume 10μL.

[0129] Example 1 Construction of genetically engineered bacteria expressing recombinant carbonyl reductase and glucose dehydrogenase

[0130] The target genes of carbonyl reductase WTEA (nucleotide sequence is SEQ ID NO: 2, amino acid sequence is SEQ ID NO: 1) and glucose dehydrogenase GDH were entrusted to a commercial company for full gene synthesis, cloned into pET28a(+) vector respectively, transferred into Escherichia coli DH5α competent cells, cultured on plates, single colonies of positive transformants were picked and plasmids were extracted and sequenced, and then the recombinant plasmids were extracted and introduced into BL21(DE3) strains, single bacteria were picked and cultured with LB to obtain genetically engineered bacteria pET28a(+)-WTEA that can be induced to express recombinant carbonyl reductase and genetically engineered bacteria pET28a(+)-GDH that express recombinant glucose dehydrogenase GDH, respectively.

[0131] Example 2 Preparation of recombinant carbonyl reductase and glucose dehydrogenase

[0132] The genetically engineered bacteria stored in glycerol in the previous step were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 220 rpm for 14 h to obtain seed culture solution. The seed culture solution was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance at a ratio of 1.5%, and then cultured at 37°C, 220 rpm until OD 600 Value>2.0. Add isopropylthiogalactoside (IPTG) at a final concentration of 1 mM, cool to 25°C to induce protein expression, continue culturing for 20 h, put the tank in place, and centrifuge (centrifugation conditions: 4000 g for 30 min) to obtain the bacteria in preparation for biotransformation.

[0133] LB liquid medium (g / L): tryptone 10.0, yeast extract 5.0, NaCl 10.0, deionized water 1 L, pH 7.0.

[0134] Fermentation medium 2 (g / L): yeast extract 24.0, soy peptone 12.0, NaCl 3.0, glycerol 5.0, K2HPO4·3H2O 2.0, MgSO4·7H2O 0.5, deionized water 1 L, pH 7.5.

[0135] Example 3 Construction and screening of carbonyl reductase WTEA mutants

[0136] The wild-type carbonyl reductase gene WTEA was mutated by directed evolution to obtain a plasmid library containing the evolved carbonyl reductase gene. It was then transferred into Escherichia coli BL21 (DE3) (Cat. No.: Kangwei Century CW0809S) and plated on LB solid medium containing 50 μg / mL kanamycin. After culturing in a 37°C oven for 14 hours, a single colony was selected into a 96-well plate containing 400 μL LB liquid medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C and 200 rpm to obtain a seed solution. Then 10 μL of the seed solution was transferred to a 96-deep-well plate containing 400 μL fermentation medium (fermentation medium 2, containing 50 μg / mL kanamycin) and cultured for 3 hours at 37°C and 200 rpm. Then, isopropylthiogalactoside (IPTG) was added at a final concentration of 1 mM, the temperature was lowered to 25°C to induce mutant expression, and the culture was continued for 20-24 hours. Then, the cells were centrifuged at 4000g for 30min and resuspended in 200μL lysis buffer (0.1M phosphate buffer containing 1000U lysozyme, pH 7.0) and lysed at 30°C for 1h. The 96-well plate was then centrifuged at 4000g for 30min at 4°C, and the clarified supernatant was used to determine the activity of the mutants. 190μL of the reaction solution (containing 0.4mM substrate, 1mM NADPH, 40μL dimethyl sulfoxide) was added to a new 96-well plate, and after adding 10μL of the supernatant, the change in NADPH was detected at 340nm. The consumption of NADPH reflects the level of the mutant enzyme activity, and the relative activity of each mutant is shown in Table 4.

[0137] Table 4: Mutants and their relative activities

[0138]

[0139]

[0140] *The activity of the wild-type carbonyl reductase gene WTEA (SEQ ID NO: 1) was set to 100%.

[0141] Example 4 Preparation of Compound 1b by Biocatalysis

[0142]

[0143] Wherein R1 is H; R2 is Boc; R3 is CH3; R4 is NO2, Ⅰ is compound 1a, and Ⅱ is compound 1b.

[0144] Take 0.1M phosphate buffer (140mL), add glucose (40g), add NADP + (0.2g), add mutant 65 cells (20g) obtained from the above fermentation, add glucose dehydrogenase (GDH) cells (6g), stir vigorously and slowly add a solution of compound 1a (20g) in DMSO (60mL), at 30°C, at intervals of 0.5h, adjust the pH to 7.5-8.0 with 5% sodium carbonate aqueous solution, and terminate the reaction when the reaction conversion rate is >98% monitored by HPLC.

[0145] Add tert-methyl ether (200 mL) for extraction, heat to 60°C for 1 h to inactivate the protein in the bacteria, add 10% diatomaceous earth, filter the bacteria, extract the aqueous layer with tert-methyl ether (100 mL), combine the organic layers, wash with water (100 mL×2), wash with 5% brine, dry with anhydrous sodium sulfate, filter, and concentrate to obtain a light yellow oil, which is compound 1b (17.6 g) with ee value 99.9% and de value 99.9%.

[0146] Example 5 Preparation of Compound 2b by Biocatalysis

[0147]

[0148] Wherein R1 is H; R2 is Boc; R3 is CH2CH3; R4 is Cl, Ⅰ is compound 2a, and Ⅱ is compound 2b.

[0149] Take 0.1M phosphate buffer (70mL), add glucose (10g), add NADP + (0.1g), add mutant 65 cells (10g) obtained from the above fermentation, add glucose dehydrogenase (GDH) cells (3g), add compound 2a (7.5g) in DMSO (30mL) in batches with vigorous stirring, at 30°C, at intervals of 0.5h, adjust the pH to 7.5-8.0 with 5% sodium carbonate aqueous solution, and terminate the reaction when the reaction conversion rate is >98% monitored by HPLC.

[0150] Methyl tert-ether (100 mL) was added for extraction, and the mixture was heated to 60°C for 1 h to inactivate the protein in the cells. 10% diatomaceous earth was added, and the cells were filtered. The aqueous layer was extracted with methyl tert-ether (50 mL). The organic layers were combined, washed with water (50 mL × 2), washed with 5% brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a light yellow oil, which was compound 2b (6.6 g) with an ee value of 99.9% and a de value of 99.9%.

[0151] Example 6 Preparation of Compound 3b by Biocatalysis

[0152]

[0153] Among them, R1 is H; R2 is Boc; R3 is CH2CH3, R4 is SO2Et, Ⅰ is compound 3a, and Ⅱ is compound 3b.

[0154] Take 0.1M phosphate buffer (70mL), add glucose (15g), NADP + (0.1g), add mutant 65 cells (10g) obtained by the above fermentation, add glucose dehydrogenase (GDH) cells (2g), add compound 3a (10g) in DMSO (30mL) in batches with vigorous stirring, at 30°C, at intervals of 0.5h, adjust the pH to 7.5-8.0 with 5% sodium carbonate aqueous solution, and terminate the reaction when the reaction conversion rate is >98% monitored by HPLC.

[0155] Methyl tert-ether (200 mL) was added for extraction, and the mixture was heated to 60°C for 1 h to inactivate the protein in the cells. 10% diatomaceous earth was added, and the cells were filtered. The aqueous layer was extracted with methyl tert-ether (100 mL). The organic layers were combined, washed with water (100 mL × 2), washed with 5% brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a light yellow oil, which was compound 3b (4.7 g) with an ee value of 99.9% and a de value of 99.9%.

[0156] Example 7 Preparation of Compound 4b by Biocatalysis

[0157]

[0158] Among them, R1 is H; R2 is Boc; R3 is CH3, R4 is SO2Me, Ⅰ is compound 4a, and Ⅱ is compound 4b.

[0159] Take 0.1M phosphate buffer (140mL), add glucose (40g), add NADP + (0.2g), add mutant 65 cells (20g) obtained from the above fermentation, add glucose dehydrogenase (GDH) cells (6g), stir vigorously and slowly add a solution of compound 4a (20g) in DMSO (60mL), at 30°C, at intervals of 0.5h, adjust the pH to 7.5-8.0 with 5% sodium carbonate aqueous solution, and terminate the reaction when the reaction conversion rate is >98% monitored by HPLC.

[0160] Add tert-methyl ether (200 mL) for extraction, heat to 60°C for 1 h to inactivate the protein, add 10% diatomaceous earth, filter the bacteria, extract the aqueous layer with tert-methyl ether (100 mL), combine the organic layers, wash with water (100 mL×2), wash with 5% brine, dry over anhydrous sodium sulfate, filter, and concentrate to obtain a light yellow oil, which is compound 4b (17.6 g) with ee value 99.9% and de value 99.9%.

[0161] Comparative Example 1 Comparison of wild-type carbonyl reductase and mutant carbonyl reductase in the biocatalytic preparation of compounds 1b, 2b, and 4b

[0162] Take 140mL of 0.1M phosphate buffer, add 40g of glucose, add 0.2g of NADP + , add 6 g of glucose dehydrogenase (GDH), add 20 g of wild-type carbonyl reductase (or mutant 47, or mutant 63, or mutant 65) obtained by fermentation, stir vigorously and slowly add DMSO (60 mL) solution containing 20 g of substrate 1a (or 20 g of substrate 2a, 20 g of substrate 4a), 30°C, at intervals of 0.5 h, adjust the pH to 7.5-8.0 with 5% sodium carbonate aqueous solution, react for 24 h to terminate the reaction, and monitor the reaction conversion rate by HPLC.

[0163] The liquid phase comparison of the chiral purity of the four isomers of racemic compound 1 and the WTEA enzyme conversion reaction to prepare compound 1b is shown in Figure 1 As shown, the retention times of the four isomers are 23 min, 28 min, 39 min, and 47 min, respectively. The retention time of compound 1b obtained by WTEA enzyme conversion reaction is 39 min, and the chiral purity (de) is 98%.

[0164] The liquid phase comparison of the chiral purity of the four isomers of the racemic compound 1b and the mutant carbonyl reductase (mutant 65) in the preparation of compound 1b is shown in Figure 2. Figure 2 As shown, the retention time of compound 1b obtained by the conversion reaction of mutant 65 is 39 min, and the chiral purity (de) is 99%.

[0165] The liquid phase diagram of the four isomers of racemic compound 2b is shown in Figure 3 As shown, the retention times of the four isomers are 8 min; 10 min; 12 min; and 16 min, respectively.

[0166] The liquid phase diagram of the chiral purity of compound 2b prepared by WTEA enzymatic conversion reaction is shown in Figure 4 As shown, the retention time of compound 2b obtained by WTEA enzymatic conversion reaction is 10 min, and the chiral purity (de) is 89%.

[0167] The liquid phase diagram of the chiral purity of compound 2b prepared by the conversion reaction of mutant carbonyl reductase (mutant 65) is shown in Figure 5 As shown, the retention time of compound 2b obtained by the conversion reaction of mutant 65 was 10 min, and the chiral purity (de) was 99%.

[0168] Table 5: Comparison of the activity and stereoselectivity of wild-type carbonyl reductase and mutant carbonyl reductase catalysis (1a, 2a, 3a)

[0169]

[0170]

[0171] Note: SEQ ID NO 1 is the wild-type carbonyl reductase; mutant 47 is derived from the patent application with publication number CN109207531A.

[0172] Comparative Example 2 Comparison of stereoselectivity of wild-type carbonyl reductase and mutant carbonyl reductase in the biocatalytic preparation of compound 1-7b

[0173] Prepare 2 mL reaction systems containing different substrates and different carbonyl reductases: substrates 1a–7a (20 mM), 10% DMSO (v / v), glucose (40 mM), NADP + (0.2mM), carbonyl reductase bacteria (50g / LWTEA or mutant 65 bacteria), glucose dehydrogenase bacteria (25g / L), phosphate buffer (0.1M, pH7.0). The reaction mixture was reacted at 30°C, 220rpm for 24 hours. After the reaction, the reaction solution was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. Finally, the stereoselectivity of the wild-type carbonyl reductase and mutant carbonyl reductase biocatalytic method for preparing compound 1-7b was detected by high performance liquid chromatography.

[0174] The liquid phase diagram of the four isomers of racemic compound 5b is shown in Figure 6 As shown, the retention times of the four isomers are 13 min; 16 min; 17 min; and 29 min, respectively.

[0175] The liquid phase diagram of the chiral purity of compound 5b prepared by WTEA enzymatic conversion reaction is shown in Figure 7 As shown, the retention time of compound 5b obtained by WTEA enzymatic conversion reaction was 16 min, and the chiral purity (ic) was 69%.

[0176] The liquid phase diagram of the chiral purity of compound 5b prepared by the conversion reaction of mutant carbonyl reductase (mutant 65) is shown in Figure 8 As shown, the retention time of compound 5b obtained by the conversion reaction of mutant 65 was 16 min, and the chiral purity (ic) was 96%.

[0177] Table 6: Comparison of stereoselectivity of wild-type carbonyl reductase and mutant carbonyl reductase (mutant 65) catalyzing substrate (1-7a) to prepare compound 1-7b

[0178]

[0179] SEQUENCE LISTING <110> Shanghai Institute of Pharmaceutical Industry China Pharmaceutical Industry Research Institute <120> A carbonyl reductase mutant and its application <130> P21014495C <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 249 <212> PRT <213> Exiguobacterium acetylicum <400> 1 Met Lys Tyr Thr Val Ile Thr Gly Ala Ser Ser Gly Ile Gly Tyr Glu 1 5 10 15 Thr Ala Lys Leu Leu Ala Gly Lys Gly Lys Ser Leu Val Leu Val Ala 20 25 30 Arg Arg Thr Ser Glu Leu Glu Lys Leu Arg Asp Glu Val Lys Gln Ile 35 40 45 Ser Pro Asp Ser Asp Val Ile Leu Lys Ser Val Asp Leu Ala Asp Asn 50 55 60 Gln Asn Val His Asp Leu Tyr Glu Gly Leu Lys Glu Leu Asp Ile Glu 65 70 75 80 Thr Trp Ile Asn Asn Ala Gly Phe Gly Asp Phe Asp Leu Val Gln Asp 85 90 95 Ile Glu Leu Gly Lys Ile Glu Lys Met Leu Arg Leu Asn Ile Glu Ala 100 105 110 Leu Thr Ile Leu Ser Ser Leu Phe Val Arg Asp His His Asp Ile Glu 115 120 125 Gly Thr Thr Leu Val Asn Ile Ser Ser Ala Gly Gly Tyr Arg Ile Val 130 135 140 Pro Asn Ala Val Thr Tyr Cys Ala Thr Lys Phe Tyr Val Ser Ala Tyr 145 150 155 160 Thr Glu Gly Leu Ala Gln Glu Leu Gln Lys Gly Gly Ala Lys Leu Arg 165 170 175 Ala Lys Val Leu Ala Pro Ala Ala Thr Glu Thr Glu Phe Ala Asp Arg 180 185 190 Ser Arg Gly Glu Ala Gly Phe Asp Tyr Ser Lys Asn Val Lys Lys Tyr 195 200 205 His Thr Ala Ala Glu Met Ala Gly Phe Leu His Gln Leu Ile Glu Ser 210 215 220 Asp Ala Ile Val Gly Ile Val Asp Gly Glu Thr Tyr Glu Phe Glu Leu 225 230 235 240 Arg Gly Pro Leu Phe Asn Tyr Ala Gly 245 <210> 2 <211> 750 <212> DNA <213> Exiguobacterium acetylicum <400> 2 atgaaataca ccgttatcac cggtgcttct tctggtatcg gttacgaaac cgctaaactg 60 ctggctggta aaggtaaatc tctggttctg gttgctcgtc gtacctctga actggaaaaa 120 ctgcgtgacg aagttaaaca gatctctccg gactctgacg ttatcctgaa atctgttgac 180 ctggctgaca accagaacgt tcacgacctg tacgaaggtc tgaaagaact ggacatcgaa 240 acctggatca acaacgctgg tttcggtgac ttcgacctgg ttcaggacat cgaactgggt 300 aaaatcgaaa aaatgctgcg tctgaacatc gaagctctga ccatcctgtc ttctctgttc 360 gttcgtgacc accacgacat cgaaggtacc accctggtta acatctcttc tgcgggtggt 420 taccgtatcg ttccgaacgc tgttacctac tgcgctacca aattctacgt ttctgcttac 480 accgaaggtc tggctcagga actgcagaaa ggtggtgcta aactgcgtgc taaagttctg 540 gctccggctg ctaccgaaac cgaattcgct gaccgtagcc gtggtgaagc tggtttcgac 600 tactctaaaa acgttaaaaa ataccacacc gctgctgaaa tggctggttt cctgcaccag 660 ctgatcgaat ctgacgctat cgttggtatc gttgacggtg aaacctacga attcgaactg 720 cgtggtccgc tgttcaacta cgctggttaa 750

Claims

1. A carbonyl reductase mutant, characterized in that: The mutation sites of the carbonyl reductase mutant are as follows compared with the amino acid sequence shown in SEQ ID NO: 1: F88V, R142M, A190V and S193A.

2. A method for preparing a compound as shown in formula II, comprising the following steps: in a liquid reaction system, the compound as shown in formula I is subjected to a reduction reaction as shown in the following formula in the presence of a coenzyme and the carbonyl reductase mutant as described in claim 1; R1 is H, or benzyl; R 1-1 is C1-C6 alkyl or benzyl; R2 is H, or benzyl; R 2-1 is C1-C6 alkyl or benzyl; R3 is , where R 3-1 is a C1-C6 alkyl group; R4 is H, NO2, halogen, C1-C6 alkyl, C1-C6 alkoxy or sulfonyl substituted by C1-C6 alkyl.

3. The method for preparing the compound of formula II as claimed in claim 2, characterized in that: The reduction reaction satisfies one or more of the following conditions: ① The halogen is F, Cl, Br or I; ② The C1-C6 alkyl group is a C1-C4 alkyl group; ③ The coenzyme is a reducing coenzyme and / or an oxidizing coenzyme; ④ The mass ratio of the coenzyme to the compound shown in formula I is 1:(1-100); ⑤ The liquid reaction system includes an enzyme for coenzyme regeneration and a cosubstrate for coenzyme regeneration; ⑥ The reaction temperature of the reduction reaction is 10°C-50°C; ⑦ The reaction time of the reduction reaction is 0.1-72 hours; ⑧ The pH of the reduction reaction is 6-10; and, ⑨ the carbonyl reductase mutant is added to the reduction reaction in the form of free enzyme, immobilized enzyme, bacterial powder or bacterial cell enzyme.

4. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl or tert-butyl.

5. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The oxidative coenzyme is NAD + and / or NADP + ; The reducing coenzyme is NADH and / or NADPH.

6. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The mass ratio of the coenzyme to the compound shown in formula I is 1:(50-100).

7. The method for preparing the compound of formula II as claimed in claim 5, characterized in that: The mass ratio of the coenzyme to the compound shown in formula I is 1:100 or 1:

75.

8. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The enzyme used for coenzyme regeneration is one or more of alcohol dehydrogenase, formate dehydrogenase and glucose dehydrogenase.

9. The method for preparing the compound of formula II as claimed in claim 8, characterized in that: The enzyme used for coenzyme regeneration is glucose dehydrogenase.

10. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The co-substrate is one or more of isopropanol, glucose and ammonium formate.

11. The method for preparing the compound of formula II as claimed in claim 10, characterized in that: The co-substrate is glucose.

12. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The liquid reaction system also includes a buffer.

13. The method for preparing the compound of formula II as claimed in claim 12, characterized in that: The liquid reaction system also includes a phosphate buffer.

14. The method for preparing the compound of formula II as claimed in claim 13, characterized in that: The phosphate buffer is 0.1 M phosphate buffer.

15. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The reaction temperature of the reduction reaction is 25°C-35°C.

16. The method for preparing the compound of formula II as claimed in claim 15, characterized in that: The reaction temperature of the reduction reaction is 30°C.

17. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The reaction time of the reduction reaction is 3-24 hours.

18. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The carbonyl reductase mutant is an enzyme in bacterial form.

19. The method for preparing the compound of formula II as claimed in claim 3, characterized in that: The reduction reaction satisfies one or more of the following conditions: ①R1 is H, , , , , or ; ②R2 is H, , , , , or ; ③R3 is , , or ; ④R4 is H, NO2, F, Cl, Br, I, methyl, methoxy or ; ⑤ The liquid reaction system further comprises a cosolvent; ⑥ The mass concentration of the co-substrate in the liquid reaction system is 5-30%; ⑦ The mass concentration of the enzyme used for coenzyme regeneration in the liquid reaction system is 1-10%; ⑧After the reduction reaction is completed, a post-treatment step is also included.

20. The method for preparing the compound of formula II as claimed in claim 19, characterized in that: The co-solvent is selected from one or more of dimethyl sulfoxide, isopropanol and toluene.

21. The method for preparing the compound of formula II as claimed in claim 20, characterized in that: The cosolvent is selected from dimethyl sulfoxide.

22. The method for preparing the compound of formula II as claimed in claim 19, characterized in that: The mass concentration of the co-substrate in the liquid reaction system is 5%-20%.

23. The method for preparing the compound of formula II as claimed in claim 22, characterized in that: The mass concentration of the co-substrate in the liquid reaction system is 16%, 8% or 12%.

24. The method for preparing the compound of formula II as claimed in claim 19, characterized in that: The mass concentration of the enzyme for coenzyme regeneration in the liquid reaction system is 1%-5%.

25. The method for preparing the compound of formula II as claimed in claim 24, characterized in that: The mass concentration of the enzyme for coenzyme regeneration in the liquid reaction system is 2.5%, 1.6% or 2.3%.

26. The method for preparing the compound of formula II as claimed in claim 19, characterized in that: The post-treatment step includes: adding an organic solvent to the aforementioned liquid reaction system, heating, filtering the bacteria, extracting, washing the organic phase with water, drying and filtering and concentrating the organic layer to obtain a compound as shown in Formula II.

27. The method for preparing the compound of formula II as claimed in claim 26, characterized in that: The organic layer is as shown in Formula II. R1 is H, R2 is , , or , R3 is , , or , R4 is F, Cl or Br.

28. The method for preparing the compound of formula II as claimed in claim 19, characterized in that: The reduction reaction satisfies one or more of the following conditions: ① The compound shown in formula I is , , , , , or ; ② The compound shown in formula II is , , , , , or ; ③ The mass concentration of the co-solvent in the liquid reaction system is 10%-50%; ④ The organic solvent is an ester solvent, an ether solvent, an alcohol solvent, an aromatic hydrocarbon solvent or a chlorinated alkane solvent; ⑤ The heating temperature is 60°C; ⑥ The heating time is 1h; 7. The water in the water washing is pure water or water containing inorganic salts; and ⑧ the drying is drying using a desiccant.

29. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The mass concentration of the co-solvent in the liquid reaction system is 20%-30%.

30. The method for preparing the compound of formula II as claimed in claim 29, characterized in that: The mass concentration of the co-solvent in the liquid reaction system is 30%, 29% or 28%.

31. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The ester solvent is ethyl acetate or isopropyl acetate.

32. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The ether solvent is methyl tert-butyl ether or 2-methyltetrahydrofuran.

33. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The alcohol solvent is n-butanol.

34. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The aromatic hydrocarbon solvent is toluene.

35. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The chlorinated alkane solvent is dichloromethane.

36. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The water used in the water washing includes pure water and / or 5% saline.

37. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The desiccant is anhydrous sodium sulfate.

38. The method for preparing the compound of formula II as claimed in claim 28, characterized in that: The post-treatment step includes: adding methyl tert-ether or ethyl acetate to the reaction solution of the aforementioned reduction reaction, heating, filtering the bacteria and extracting, washing the organic phase with pure water, washing with 5% salt water, drying with anhydrous sodium sulfate and filtering and concentrating the organic layer to obtain the compound shown in formula II.

39. Use of the carbonyl reductase mutant according to claim 1 in reducing carbonyl groups.

40. The use according to claim 39, characterized in that The reaction substrate and reaction conditions used are the reaction substrate and reaction conditions in the method for preparing the compound shown in Formula II as described in any one of claims 2 to 38.

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