WO2019153634A1 - 醇脱氢酶突变体及其在双芳基手性醇合成中的应用 - Google Patents

醇脱氢酶突变体及其在双芳基手性醇合成中的应用 Download PDF

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WO2019153634A1
WO2019153634A1 PCT/CN2018/094507 CN2018094507W WO2019153634A1 WO 2019153634 A1 WO2019153634 A1 WO 2019153634A1 CN 2018094507 W CN2018094507 W CN 2018094507W WO 2019153634 A1 WO2019153634 A1 WO 2019153634A1
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amino acid
substitution
acid sequence
seq
mutant
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PCT/CN2018/094507
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倪晔
王岳
戴威
许国超
周婕妤
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江南大学
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Priority to US16/517,792 priority patent/US10711291B2/en
Priority to US16/521,067 priority patent/US11078465B2/en
Publication of WO2019153634A1 publication Critical patent/WO2019153634A1/zh

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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)

Definitions

  • the invention relates to an alcohol dehydrogenase mutant and its application in the synthesis of a bisaryl chiral alcohol, and belongs to the technical field of bioengineering.
  • Chiral bisaryl alcohol compounds are key chiral intermediates for the synthesis of numerous drugs and fine chemicals, wherein chiral (4-chlorophenyl)-(pyridin-2-yl)-methanol (CPMA) is a synthetic antihistamine.
  • CPMA (4-chlorophenyl)-(pyridin-2-yl)-methanol
  • a key chiral intermediate for the drug betahistine is mainly achieved by the following five techniques:
  • the above reaction has the problems of high cost of the noble metal ligand catalyst, low substrate concentration, high pressure conditions for the reaction, many operation steps, and low optical purity of the material, which cannot meet the requirements of the optical purity of the drug, and is not favorable for industrialization. produce.
  • Biocatalysis refers to the process of chemical conversion using enzymes or biological organisms (cells, organelles, tissues, etc.) as a catalyst.
  • the action conditions are mild, and are completed in an environment of normal temperature, neutrality, and water.
  • Unique advantages It meets the goals of industrial development such as “sustainable development”, “green chemistry” and “environmentally friendly manufacturing”.
  • the use of alcohol dehydrogenase to asymmetrically reduce the carbonyl group in the latent ketone has the advantages of high stereoselectivity and mild reaction conditions, and has important economic, social and ecological significance.
  • the biological asymmetric reduction method can be mainly realized by the following four technologies:
  • Li Zhe et al. studied the asymmetric reduction of a series of diaryl ketones by a carbonyl reductase PasCR derived from Pichia pastoris GS115.
  • the substrate concentration was 10 mM and the conversion rate was only 50%.
  • the present invention provides a series of alcohol dehydrogenase mutant proteins, a nucleic acid sequence encoding the mutant protein, and a recombinant expression vector containing the nucleic acid sequence, in view of the problem of low stereoselectivity of the alcohol dehydrogenase in the prior art. And recombinantly expressing the transformant, and the alcohol dehydrogenase mutant protein or a recombinant transformant expressing the alcohol dehydrogenase mutant protein as a catalyst for asymmetric reduction, preparation of an optical chiral bisaryl alcohol.
  • a first object of the invention is to provide a higher reactivity and stereoselective alcohol dehydrogenase mutant.
  • the alcohol dehydrogenase mutant is one or more amino acid positions in which the amino acid sequence is the alcohol dehydrogenase of SEQ ID NO.
  • the amino acid sequence of the alcohol dehydrogenase mutant comprises: amino acid glutamic acid at position 214 and amino acid at position 237 in the amino acid sequence shown in SEQ ID No. An amino acid sequence obtained by mutating one or two sites in serine.
  • the mutant comprises the substitution of the glutamic acid at position 214 of the amino acid sequence of SEQ ID NO. 2 with valine (E214V), designated M1.
  • the mutant comprises the substitution of the glutamic acid at position 214 of the amino acid sequence of SEQ ID NO. 2 with tyrosine (E214Y), designated M2.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID NO. 2 with isoleucine (E214I), designated M3.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID NO. 2 with glycine (E214G), designated M4.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID NO. 2 with glutamine (E214Q), designated M5.
  • the mutant comprises the 214th glutamic acid (E214S) having the amino acid sequence of the amino acid sequence of SEQ ID NO. 2, which is designated M6.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID NO. 2 with asparagine (E214N), designated M7.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID NO. 2 with arginine (E214R) and designating it M8.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with valine, and at the same time 237
  • the serine was replaced with alanine (E214V/S237A) and designated M9.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with tyrosine, and at position 237 The serine was replaced with alanine (E214Y/S237A) and designated M10.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with isoleucine, and at position 237 The serine was replaced with alanine (E214I/S237A) and designated M11.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with glycine, and replacing the serine at position 237 Cysteine (E214G/S237C), named M12.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with glutamine, and the 237th The serine was replaced with cysteine (E214Q/S237C) and designated M13.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with serine, and replacing the serine at position 237 Cysteine (E214S/S237C), named M14.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with asparagine, and at position 237 The serine was replaced with cysteine (E214N/S237C) and designated M15.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID No. 2 with arginine, and at the same time The serine was replaced with cysteine (E214R/S237C) and designated M16.
  • the recombinant strain expressing the mutant is expressed.
  • the recombinant bacteria is constructed by cloning a nucleic acid molecule encoding the mutant into a recombinant vector, and transforming the resulting recombinant vector into a transformant to obtain a recombinant expression transformant.
  • the resulting recombinant expression transformant is cultured, and the protein can be isolated and purified.
  • the host of the recombinant strain is Escherichia coli, and the plasmid is pET28a (+).
  • the host of the recombinant strain is E. coli BL21 (DE3).
  • Another object of the present invention is to provide a method for producing an alcohol dehydrogenase by the recombinant bacteria, the specific steps are as follows: inoculating the recombinant bacteria into an LB medium containing 40-60 ⁇ g/mL kanamycin sulfate, 30 ⁇ 40°C, 100 ⁇ 200 rpm shaker culture, when the absorbance OD 600 of the culture solution reaches 0.5-1.0, 0.05-1.0 mM isopropyl- ⁇ -D-hexa-fromogalactopyranoside (IPTG) is added for induction. At a temperature of 16 to 30 ° C, a mutant expressing a recombinant alcohol dehydrogenase can be obtained by inducing 5 to 10 hours.
  • IPTG isopropyl- ⁇ -D-hexa-fromogalactopyranoside
  • the use of the mutant as a catalyst in the preparation of an optically pure chiral bisaryl alcohol in the asymmetric reduction of a latent chiral carbonyl compound is not limited.
  • the latent chiral carbonyl compound is 4-chlorophenyl--2-pyridyl-methanone, phenyl-2-pyridyl-methanone, 4-chlorophenyl- Phenyl ketone, 4-bromophenyl-phenyl ketone, 4-fluorophenyl-phenyl ketone, 4-methoxyphenyl-phenyl ketone, 4-nitrophenyl-phenyl ketone ,2-acetylpyridine, acetophenone, 4-chloroacetophenone, 4-chlorobenzoylmethyl chloride, ethyl 2-oxo-4-phenyl-butyrate, ethyl 4-chloroacetoacetate, benzene Methyl formylformate.
  • a method for producing chiral CPMA by using alcohol dehydrogenase the specific steps of the method are as follows: constructing a reaction system, the concentration of CPMK is 10-500 mM, and the amount of the dehydrogenase mutant according to any one of claims 1-3 is 1.
  • NADP + dosage is 0.1 ⁇ 1.0mM
  • the coenzyme circulation system contains glucose dehydrogenase GDH and D-glucose, wherein the glucose dehydrogenase GDH dosage is 1 ⁇ 10kU / L, D-
  • the dosage of glucose is 20-1000 mM, the concentration of phosphate buffer is 0.1-0.2 M; the reaction is carried out at 30-35 ° C, pH 6-8 for 1-24 h, after the asymmetric reduction reaction, the organic solvent extraction method can be followed.
  • the chiral CPMA was extracted from the reaction solution.
  • the coenzyme circulation system may also be phosphite/phosphite dehydrogenase (FTDH), formate/formate dehydrogenase (FDH), lactic acid/lactate dehydrogenase (LDH) ) or glycerol/glycerol dehydrogenase.
  • FTDH phosphite/phosphite dehydrogenase
  • FDH formate/formate dehydrogenase
  • LDH lactic acid/lactate dehydrogenase
  • glycerol/glycerol dehydrogenase glycerol/glycerol dehydrogenase
  • the chromatographic method comprises: taking 100 ⁇ L of the reaction solution, adding 500 ⁇ L of ethyl acetate, shaking for 1 to 2 minutes, centrifuging at 12,000 rpm for 2-5 minutes, taking the supernatant into a centrifuge tube, waiting for organic The phase is naturally complete, 500 ⁇ L of chromatographically pure ethanol is added, and the conversion and ee values are analyzed by chiral liquid chromatography and gas chromatography.
  • the CPMK liquid chromatographic conditions are as follows: Daicel Chiralcel OB-H (5 ⁇ m, 250 mm ⁇ 4.6 mm) liquid chromatography column, the mobile phase is n-hexane: ethanol: ethanolamine (90:10:0.01, v/v/v) , flow rate 0.8mL / min, column temperature 30 ° C, UV detection wavelength 254nm, injection volume 10 ⁇ L, (S)- and (R)-CPMA retention time are 12.54min and 13.57min; p-nitrobenzophenone
  • the alcohol dehydrogenase mutation obtained by the present invention has high activity on various carbonyl compounds, and can catalytically reduce a plurality of aliphatic or aryl-substituted ketone substrates, especially bis-aryl groups having a large steric hindrance. Ketone substrate. Molecular engineering of KpADH by a combination of mutations increases the stereoselectivity of the enzyme, which will make it more industrially useful.
  • the positive progress of the present invention is that the alcohol dehydrogenase single point mutants E214Y, E214V and E214I of the present invention have a higher (R)-CPMA pair than the wild type alcohol dehydrogenase KpADH.
  • the combination mutants E214G/S237C, E214Q/S237C, E214S/S237C, E214N/S237C and E214R/S237C all achieved the inversion from the R configuration to the S configuration, and these mutation points are available for later study.
  • the alcohol dehydrogenase mutant obtained by the invention is particularly suitable for asymmetric reduction of bisaryl ketone, and has good industrial application prospects.
  • Figure 1 is a diagram showing the full-plasmid PCR nucleic acid electrophoresis pattern of an alcohol dehydrogenase mutant
  • Lanes 1 to 8 full-plasmid bands at an annealing temperature of 55 ° C for M1 to M8 mutants;
  • Lanes 9 to 16 full-plasmid bands at 50 ° C annealing temperature of M9-M16 mutants
  • Figure 2 is a SDS-PAGE electropherogram of the expression and purification results of the dehydrogenase mutant
  • Lanes 1-20 are alcohol dehydrogenase mutants M1 to M16, respectively;
  • Figure 4 is a chiral liquid chromatogram of the reduction product of p-nitrobenzophenone catalyzed by alcohol dehydrogenase mutant M10;
  • Figure 5 is a chiral liquid chromatogram of the acetophenone reduction product catalyzed by the alcohol dehydrogenase mutant M12;
  • Figure 6 is a chiral liquid chromatogram of the reduction product of 4 chloroacetophenone catalyzed by alcohol dehydrogenase mutant M11;
  • Figure 7 is a chiral liquid chromatogram of the reduction product of tetrachloroformyl chloride by the alcohol dehydrogenase mutant M14.
  • Example 1 Method for measuring the activity of alcohol dehydrogenase:
  • the total reaction system is 200 ⁇ L, including: 1.0 mM NADPH, 1.0 mM substrate CPMK, sodium phosphate buffer (PBS, l00 mM, pH 7.0), thoroughly mixed, incubated at 30 ° C for 2 min, adding appropriate amount of enzyme solution, detecting 340 nm light The change in absorption value.
  • the enzyme activity was calculated by the following formula:
  • Enzyme activity (U) EW ⁇ V ⁇ 10 3 /(6220 ⁇ l)
  • EW is the change in absorbance at 340 nm in 1 minute
  • V is the volume of the reaction solution in mL
  • 6220 is the molar extinction coefficient of NADPH in units of L/(mol ⁇ cm)
  • 1 is the optical path distance, unit Is cm.
  • One unit of activity (U) corresponds to the amount of enzyme required to catalyze the oxidation of 1 ⁇ mol of NADPH per minute under the above conditions.
  • Example 2 Construction of an alcohol dehydrogenase mutant gene and recombinant expression transformants
  • the full-plasmid PCR method was used to perform site-directed mutagenesis of the amino acid residue at position 237.
  • the primers were designed as follows (both described in the 5'-3' direction) and underlined for the mutation site:
  • the PCR reaction system is: PCR reaction system (50 ⁇ L) including KOD enzyme (2.5 U/mL) 1.0 ⁇ L, template (5-50 ng) 1.0 ⁇ L, dNTP 4.0 ⁇ L, 10 ⁇ reaction buffer 5.0 ⁇ L, upstream and downstream primers 1.0 each ⁇ L, ddH 2 O was made up to 50 ⁇ L.
  • the PCR amplification procedure was: (1) denaturation at 94 °C for 3 min, (2) denaturation at 94 °C for 30 sec, (3) annealing at 54 °C for 30 sec, (4) extension at 72 °C for 150 sec, and repeating steps (2) to (4) for 10-. 15 cycles, the last 72 ° C extension for 10 min, 4 ° C to preserve the PCR amplification products.
  • DpnI restriction enzyme was added to the reaction mixture and incubated at 37 ° C for 1 h. 10 ⁇ L of the digested PCR reaction solution was transferred to 50 ⁇ L of E. coli BL21 (DE3) competent cells by CaCl 2 thermal transformation. The samples were uniformly coated on an LB agar plate containing 50 ⁇ g/ml kanamycin sulfate, and cultured in an inverted manner at 37 ° C for 12 hours.
  • the recombinant Escherichia coli carrying the stereoselective improvement mutant was inoculated into LB medium containing kanamycin sulfate (50 ⁇ g/mL) at 2% transfer rate, and cultured at 37 ° C, shaking at 200 rpm, and the absorbance of the culture solution.
  • kanamycin sulfate 50 ⁇ g/mL
  • IPTG isopropyl- ⁇ -D-hexa-fromogalactomannan
  • the induction temperature was 25 ° C.
  • centrifugation at 8000 rpm for 10 min to obtain high-efficiency expression of recombinant alcohol dehydrogenation.
  • the cells of the enzyme mutant were suspended in potassium phosphate buffer (100 mM, pH 6.0) and sonicated.
  • the column used for purification was a nickel affinity column HisTrap FF crude, which was performed by affinity chromatography using a histidine tag on the recombinant protein.
  • the nickel column was equilibrated with the A solution, the crude enzyme solution was loaded, and the breakthrough peak was further eluted using the A solution. After the equilibration, the gradient was washed with the B solution (20 mM sodium phosphate, 500 mM NaCl, 1000 mM imidazole, pH 7.4).
  • the recombinant protein that binds to the nickel column is eluted to obtain a recombinant alcohol dehydrogenase mutant.
  • the purified protein was assayed for viability (CPMK as substrate, NADPH as coenzyme) and SDS-PAGE analysis. After purifying the nickel column, a single band was displayed at around 45 kDa, and the amount of heteroprotein was small, indicating that the column purification effect was better.
  • the purified alcohol dehydrogenase protein was then replaced with Tris-HCl (100 mM, pH 7.0) buffer using a Hi Trap Desalting Desalting Column (GE Healthcare).
  • the single point mutation of E214 site uses a site-directed mutagenesis strategy to replace glutamate with the other 19 amino acids.
  • the results of the new mutants are shown in Table 3.
  • the Km of KpADH is 0.76 mM –1 and the reduction product configuration is In the R configuration, the ee value was 81.7%, and only the K m of E214G, E214V and E214I in the mutant was significantly reduced, being 0.25 mM, 0.42 mM and 0.41 mM, respectively.
  • E214V, E214Y and E214I were used as templates to replace the serine at position 237 with glycine, and the serine at position 237 with E214G, E214Q, E214S, E214N and E214R as templates.
  • the results of the new mutants were replaced by cysteine.
  • Kcat/Km of mutant enzymes E214V/S237A, E214Y/S237A, E214I/S237A is slightly improved compared to WT, but asymmetric synthesis ( The stereoselectivity of R)-CPMA was significantly improved, 98.5%, 99.1% and 98.3%, respectively; the products synthesized by E214G/S237C, E214Q/S237C, E214S/S237C, E214N/S237C and E214R/S237C were (S)-CPMA, in which E214G/S237C has the highest selectivity, reaching 75.5% (S), and the catalytic efficiency Kcat/Km is also slightly improved compared to WT.
  • the above combined mutants have high guiding value for studying the modification of the asymmetric synthesis of R- and S- by the enzyme.
  • the alcohol dehydrogenase mutant obtained in Example 3 was investigated for the reduction of latent chiral carbonyl compounds.
  • the alcohol dehydrogenase shown has a higher activity on the ester substrate, such as ethyl 2-oxo-4-phenyl-butyrate, ethyl 4-chloroacetoacetate and benzoic acid.
  • Methyl formate, in which ethyl 4-chloroacetoacetate, WT has the highest activity, reaching 41.84 U/mg; the presence of chlorine atoms in the substrate can enhance the activity, such as p-acetophenone, 4-chlorophenyl
  • the comparison of ketone and 4-chlorophenacyl chloride shows that acetophenone has the lowest activity and 4-chlorophenacyl chloride has the highest activity.
  • the enzyme activity of mutant E214I/S237A on 4-chlorophenacyl chloride It reached 30.09 U/mg.
  • E214G/S237C, E214Q/S237C, E214S/S237C, E214N/S237C and E214R/S237C asymmetrically reduce the substrate acetophenone to obtain the R configuration product with high stereoselectivity, of which E214G/ The ee value of S237C reached 99.5%; alcohol dehydrogenase and its mutant showed high stereoselectivity to the substrate 4-chloroacetophenone, among which E214I/S237A was the highest, and asymmetric reduction gave the R configuration.
  • the selectivity was 99.4%; the E214S/S237C asymmetric reduction substrate 4-chloro-benzoyl chloride gave the highest ee of the R configuration product, 99.6%; the mutant E214Y/S237A asymmetric reduction substrate to bromodiphenyl
  • the steric selectivity of ketone and p-nitrobenzophenone to S configuration products was the highest, 98.5% and 99.2%, respectively; E214N/S237C asymmetric reduction substrate p-methoxybenzophenone obtained S configuration
  • the stereoselectivity reached 99.1%, and all of the above products can be recrystallized to have a selectivity of >99.9%, which has high utilization value.
  • Example 5 Alcohol dehydrogenase mutant asymmetric reduction CPMK preparation (R)-CPMA
  • a 20 mL biocatalytic system was established: 100 mM potassium phosphate buffer (pH 7.0), and the alcohol dehydrogenase mutant E214Y/S237A obtained in Example 2 and wild KpADH 10 g/L, CPMK 100 mM, 200 mM and 500 mM (substrate batch) were added. Add to).
  • the mutant E214G/S237C and the substrate acetophenone, the mutant E214I/S237A and the substrate 4-chloroacetophenone, the mutant E214S/S237C and the substrate acetophenone, and the mutant E214G/S237C were established in the same manner.
  • the alcohol dehydrogenase mutant of the present invention not only has very good performance in high-efficiency, high stereoselective asymmetric reduction of CPMK, but also has high catalysis for other aryl ketone substrates. Efficiency and high stereo selectivity.

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Abstract

醇脱氢酶突变体及其在双芳基手性醇合成中的应用。将本发明的所述的醇脱氢酶与葡萄糖脱氢酶或甲酸脱氢酶进行偶联，可用于多种抗组胺药物手性双芳基醇中间体的合成。

Description

醇脱氢酶突变体及其在双芳基手性醇合成中的应用 技术领域
本发明涉及醇脱氢酶突变体及其在双芳基手性醇合成中的应用，属于生物工程技术领域。
背景技术
手性双芳基醇化合物是合成众多药物和精细化学品的关键手性中间体，其中手性(4-氯苯基)-(吡啶-2-基)-甲醇(CPMA)是合成抗组胺药物倍他司汀的关键手性中间体。以潜手性(4-氯苯基)-(吡啶-2-基)-甲酮(CPMK)为原料，通过化学法不对称还原合成手性CPMA主要由以下五种技术实现：
1.在底物浓度为1.0mM条件下，以trans-RuCl 2[(R)-xylbinap][(R)-daipen]为催化剂，在压力为40-60psi充氮气条件下室温反应24h，还原获得(S)-(4-氯苯基)-(吡啶-2-基)-甲醇((S)-CPMA)，其ee值为60.6％，产率为98％。(C.Y.Chen,et al.,Org.Lett.,2003,5,5039-5042)。
2.以(S)-[Ru(BINAP)Cl 2] 2(NE 3)为催化剂，加压，通氢气，还原得到(S)-CPMA)，ee值为99％。(赵志全等,中国医药工业杂志,2006,37,726-727)。
3.以CPMK为原料，在底物浓度仅为0.2mM条件下以(S,S)-6-CHOONa为催化剂，在50℃条件下反应24h，还原获得的(R)-(4-氯苯基)-(吡啶-2-基)-甲醇((R)-CPMA)，其ee值为40.8％，产率为90％。(B.G.Wang,Org.Lett.,2017,19,2094-2097)。
4.以CPMK为原料，采用三步反应，1)先用三氟甲磺酸酐等进行保护，2)再用催化剂钯配位体及Me-CBS等还原羰基到S构型羟基，3)在三苯基磷钯作用下脱保护，得到(S)-CPMA。(中国专利CN101848893A)。
5.以手性BINAL-H为手性还原剂，在底物浓度为400mM CMPK条件下定向合成单一构型CPMA。进行乙酸乙酯-石油醚重结晶后，(R)-CPMA收率88.2％，纯度96.2％，(S)-CPMA收率87.4％，纯度95.7％。(中国专利CN103121966A)。
由此可见，上述反应存在贵金属配位体催化剂成本较高、底物浓度低、反应需要高压条件、操作步骤较多、物光学纯度低的问题，不能满足药物对光学纯度的要求，不利于工业化生产。
生物催化是指利用酶或者生物有机体(细胞、细胞器、组织等)作为催化剂进行化学转化的过程，作用条件温和，在常温、中性和水等环境中完成，对于手性活性药物成分的合成具有独特的优点。符合“可持续发展”、“绿色化学”、“环境友好制造”等工业发展的目标。与化学合成方法相比，使用醇脱氢酶将潜手性酮中的羰基进行不对称还原的反应具有立体选 择性高、反应条件温和等优势，具有重要的经济、社会价值和生态意义。生物不对称还原法主要可通过以下四种技术实现：
1. 2007年，Truppo等筛选了一系列商品化酮还原酶KRED后，发现虽然有一些酮还原酶对双芳基底物有还原能力，但立体选择性一般，且底物谱较窄，底物中的取代基团对立体选择性的影响较大。仅KRED124可以不对称还原CPMK生成(R)-CPMA，ee值为94％，转化率98％，且需要外加葡萄糖脱氢酶提供辅酶循环。(M.D.Truppo,Org.Lett.,2007,9,335-338)。
2. 2009年，朱敦明等发现来游于Sporobolomyces salmonicolor的重组羰基还原酶SsCR及其突变体可以立体选择性还原不同双芳基酮底物(8-99％ee)。在葡萄糖脱氢酶的协助下，还原CPMK生成(R)-CPMA，转化率62％，对映选择性为88％(R)。(D.M.Zhu,Org.Lett.,2008,10,525-528)。
3. 2012年，周婕妤等通过传统富集培养筛选到一株克鲁维酵母Kluyveromyces sp.CCTCCM2011385，可催化还原CPMK生成(S)-CPMA(87％ee)。然而活性酶在野生菌中的含量低，最高仅能催化2g/L底物，产物浓度较低，分离成本高，因而不能满足应用的需要。(Y.Ni,Process Biochem.,2012,47,1042-1048；中国专利CN102559520A)。
4. 2013年，李哲等研究了一个来源于毕赤酵母GS115的羰基还原酶PasCR对一系列双芳香基甲酮类化合物的不对称还原，底物浓度为10mM，转化率最高只有50％。(李哲等,生物工程学报,2013,29,68-77)。
由此可知，采用生物不对称还原法制备手性CPMA的立体选择性较难达到医药的要求大于95％的对映体过量值，尤其缺乏合成制备(S)-CPMA的还原酶，因此亟需开发高效、高立体选择性的生物酶催化剂。
发明内容
本发明针对现有技术中的醇脱氢酶立体选择性较低的问题，提供了一系列醇脱氢酶的突变体蛋白质、编码该突变体蛋白质的核酸序列，含有该核酸序列的重组表达载体和重组表达转化体，以及该醇脱氢酶突变体蛋白质或表达该醇脱氢酶突变体蛋白质的重组转化体作为催化剂在不对称还原、制备光学手性双芳基醇中应用。
本发明的第一个目的是提供一种更高的反应活性和立体选择性的醇脱氢酶突变体。
在本发明的一种实施方式中，所述醇脱氢酶突变体是将氨基酸序列为SEQ ID NO.2所示醇脱氢酶的一个或多个氨基酸位点进行突变。
在本发明的一种实施方式中，所述醇脱氢酶突变体的氨基酸序列包括：在SEQ ID No.2所示的氨基酸序列上将第214位的氨基酸谷氨酸、第237位的氨基酸丝氨酸中的一个或两个位点进行突变而得到的氨基酸序列。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214位的谷氨酸替换为缬氨酸(E214V)，命名为M1。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214位的谷氨酸替换为酪氨酸(E214Y)，命名为M2。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214位的谷氨酸替换为异亮氨酸(E214I)，命名为M3。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214位的谷氨酸替换为甘氨酸(E214G)，命名为M4。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214位的谷氨酸替换为谷氨酰胺(E214Q)，命名为M5。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214为谷氨酸替换为丝氨酸(E214S)，命名为M6。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214位的谷氨酸替换为天冬酰胺(E214N)，命名为M7。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列为SEQ ID NO.2所示氨基酸序列的第214位的谷氨酸替换为精氨酸(E214R)，命名为M8。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为缬氨酸，同时将237位点的丝氨酸替换成丙氨酸(E214V/S237A)，命名为M9。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为酪氨酸，同时将第237位的丝氨酸替换成丙氨酸(E214Y/S237A)，命名为M10。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为异亮氨酸，同时将第237位的丝氨酸替换成丙氨酸(E214I/S237A)，命名为M11。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为甘氨酸，同时将第237位的丝氨酸替换成半胱氨酸 (E214G/S237C)，命名为M12。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为谷氨酰胺，同时将第237位的丝氨酸替换成半胱氨酸(E214Q/S237C)，命名为M13。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为丝氨酸，同时将第237位的丝氨酸替换成半胱氨酸(E214S/S237C)，命名为M14。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为天冬酰胺，同时将第237位的丝氨酸替换成半胱氨酸(E214N/S237C)，命名为M15。
在本发明的一种实施方式中，所述突变体包括将氨基酸序列如SEQ ID No.2所示醇脱氢酶的第214位的谷氨酸替换为精氨酸，同时将第237位的丝氨酸替换成半胱氨酸(E214R/S237C)，命名为M16。
在本发明的一种实施方式中，表达所述突变体的重组菌。
在本发明的一种实施方式中，所述重组菌的构建方法：将编码所述突变体的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述蛋白质。
在本发明的一种实施方式中，所述重组菌的宿主为大肠杆菌(Escherichia coli)，所述质粒为pET28a(+)。
在本发明的一种实施方式中，所述重组菌的宿主为E.coli BL21(DE3)。
本发明的另一个目的是，提供一种所述重组菌生产醇脱氢酶的方法，具体步骤如下：将重组菌接种至含有40-60μg/mL硫酸卡那霉素的LB培养基中，30～40℃，100～200rpm摇床培养，培养液的吸光度OD 600达到0.5～1.0时，加入0.05～1.0mM的异丙基-β-D-六代呋喃半乳糖苷(IPTG)进行诱导，诱导温度为16～30℃，诱导5～10h即可获得高效表达重组醇脱氢酶的突变体。
在本发明的一种实施方式中，所述突变体作为催化剂在不对称还原潜手性羰基化合物制备光学纯手性双芳基醇中的应用。
在本发明的一种实施方式中，所述潜手性羰基化合物为4-氯苯基--2-吡啶基-甲酮，苯基-2-吡啶基-甲酮，4-氯苯基-苯基甲酮，4-溴苯基-苯基甲酮，4-氟苯基-苯基甲酮，4-甲氧苯基-苯基甲酮，4-硝基苯基-苯基甲酮，2-乙酰吡啶，苯乙酮，4-氯苯乙酮，4-氯苯酰甲基氯，2- 氧代-4-苯基-丁酸乙酯，4-氯乙酰乙酸乙酯，苯甲酰甲酸甲酯。
一种应用醇脱氢酶生产手性CPMA的方法，所述方法的具体步骤如下：构建反应体系，CPMK浓度为10-500mM，权利要求1-3任一所述脱氢酶突变体用量为1-10kU/L，NADP +用量为0.1～1.0mM，加入辅酶循环系统，辅酶循环系统中含有葡萄糖脱氢酶GDH和D-葡萄糖，其中葡萄糖脱氢酶GDH用量为1～10kU/L，D-葡萄糖用量为20～1000mM，磷酸盐缓冲液的浓度为0.1-0.2M；在30～35℃，pH 6～8的条件下反应1～24h，不对称还原反应结束后，可按照有机溶剂萃取方法从反应液中提取手性CPMA。
在本发明的一种实施方式中，所述辅酶循环系统还可以是亚磷酸盐/亚磷酸盐脱氢酶(FTDH)、甲酸/甲酸脱氢酶(FDH)、乳酸/乳酸脱氢酶(LDH)或甘油/甘油脱氢酶。
在本发明的一种实施方式中，所述的色谱分析方法为：取100μL反应液，加入500μL乙酸乙酯，震荡1～2min,12000rpm离心2-5min，取上清到离心管中，待有机相自然会发完全，加入500μL色谱纯乙醇，进行手性液相色谱和气相色谱分析转化率和ee值。对CPMK液相色谱条件具体如下：Daicel Chiralcel OB-H(5μm,250mm×4.6mm)液相色谱柱，流动相为正已烷:乙醇:乙醇胺(90:10:0.01,v/v/v)，流速0.8mL/min，柱温30℃，紫外检测波长254nm，进样量10μL，(S)-和(R)-CPMA保留时间分别为12.54min和13.57min；对对硝基二苯甲酮液相色谱条件具体如下：Daicel Chiralcel OJ-H(5μm,250mm×4.6mm)液相色谱柱，流动相为正己烷：异丙醇:乙醇胺(90:10:0.01,v/v/v)，流速＝0.8ml/min，柱温30℃，紫外检测波长254nm，进样量10μL，(S)-和(R)-保留时间分别为74.49min和95.56min；对苯乙酮的气相色谱条件如下：CP7502-Chirasil-DEX CB手性气相柱，100℃保持2min，以4℃/min升至180℃，保持2分钟，进样量2μL，(S)-和(R)-保留时间分别为10.15min和10.85min；对对氯苯乙酮的气相色谱条件如下：CP7502-Chirasil-DEX CB手性气相柱，100℃保持2min，以7℃/min升至180℃，保持2分钟，进样量2μL，(S)-和(R)-保留时间分别为9.26min和9.94min；对对氯苯甲酰基氯的气相色谱条件如下：CP7502-Chirasil-DEX CB手性气相柱，100℃保持2min，以5℃/min升至180℃，保持2分钟，进样量2μL，(S)-和(R)-保留时间分别为16.68min和17.55min。
本发明的有益效果为：
(1)本发明得到的醇脱氢酶突变对多种羰基化合物具有较高的活力，可以催化还原多种脂肪族或芳基取代的酮底物，尤其是空间位阻较大的双芳基酮底物。通过组合突变手段对KpADH进行分子改造，提高该酶的立体选择性，这将使其具有更高的工业应用价值。
(2)本发明的积极进步结果在于：与野生型醇脱氢酶KpADH相比，本发明所述醇脱氢酶单点突变体E214Y,E214V和E214I具有更高的(R)-CPMA的对映体选择性，其ee由野生型的82％(R)提高至91％(R)以上，其中S237V提高到了95.3％(R)，组合突变体E214V/S237A、E214Y/S237A、E214I/S237A的的(R)-CPMA的对映体选择性都提高到了97％以上，并且通过重结晶都能得到対映体纯度>99.9％的R型产物，具有很高的价值；E214G、E214Q、E214S、E214N和E214R具有反转的(S)-CPMA立体选择性的潜力，其ee由野生型的82％(R)降低至60％(R)以下，其中E214G单点突变就实现了从R构型到S构型的翻转，组合突变体E214G/S237C、E214Q/S237C、E214S/S237C、E214N/S237C和E214R/S237C都实现了从R构型到S构型的翻转，这些突变点可供以后研究的重要参考位点。本发明所获得的醇脱氢酶突变体特别适用于双芳基酮的不对称还原，具有良好的工业应用前景。
附图说明
图1为醇脱氢酶突变体全质粒PCR核酸电泳图；
泳道1～8：M1～M8突变株55℃退火温度下全质粒条带；
泳道9～16：M9～M16突变株50℃退火温度下全质粒条带；
图2为脱氢酶突变体表达及纯化结果的SDS-PAGE电泳图；
泳道1～20分别为醇脱氢酶突变体M1～M16；
图3为醇脱氢酶突变体M10催化CPMK还原产物手性液相色谱图；
图4为醇脱氢酶突变体M10催化对硝基二苯甲酮还原产物手性液相色谱图；
图5为醇脱氢酶突变体M12催化苯乙酮还原产物手性液相色谱图；
图6为醇脱氢酶突变体M11催化4氯苯乙酮还原产物手性液相色谱图；
图7为醇脱氢酶突变体M14催化4氯本甲酰基氯还原产物手性液相色谱图。
具体实施方式
下面通过具体实施例对本发明进行详细说明，但并不因此将本发明限制在所述的实施例范围之中。下述实施例中未注明具体实验条件的实验方法，可按照常规方法和条件，或按照说明书进行选择。
实施例1：醇脱氢酶的活力测定方法：
总反应体系为200μL，包括：1.0mM NADPH,1.0mM底物CPMK，磷酸钠缓冲液(PBS, l00mM,pH 7.0),充分混匀，30℃保温2min，加入适量的酶液，检测340nm下光吸收值的变化。
用下式计算得到酶活力：
酶活力(U)＝EW×V×10 3/(6220×l)
式中，EW为1分钟内340nm处吸光度的变化；V为反应液的体积，单位为mL；6220为NADPH的摩尔消光系数，单位为L/(mol·cm)；1为光程距离，单位为cm。1个活力单位(U)对应于上述条件下每分钟催化氧化lμmol NADPH所需的酶量。
光学纯度ee的测定方法：
Figure PCTCN2018094507-appb-000001
As：液相色谱获得的(S)-CPMA的摩尔浓度；A R：液相色谱获得的(R)-CPMA的摩尔浓度；
实施例2：醇脱氢酶突变体基因和重组表达转化体的构建
采用全质粒PCR方法对位于第237位的氨基酸残基进行定点突变，其引物设计如下(均按5’-3’方向描述)，下划线代表突变位点：
表1 定点突变引物设计表
引物名称 序列
E214A-F AGAAACTAAAT GCAACTTGTG
E214A-R TCACAAGT TGCATTTAGTTTC
E214T-F AGAAACTAAAT ACCACTTGTG
E214T-R TCACAAGT GGAATTTAGTTTC
E214R-F AGAAACTAAAT GCAACTTGTG
E214R-R TCACAAGT TGCATTTAGTTTC
E214N-F AGAAACTAAAT AATACTTGTG
E214N-R TCACAAGT ATTATTTAGTTTC
E214D-F AGAAACTAAAT GATACTTGTG
E214D-R TCACAAGT ATCATTTAGTTTC
E214C-F AGAAACTAAAT TGTACTTGTG
E214C-R TCACAAGT ACAATTTAGTTTC
E214E-F AGAAACTAAAT GAAACTTGTG
E214E-R TCACAAGT TTCATTTAGTTTC
E214Q-F AGAAACTAAAT CAGACTTGTG
E214Q-R TCACAAGT CTGATTTAGTTTC
E214G-F AGAAACTAAAT GGTACTTGTG
E214G-R TCACAAGT CCAATTTAGTTTC
E214H-F AGAAACTAAAT CATACTTGTG
E214H-R TCACAAGT ATGATTTAGTTTC
E214I-F AGAAACTAAAT ATTACTTGTG
E214I-R TCACAAGT AATATTTAGTTTC
E214L-F AGAAACTAAAT CTGACTTGTG
E214L-R TCACAAGT CAGATTTAGTTTC
E214K-F AGAAACTAAAT AAAACTTGTG
E214K-R TCACAAGT TTTATTTAGTTTC
E214M-F AGAAACTAAAT ATGACTTGTG
E214M-R TCACAAGT CATATTTAGTTTC
E214F-F AGAAACTAAAT TTTACTTGTG
E214F-R TCACAAGT AAAATTTAGTTTC
E214P-F AGAAACTAAAT CCGACTTGTG
E214P-R TCACAAGT CGGATTTAGTTTC
E214W-F AGAAACTAAAT TGGACTTGTG
E214W-R TCACAAGT CCTATTTAGTTTC
E214Y-F AGAAACTAAAT TATACTTGTG
E214Y-R TCACAAGT ATAATTTAGTTTC
E214V-F AGAAACTAAAT GTTACTTGTG
E214V-R TCACAAGT AACATTTAGTTTC
S237A-F ACTCACTTC GCACAATTCATT
S237A-R AATGAATTG TGCGAAGTGAGT
S237C-F ACTCACTTC TGTCAATTCATT
S237C-R AATGAATTG ACAGAAGTGAGT
PCR反应体系为：PCR反应体系(50μL)包括KOD酶(2.5U/mL)l.0μL，模板(5-50ng)l.0μL，dNTP 4.0μL，10×reaction buffer 5.0μL，上下游引物各1.0μL，ddH 2O补足至50μL。
PCR扩增程序为：(1)94℃变性3min，(2)94℃变性30sec，(3)54℃退火30sec，(4)72℃延伸150sec，重复步骤(2)～(4)进行10-15个循环，最后72℃延伸10min，4℃保存PCR扩增产物。
PCR结束后，添加DpnI限制性内切酶于反应混合物中并置于37℃孵育1h，用CaCl 2热转化法将10μL消化后PCR反应液转入50μL E.coli BL21(DE3)感受态细胞，并均匀涂布于含有50μg/ml硫酸卡那霉素的LB琼脂平板，37℃倒置培养12h。
实施例3：醇脱氢酶及其突变体的表达及纯化
将携带立体选择性改善突变体的重组大肠杆菌按2％的转接量接种至含有硫酸卡那霉素(50μg/mL)的LB培养基中，37℃，200rpm摇床培养，培养液的吸光度OD 600达到0.8时，加入0.2mM的异丙基-β-D-六代呋喃半乳糖甘(IPTG)进行诱导，诱导温度为25℃，诱导8h后，8000rpm离心10min获得高效表达重组醇脱氢酶突变体的菌体，将收集的菌体悬浮于磷酸钾缓冲液(100mM，pH 6.0)中，超声破碎。
纯化所使用的柱子为镍亲和柱HisTrap FF crude，利用重组蛋白上的组氨酸标签进行亲和层析来完成。首先使用A液将镍柱平衡，粗酶液上样，继续使用A液将穿透峰洗脱下来，待平衡后用B液(20mM磷酸钠，500mM NaCl,l000mM咪唑，pH 7.4)进行梯度洗脱，将结合 到镍柱上的重组蛋白洗脱下来，获得重组醇脱氢酶突变体。对纯化后的蛋白进行活力测定(CPMK为底物，NADPH为辅酶)及SDS-PAGE分析。镍柱纯化后，在45kDa左右显示单条带，且杂蛋白较少，说明柱纯化效果较好。之后使用Hi Trap Desalting脱盐柱(GE Healthcare)将纯化后的醇脱氢酶蛋白置换到Tris-HCl(l00mM，pH 7.0)缓冲液中。
实施例4：醇脱氢酶突变体的动力学和立体选择性分析
测定KpADH在不同底物浓度和辅酶浓度情况下的活力，并根据活力和底物浓度的倒数做出双倒数曲线，计算动力学参数。
利用随机突变筛选得到了S237A和S237C两个对于不对称合成R-和S-的改造有价值的突变体，并且以这两点为模板作随机突变，突变株表征结果如表2所示：突变体E214V/S237A不对称还原底物得到(R)-CPMA，e.e.值达到98.5％；而突变体E214G/S237C不对称还原底物得到(S)-CPMA，该实现了立体选择性的翻转，e.e.值为75.5％。其他突变体的立体选择性与WT相比，没有太大改善，结合表2与表3数据可知，醇脱氢酶的214位点是决定选择性的提高和翻转非常重要的位点。
表2 醇脱氢酶随机突变体动力学参数及立体选择性
Figure PCTCN2018094507-appb-000002
E214位点单点采用定点突变策略，将谷氨酸替换成其他19种氨基酸，构建新的突变体后表征结果如下表3所示，KpADH的Km为0.76mM –1，其还原产物构型为R构型，ee值为81.7％，突变体中仅E214G、E214V和E214I的K m明显降低，分别为0.25mM，0.42mM和0.41mM。突变体E214Y，E214V，E214I和E214F不对称合成(R)-CPMA的立体选择性有明 显提高，分别为93.8％，95.3％，93.0％和91.9％；突变体E214G，E214Q，E214S，E214N和E214R表现出明显降低的立体选择性，其中E214G不对称还原生成的是(S)-CPMA，e.e.值为25.6(S)，其他4个突变株依旧是合成(R)-CPMA，分别是58.5％，14.2％，58.1％和42.5％。
表3 醇脱氢酶突变体E214位点单点突变体的动力学参数及立体选择性
Figure PCTCN2018094507-appb-000003
为了提高该酶R-和S-立体选择性，以E214V，E214Y和E214I为模板，将237位点的丝氨酸替换成甘氨酸，以E214G，E214Q、E214S、E214N和E214R为模板将237位点的丝氨酸替换成半胱氨酸，构建新的突变体表征结果如下表4所示：突变体酶E214V/S237A、E214Y/S237A、E214I/S237A的Kcat/Km相比WT略有提高，但不对称合成(R)-CPMA的立体选择性都有较明显的提高，分别为98.5％，99.1％和98.3％；E214G/S237C、E214Q/S237C、E214S/S237C、E214N/S237C和E214R/S237C合成的产物都是(S)-CPMA，其中E214G/S237C的选择性最高，达到了75.5％(S)，并且催化效率Kcat/Km相比WT也略有提高。上述组合突变体对于研究该酶不对称合成R-和S-的改造具有很高的指导价值。
表4 醇脱氢酶组合突变体动力学参数及立体选择性
Figure PCTCN2018094507-appb-000004
实施例5：醇脱氢酶突变体的底物特异性分析
考察了实例3得到的醇脱氢酶突变体还原潜手性羰基化合物的情况，所考察的潜手性羰基化合物包括4-氯苯基--2-吡啶基-甲酮((4-chlorophenyl)-(pyridin-2-yl)-methanone,CPMK)，苯基-2-吡啶基-甲酮(phenyl-(pyridin-2-yl)-methanone)，4-氯苯基-苯基甲酮((4-chlorophenyl)-(phenyl)-methanone)，4-溴苯基-苯基甲酮((4-bromophenyl)(phenyl)methanone)，4-氟苯基-苯基甲酮((4-fluorophenyl)(phenyl)methanone)，4-甲氧苯基-苯基甲酮(4-methoxyphenyl)-(phenyl)-methanone)，4-硝基苯基-苯基甲酮((4-nitrophenyl)(phenyl)methanone)，2-乙酰吡啶(1-(pyridin-2-yl)ethanone)，苯乙酮(acetophenone)，4-氯苯乙酮(4'-Chloroacetophenone)，4-氯苯酰甲基氯(4-Chlorophenacyl Chloride)，2-氧代-4-苯基-丁酸乙酯(ethyl 2-oxo-4-phenylbutanoate，OPBE)，4-氯乙酰乙酸乙酯(ethyl 4-chloro-3-oxobutanoate)，苯甲酰甲酸甲酯(methyl 2-oxo-2-phenylacetate)。
由表5可知，所示醇脱氢酶对酯类底物表现出了较高的活力，如2-氧代-4-苯基-丁酸乙酯，4-氯乙酰乙酸乙酯和苯甲酰甲酸甲酯，其中对4-氯乙酰乙酸乙酯，WT表的活力最高，达到了41.84U/mg；底物中氯原子的存在能够提高活力，如对苯乙酮，4-氯苯乙酮和4-氯苯酰甲基氯对比可知道，苯乙酮的活力最低，4-氯苯酰甲基氯活力最高，其中突变体E214I/S237A对4-氯苯酰甲基氯的酶活力达到了30.09U/mg。
表5 醇脱氢酶突变体的底物特异性(比活力，U/mg)
Figure PCTCN2018094507-appb-000005
由表6可知，E214G/S237C、E214Q/S237C、E214S/S237C、E214N/S237C和E214R/S237C不对称还原底物苯乙酮得到R构型产物，并具有较高的立体选择性，其中E214G/S237C的ee值达到了99.5％；醇脱氢酶及其突变体对底物4-氯苯乙酮都表现出较高的立体选择性，其中E214I/S237A最高，不对称还原得到R构型的选择性为99.4％；E214S/S237C不对称还原底物4-氯-苯甲酰基氯得到R构型产物的ee最高，为99.6％；突变体E214Y/S237A不对称还原底物对溴二苯甲酮和对硝基二苯甲酮的到S构型产物的立体选择性最高，分别为98.5％和99.2％；E214N/S237C不对称还原底物对甲氧基二苯甲酮得到S构型的立体选择性达到了99.1％，上述产物均可以通过重结晶使得选择性>99.9％，具有较高的利用价值。
表6 醇脱氢酶突变体对不同底物的立体选择性
Figure PCTCN2018094507-appb-000006
实例5：醇脱氢酶突变体不对称还原CPMK制备(R)-CPMA
建立了20mL的生物催化体系：100mM磷酸钾缓冲液(pH 7.0)，加入实例2获得的醇脱氢酶突变体E214Y/S237A和野生KpADH 10g/L，CPMK 100mM，200mM和500mM(底物分批添加)。同时以同样的方法建立突变体E214G/S237C和底物苯乙酮，突变体E214I/S237A和底物4-氯苯乙酮，突变体E214S/S237C和底物苯乙酮，突变体E214G/S237C和底物苯乙酮，突变体E214G/S237C和底物4-氯苯甲酰基氯，E214Y/S237A和对溴二苯甲酮，E214Y/S237A和对硝基二苯甲酮及E214N/S237C和对甲氧基苯甲酮8个反应。在30℃和200rpm条件下反应12h，恒定pH7.5。反应过程中的转化率分析结果见表7到表11，其中不对称合成CPMA的反应中，野生型KpADH最终还原产物为(R)-CPMA，ee值为82％；突变体E214Y/S237A最终还原产物为(R)-CPMA，ee值为99.1％。将所得(R)-CPMA粗品重新溶解于乙醇中，加入相应的产物(R)-CPMA纯品为晶种于4℃重结晶，最终获得光学纯度均>99.9％ee的产品；E214G/S237C不对称还原苯乙酮，E214I/S237A不对称还原4-氯苯乙酮，E214S/S237C 不对称还原4-氯本酰甲基氯都通过重结晶得到了光学纯度均>99.9％ee的产品；E214Y/S237A不对称还原对硝基二苯甲酮，由于酶活力太低，底物可溶性差，仅能在24h还原20mM终浓度的底物，通过重结晶也能得到光学纯度>99.9％ee的产品；对于底物对溴二苯甲酮和对甲氧基二苯甲酮由于活力太低，可溶性太差，底物24h后依然不能彻底转化。
表7 野生型醇脱氢酶KpADH催化CPMK的不对称还原
Figure PCTCN2018094507-appb-000007
表8 醇脱氢酶突变体E214Y/S237A催化CPMK不对称还原
Figure PCTCN2018094507-appb-000008
表9 醇脱氢酶突变体E214G/S237C催化苯乙酮不对称还原
Figure PCTCN2018094507-appb-000009
表10 醇脱氢酶突变体E214S/S237C催化4-氯本甲酰基氯不对称还原
Figure PCTCN2018094507-appb-000010
表11 醇脱氢酶突变体E214I/S237A催化4-氯苯乙酮不对称还原
Figure PCTCN2018094507-appb-000011
表12 醇脱氢酶突变体E214Y/S237A催化4-硝基苯乙酮不对称还原
Figure PCTCN2018094507-appb-000012
由此可知，本发明所述醇脱氢酶突变体不仅对在CPMK的高效、高立体选择性不对称还原方面具有非常好的性能，并且对其他芳基酮类底物也具有较高的催化效率和高立体选择性。
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明，熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Figure PCTCN2018094507-appb-000013
Figure PCTCN2018094507-appb-000014
Figure PCTCN2018094507-appb-000015
Figure PCTCN2018094507-appb-000016
Figure PCTCN2018094507-appb-000017
Figure PCTCN2018094507-appb-000018
Figure PCTCN2018094507-appb-000019
Figure PCTCN2018094507-appb-000020
Figure PCTCN2018094507-appb-000021
Figure PCTCN2018094507-appb-000022
Figure PCTCN2018094507-appb-000023
Figure PCTCN2018094507-appb-000024

Claims (10)

  1. 一种醇脱氢酶的突变体，其特征在于，所述突变体的氨基酸序列包括：在SEQ ID NO.2所示氨基酸序列上进行一个或多个氨基酸位点的突变而得到的氨基酸序列。
  2. 根据权利要求1所述突变体，其特征在于，所述突变体的氨基酸序列包括：在SEQ ID No.2所示的氨基酸序列上将第214位的氨基酸谷氨酸、第237位的氨基酸丝氨酸中的一个或两个位点进行突变而得到的氨基酸序列。
  3. 根据权利要求1或2所述突变体，其特征在于，所述突变体的氨基酸序列包括以下M1～M16中的任意一种替换而得到的氨基酸序列：
    M1是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为缬氨酸；
    M2是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为酪氨酸；
    M3是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为异亮氨酸；
    M4是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为甘氨酸；
    M5是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为谷氨酰胺；
    M6是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为丝氨酸；
    M7是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为天冬酰胺；
    M8是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为谷氨酰胺；
    M9是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为缬氨酸，同时将第237位的丝氨酸替换成丙氨酸；
    M10是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为酪氨酸，同时将第237位的丝氨酸替换成丙氨酸；
    M11是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为异亮氨酸，同时将第237位的丝氨酸替换成丙氨酸；
    M12是将SEQ ID No.2所氨基酸序列的第214位谷氨酸替换为甘氨酸，同时将第237位的丝氨酸替换成半胱氨酸；
    M13是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为谷氨酰胺，同时将第237位的丝氨酸替换成半胱氨酸；
    M14是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为丝氨酸，同时将第237位的丝氨酸替换成半胱氨酸；
    M15是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为天冬酰胺，同时将第237位的丝氨酸替换成半胱氨酸；
    M16是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为精氨酸，同时将第237位的丝氨酸替换成半胱氨酸。
  4. 编码权利要求1-3任一所述突变体的核苷酸序列。
  5. 表达权利要求1-3任一所述突变体的重组菌。
  6. 构建权利要求5所述重组菌的方法，其特征在于，具体步骤如下：将编码所述突变体的核苷酸序列克隆到重组载体中，将所得重组载体转化到宿主中，得到重组转化体，通过培养所得重组表达转化体，即可分离纯化获得所述突变体。
  7. 根据权利要求6所述方法，其特征在于，所述重组菌的宿主为大肠杆菌(Escherichia coli)，所述质粒为pET28a(+)。
  8. 根据权利要求6或7所述方法，其特征在于，重组菌的宿主为E.coli BL21(DE3)。
  9. 应用权利要求5所述重组菌生产醇脱氢酶突变体的方法，其特征在于，所述方法为：将重组菌接种至含有40-60μg/mL硫酸卡那霉素的LB培养基中，30～40℃，100～200rpm摇床培养，培养液的吸光度OD 600达到0.5～1.0时，加入0.05～1.0mM的异丙基-β-D-六代呋喃半乳糖苷(IPTG)进行诱导，诱导温度为16～30℃，诱导5～10h即可获得高效表达重组醇脱氢酶的突变体。
  10. 一种应用醇脱氢酶生产手性CPMA的方法，其特征在于，所述方法的具体步骤如下：构建反应体系，CPMK浓度为10-500mM，权利要求1-3任一所述脱氢酶突变体用量为1-10kU/L，NADP +用量为0.1～1.0mM，加入辅酶循环系统，辅酶循环系统中含有葡萄糖脱氢酶GDH和D-葡萄糖，其中葡萄糖脱氢酶GDH用量为1～10kU/L，D-葡萄糖用量为20～1000mM，磷酸盐缓冲液的浓度为0.1-0.2M；在30～35℃，pH 6～8的条件下反应1～24h，不对称还原反应结束后，可按照有机溶剂萃取方法从反应液中提取手性CPMA；所述辅酶循环系统还可以是亚磷酸盐/亚磷酸盐脱氢酶(FTDH)、甲酸/甲酸脱氢酶(FDH)、乳酸/乳酸脱氢酶(LDH)或甘油/甘油脱氢酶。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112522224A (zh) * 2020-12-28 2021-03-19 华东理工大学 一种活性和立体选择性提高的醇脱氢酶突变体及其重组载体、基因工程菌以及应用

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108359649A (zh) 2018-02-12 2018-08-03 江南大学 醇脱氢酶突变体及其在双芳基手性醇合成中的应用
CN108504641A (zh) 2018-02-12 2018-09-07 江南大学 醇脱氢酶突变体及其在双芳基手性醇合成中的应用
CN111100851B (zh) * 2018-10-25 2022-07-26 中国科学院天津工业生物技术研究所 醇脱氢酶突变体及其在手性双芳基醇化合物合成中的应用
CN109402076A (zh) * 2018-10-30 2019-03-01 江南大学 一种醇脱氢酶突变体及其在辅酶再生中的应用
CN109776400B (zh) * 2019-03-11 2020-07-07 浙江工业大学 一种(r)-苯基(吡啶-2-基)甲醇衍生物的制备方法
CN109943543B (zh) * 2019-03-28 2020-12-11 上海健康医学院 醇脱氢酶突变体及其制备方法和应用
CN112680425B (zh) * 2021-01-13 2022-06-10 江南大学 一种醇脱氢酶突变体及其应用
CN114277007A (zh) * 2021-12-31 2022-04-05 江南大学 一种醇脱氢酶突变体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103642765A (zh) * 2013-12-25 2014-03-19 南京工业大学 醇脱氢酶突变体及其应用
CN104531628A (zh) * 2014-12-23 2015-04-22 凯莱英医药集团（天津）股份有限公司 醇脱氢酶突变体及其应用
CN105936909A (zh) * 2016-05-31 2016-09-14 江南大学 一种醇脱氢酶及其基因、重组酶和合成手性双芳基仲醇的应用
CN105936895A (zh) * 2016-05-31 2016-09-14 江南大学 一种醇脱氢酶突变体及其基因和在制备手性双芳基醇中的应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107099516B (zh) * 2017-06-05 2020-04-07 华东理工大学 7β-羟基甾醇脱氢酶突变体及其在熊脱氧胆酸合成中的应用
CN108359649A (zh) * 2018-02-12 2018-08-03 江南大学 醇脱氢酶突变体及其在双芳基手性醇合成中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103642765A (zh) * 2013-12-25 2014-03-19 南京工业大学 醇脱氢酶突变体及其应用
CN104531628A (zh) * 2014-12-23 2015-04-22 凯莱英医药集团（天津）股份有限公司 醇脱氢酶突变体及其应用
CN105936909A (zh) * 2016-05-31 2016-09-14 江南大学 一种醇脱氢酶及其基因、重组酶和合成手性双芳基仲醇的应用
CN105936895A (zh) * 2016-05-31 2016-09-14 江南大学 一种醇脱氢酶突变体及其基因和在制备手性双芳基醇中的应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112522224A (zh) * 2020-12-28 2021-03-19 华东理工大学 一种活性和立体选择性提高的醇脱氢酶突变体及其重组载体、基因工程菌以及应用

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