WO2019153633A1 - 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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WO2019153633A1
WO2019153633A1 PCT/CN2018/094506 CN2018094506W WO2019153633A1 WO 2019153633 A1 WO2019153633 A1 WO 2019153633A1 CN 2018094506 W CN2018094506 W CN 2018094506W WO 2019153633 A1 WO2019153633 A1 WO 2019153633A1
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amino acid
mutant
acid sequence
dehydrogenase
serine
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倪晔
周婕妤
许国超
王岳
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江南大学
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Priority to US16/521,636 priority patent/US10865390B2/en
Publication of WO2019153633A1 publication Critical patent/WO2019153633A1/zh

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • 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)

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  • 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 amino acid sequence of the mutant comprises: amino acid glutamine at position 136 and amino acid phenylalanine at position 161 in the amino acid sequence shown in SEQ ID No.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID NO. 2 with glycine, and is named M1.
  • the mutant comprises replacing the glutamic acid at position 214 of the alcohol dehydrogenase represented by SEQ ID NO. 2 with a proline, designated M2.
  • 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 with a half. Cystine, named M3.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID No. 2 with glycine, and replacing the serine at position 237 with cysteine.
  • the 136th glutamine was replaced by asparagine, named M4;
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID No. 2 with glycine, and replacing the serine at position 237 with cysteine.
  • the 136th glutamine was replaced by asparagine, and the 196th serine was replaced by glycine, which was named M5.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID No. 2 with glycine, and replacing the serine at position 237 with cysteine.
  • the 136th glutamine was replaced by asparagine
  • the 196th serine was replaced by glycine
  • the 161th phenylalanine was replaced by proline, which was named M6.
  • the mutant comprises replacing the glutamic acid at position 214 of the amino acid sequence of SEQ ID No. 2 with a proline, and replacing the threonine at position 215 with a serine. , named M7;
  • the mutant is substituted with an asparagine at position 136 of the alcohol dehydrogenase having the amino acid sequence of SEQ ID NO. 1, and the 161th phenylpropanoid
  • the acid was replaced by proline, which was named M8.
  • the mutant comprises replacing the serine at position 196 of the alcohol dehydrogenase represented by SEQ ID NO. 1 with glycine, and replacing the serine at position 237 with cysteine.
  • Acid named M9.
  • 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)-(pyridin-2-yl)-methanone, phenyl-(pyridin-2-yl)-methanone , 4-chlorophenyl-phenyl ketone, 4-fluorophenyl-phenyl ketone, 4-bromophenyl-phenyl ketone, 4-methoxyphenyl-phenyl ketone, acetophenone, 4-chloroacetophenone, 4-chlorobenzoyl chloride.
  • 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 analysis method of the (R)- and (S)-CPMA is: taking 100 ⁇ L of the reaction solution, adding 500 ⁇ L of ethyl acetate, shaking for 1 to 2 minutes, and centrifuging at 12,000 rpm for 2-5 minutes. The supernatant was taken to a centrifuge tube, and the organic phase was naturally completely formed. 500 ⁇ L of chromatographically pure ethanol was added, and the conversion and ee values were analyzed by chiral liquid chromatography.
  • the chromatographic conditions are as follows: Daicel Chiralcel OB-H (5 ⁇ m, 250 mm ⁇ 4.6 mm) liquid chromatography column, mobile phase is n-hexane: ethanol: ethanolamine (90:10:0.01, v/v/v), flow rate 1 mL /min, column temperature 30 ° C, UV detection wavelength 254 nm, injection volume 10 ⁇ L, (R)- and (S)-CPMA retention time were 11.14 min and 12.34 min, respectively.
  • 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 alcohol dehydrogenase mutant M6 of the present invention has an inverted S-stereoselectivity to the substrate CPMK, and the ee value of the product CPMA is 82 of the wild type. %(R) is reversed to 97.8%(S), and M7 has higher R-stereoselectivity for substrate CPMK, and the ee value of product CPMA is increased from 82% (R) to 99% (R) of wild type. the above.
  • the alcohol dehydrogenase mutant obtained by the invention is particularly suitable for asymmetric reduction of bisaryl ketone, and has good industrial application prospects.
  • FIG. 1 Whole plasmid PCR nucleic acid electrophoresis pattern of wild type and alcohol dehydrogenase mutants M1 to M7;
  • Alcohol dehydrogenase mutant M7 catalyzes a chiral liquid chromatogram of CPMK reduction products.
  • 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, 100 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 amino acid residues at positions 136, 161, 196, 214, 215 and 237 were subjected to site-directed mutagenesis using a full-plasmid PCR method to construct an iterative combination mutant.
  • the primers are designed as follows (both in the 5'-3' direction, underlined for the mutation site:
  • E214G-R AATTTCACAAGT ACC ATTTAG
  • E214V-R AATTTCACAAGT AAC ATTTAG
  • F161V-R ACAATAAGCAGT AAC GACATT
  • E214V/T215S-F AAGAAACTAAAT GTTAGC TGTGAA
  • E214V/T215S-R GATAATTTCACA GCTAAC ATTTAG
  • F161V-R ACAATAAGCAGT AAC GACATT
  • the PCR reaction system was: 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 ⁇ 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-galfuran-galactoside
  • 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 ee values of the products were 51.8%, 88.0%, 93.5% and 97.8%, respectively.
  • the reduction products of the control examples M8 and M9 were in the R configuration, and the ee value of the product was not much different from that of the wild type KpADH, and the mutation had no effect on the stereoselectivity of the enzyme.
  • the alcohol dehydrogenase mutant obtained in Example 2 was investigated for the reduction of latent chiral carbonyl compounds.
  • (4-fluorophenyl)-(phenyl)-methanone has the same stereoselectivity as WT and the ee value of the product exceeds 99%.
  • the combination mutants can obtain high R- and S-stereoselectivity for aryl ketones, especially large sterically hindered bisaryl ketone substrates, which can be used to prepare R- and S-configurations.
  • Example 6 Alcohol dehydrogenase mutant asymmetric reduction CPMK preparation (S)-CPMA and (R)-CPMA
  • a 20 mL biocatalytic system was established: 100 mM potassium phosphate buffer (pH 7.0), the alcohol dehydrogenase mutant M6 obtained in Example 2, M7 and wild KpADH 10 g/L, CPMK 100 mM, 200 mM and 500 mM (substrate batch) Add to).
  • the reaction was carried out at 30 ° C and 200 rpm for 12 h with a constant pH of 7.5.
  • the ee value is 99.7%.
  • the obtained crude products of (R)-CPMA and (S)-CPMA were redissolved in ethanol, and the corresponding products (R)-CPMA and (S)-CPMA pure were added as seed crystals to recrystallize at 4 ° C to obtain optical purity. Products with >99.9% ee.
  • alcohol dehydrogenase mutant enzymes M6 and M7 of the present invention have very good performance in the efficient, highly stereoselective asymmetric reduction of CPMK.

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Abstract

提供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。该醇脱氢酶突变体具有优良的催化活性和立体选择性，可高效催化制备一系列R-和S-构型的手性双芳基醇。将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。与现有报道相比，该醇脱氢酶不对称催化还原制备双芳基手性醇的方法具有操作简便、底物浓度高、反应完全、产品纯度高的优势，具有很强的工业应用前景。

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136位的氨基酸谷氨酰胺、第161位的氨基酸苯丙氨酸、第196位的氨基酸 丝氨酸、第214位的氨基酸谷氨酸、第215位的氨基酸苏氨酸或第237位的氨基酸丝氨酸中的一个或多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所示的醇脱氢酶的第214位谷氨酸替换为甘氨酸，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所示的醇脱氢酶的第214位谷氨酸替换为缬氨酸，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所示的醇脱氢酶的第214位谷氨酸替换为甘氨酸，同时第237位丝氨酸替换为半胱氨酸，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位谷氨酸替换为甘氨酸，同时第237位丝氨酸替换为半胱氨酸，第136位谷氨酰胺替换为天冬酰胺，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位谷氨酸替换为甘氨酸，同时第237位丝氨酸替换为半胱氨酸，第136位谷氨酰胺替换为天冬酰胺，第196位丝氨酸替换为甘氨酸，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位谷氨酸替换为甘氨酸，同时第237位丝氨酸替换为半胱氨酸，第136位谷氨酰胺替换为天冬酰胺，第196位丝氨酸替换为甘氨酸，第161位苯丙氨酸替换为缬氨酸，命名为M6.
在本发明的一种实施方式中，所述突变体包括将序列为SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为缬氨酸，同时第215位苏氨酸替换为丝氨酸，命名为M7；
在本发明的一种实施方式中，所述突变体是将氨基酸序列为SEQ ID NO.1所示的醇脱氢酶的第136位谷氨酰胺替换为天冬酰胺，同时第161位苯丙氨酸替换为缬氨酸，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为SEQ ID NO.1所示的醇脱氢酶的第196位丝氨酸替换为甘氨酸，同时第237位丝氨酸替换为半胱氨酸，命名为M9。
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)-(吡啶-2-基)-甲酮、苯基-(吡啶-2-基)-甲酮、4-氯苯基-苯基甲酮、4-氟苯基-苯基甲酮、4-溴苯基-苯基甲酮、4-甲氧苯基-苯基甲酮、苯乙酮、4-氯苯乙酮、4-氯苯甲酰基氯。
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(R)-和(S)-CPMA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，进行手性液相色谱分析转化率和ee值。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l mL/min，柱温30℃，紫外检测波长254nm，进样量10μL，(R)-和(S)-CPMA保留时间分别为11.14min和12.34min。
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)与野生型醇脱氢酶KpADH相比，本发明所述醇脱氢酶突变体M6对底物CPMK具有反转的S-立体选择性，其产物CPMA的ee值由野生型的82％(R)反转至97.8％(S)，M7对底物CPMK具有更高的R-立体选择性，其产物CPMA的ee值由野生型的82％(R)提高至99％(R)以上。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野生型和醇脱氢酶突变体M1～M7的全质粒PCR核酸电泳图；
图2醇脱氢酶突变体M1～M7梯度洗脱蛋白电泳图；
图3醇脱氢酶突变体M6催化CPMK还原产物手性液相色谱图；
图4醇脱氢酶突变体M7催化CPMK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 PCTCN2018094506-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136位、161位、196位、214位、215位、237位的氨基 酸残基进行定点突变，构建迭代组合突变体。其引物设计如下(均按5’-3’方向描述，下划线代表突变位点：
M1(以pET28a-KpADH重组质粒为模板)
E214G-F:AAGAAACTAAATGGTACTTGT
E214G-R:AATTTCACAAGT ACCATTTAG
M2(以pET28a-KpADH重组质粒为模板)
E214V-F:AAGAAACTAAAT GTTACTTGT
E214V-R:AATTTCACAAGT AACATTTAG
M3(以M1重组质粒为模板)
S237C-F:AAGACTCACTTC TGTCAATTC
S237C-R:ATCAATGAATTG ACAGAAGTG
M4(以M3重组质粒为模板)
Q136N-F:ACCCCACATAGA AATAATGAT
Q136N-R:AGTTGGATCATT ATTTCTATG
M5(以M4重组质粒为模板)
S196G-F:ACTATCCACCCA GGTTTCGTT
S196G-R:TCCGAAAACGAA ACCTGGGTG
M6(以M5重组质粒为模板)
F161V-F:TATGAAAATGTC GTTACTGCT
F161V-R:ACAATAAGCAGT AACGACATT
M7(以pET28a-KpADH重组质粒为模板)
E214V/T215S-F:AAGAAACTAAAT GTTAGCTGTGAA
E214V/T215S-R:GATAATTTCACA GCTAACATTTAG
M8(以pET28a-KpADH Q136N重组质粒为模板)
F161V-F:TATGAAAATGTC GTTACTGCT
F161V-R:ACAATAAGCAGT AACGACATT
M9(以pET28a-KpADH S196G重组质粒为模板)
S237C-F:AAGACTCACTTC TGTCAATTC
S237C-R:ATCAATGAATTG ACAGAAGTG
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.0 μL，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-六代呋喃半乳糖苷(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。
由表2可知KpADH对CPMK的k cat/K m为28.9s –1·mM –1，其还原产物构型为R构型，ee值为82.5％。突变体M2和M7合成(R)-CPMA立体选择性提高，产物ee值分别为92.3％和99.1％。突变体M1表现为降低的立体选择性，还原产物构型同为R构型，产物的ee值分别为3.29％。突变体M3，M4，M5和M6表现为反转的立体选择性，还原产物为S构型，产物的ee值分别为51.8％，88.0％，93.5％和97.8％。对照例M8和M9还原产物为R构型，产物的ee值与野生型KpADH相差不大，突变后对酶的立体选择性没有影响。
表2 醇脱氢酶突变体的动力学参数及立体选择性
Figure PCTCN2018094506-appb-000002
实施例:5：醇脱氢酶突变体的底物特异性分析
考察了实例2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-吡啶-2-基-甲酮((4-chlorophenyl)-(pyridin-2-yl)-methanone,CPMK)，苯基-吡啶-2-基-甲酮(phenyl-(pyridin-2-yl)-methanone)，4-氯苯基-苯基甲酮((4-chlorophenyl)-(phenyl)-methanone)，4-氟苯基-吡啶-2-基-甲酮(4-fluorophenyl)-(phenyl)-methanone，4-溴苯基-吡啶-2-基-甲酮(4-brormophenyl)-(phenyl)-methanone，4-甲氧苯基-苯基甲酮(4-methoxyphenyl)-(phenyl)-methanone，苯乙酮(acetophenone)，4-氯苯乙酮(4'-Chloroacetophenone)，4-氯苯甲酰基氯(2-phenylacetyl chloride)。
表3 醇脱氢酶突变体的底物特异性
Figure PCTCN2018094506-appb-000003
由表3可知，经过迭代组合突变得到的组合突变株M6与WT相比，对4-氯苯基-吡啶-2-基-甲酮((4-chlorophenyl)-(pyridin-2-yl)-methanone,CPMK)，4-溴苯基-吡啶-2-基-甲酮
(4-brormophenyl)-(phenyl)-methanone和4-甲氧基-苯基甲酮
((4-chlorophenyl)-(phenyl)-methanone)具有翻转的立体选择性且产物的ee值均在95％以上；对4-氯苯甲酰基氯(2-phenylacetyl chloride)突变株M6具有与野生型相同的立体选择性且产物的ee值均在99％以上；突变株M7对4-氯苯基-吡啶-2-基-甲酮
((4-chlorophenyl)-(pyridin-2-yl)-methanone,CPMK)，和4-溴苯基-吡啶-2-基-甲酮
(4-fluorophenyl)-(phenyl)-methanone具有与WT相比相同的立体选择性且产物的ee值超过99％。实验证明通过迭代组合突变获得组合突变菌株对芳基酮，特别是大位阻的双芳基酮底物具有较高的R-和S-立体选择性，可作为制备R-和S-构型手性芳基醇中间体的生物催化剂。
实施例6：醇脱氢酶突变体不对称还原CPMK制备(S)-CPMA和(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M6，M7和野生KpADH 10g/L，CPMK 100mM，200mM和500mM(底物分批添加)。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4，表5和表6，可知野生型脱氢酶和突变体M6，M7均能不对称还原100mM和200mM CPMK。当CPMK浓度为200mM时，野生型KpADH和两种突变体(M6和M7)分别需要12h和24h以达到接近99.9％的转化率。野生型KpADH最终还原产物为(R)-CPMA，ee值为82％；突变体M6最终还原产物为(S)-CPMA，ee值为99.5％；突变体M7最终还原产物为(R)-CPMA，ee值为99.7％。将所得(R)-CPMA和(S)-CPMA粗品重新溶解于乙醇中，加入相应的产物(R)-CPMA和(S)-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。
表4 野生型醇脱氢酶KpADH催化CPMK的不对称还原
Figure PCTCN2018094506-appb-000004
表5 醇脱氢酶突变体M6催化CPMK不对称还原
Figure PCTCN2018094506-appb-000005
表6 醇脱氢酶突变体M7催化CPMK不对称还原
Figure PCTCN2018094506-appb-000006
由此可知，本发明所述醇脱氢酶突变体酶M6和M7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。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2018094506-appb-000007
Figure PCTCN2018094506-appb-000008
Figure PCTCN2018094506-appb-000009
Figure PCTCN2018094506-appb-000010
Figure PCTCN2018094506-appb-000011
Figure PCTCN2018094506-appb-000012
Figure PCTCN2018094506-appb-000013
Figure PCTCN2018094506-appb-000014

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136位的氨基酸谷氨酰胺、第161位的氨基酸苯丙氨酸、第196位的氨基酸丝氨酸、第214位的氨基酸谷氨酸、第215位的氨基酸苏氨酸、第237位的氨基酸丝氨酸中的一个或多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～M7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位谷氨酸替换为甘氨酸；
    M2是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为缬氨酸；
    M3是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为甘氨酸，同时第237位丝氨酸替换为半胱氨酸；
    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，同时第237位丝氨酸替换为半胱氨酸，第136位谷氨酰胺替换为天冬酰胺；
    M5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，同时第237位丝氨酸替换为半胱氨酸，第136位谷氨酰胺替换为天冬酰胺，第196位丝氨酸替换为甘氨酸；
    M6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，同时第237位丝氨酸替换为半胱氨酸，第136位谷氨酰胺替换为天冬酰胺，第196位丝氨酸替换为甘氨酸，第161位苯丙氨酸替换为缬氨酸；
    M7是将SEQ ID No.2所示氨基酸序列的第214位谷氨酸替换为缬氨酸，同时第215位苏氨酸替换为丝氨酸。
  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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