WO2020224232A1 - 一株不动杆菌及其在生产手性3-环己烯-1-甲酸中的应用 - Google Patents

一株不动杆菌及其在生产手性3-环己烯-1-甲酸中的应用 Download PDF

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WO2020224232A1
WO2020224232A1 PCT/CN2019/120238 CN2019120238W WO2020224232A1 WO 2020224232 A1 WO2020224232 A1 WO 2020224232A1 CN 2019120238 W CN2019120238 W CN 2019120238W WO 2020224232 A1 WO2020224232 A1 WO 2020224232A1
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cyclohexene
acinetobacter
carboxylic acid
application according
chiral
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倪晔
窦哲
许国超
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/205—Bacterial isolates
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    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C12Y114/19001—Stearoyl-CoA 9-desaturase (1.14.19.1), i.e. DELTA9-desaturase

Definitions

  • the invention relates to an Acinetobacter strain and its application in the production of chiral 3-cyclohexene-1-carboxylic acid, and belongs to the technical field of microorganisms.
  • 3-cyclohexene-1-carboxylic acid is an important chemical reagent and organic intermediate, which is widely used in many fields such as medicine and chemical industry.
  • (S)-3-cyclohexene-1-carboxylic acid is an important starting material for 3,4-diaminocyclohexanecarboxylic acid derivatives, an intermediate of edoxaban, an inhibitor of blood coagulation factor Xa.
  • (R)-3-cyclohexene-1-carboxylic acid can also be used to synthesize a variety of drug intermediates, such as the intermediates of anti-tumor drugs (+)-Phyllanthocin, and C24-C34 of FK 506 (Puleke Fu) Fragment, the starting material of oseltamivir phosphate (Tamiflu).
  • drug intermediates such as the intermediates of anti-tumor drugs (+)-Phyllanthocin, and C24-C34 of FK 506 (Puleke Fu) Fragment, the starting material of oseltamivir phosphate (Tamiflu).
  • Cihangir Tanyeli et al. (Tetrahedron Asymmetry, 2004, 15, 2057-2060) describe a commercial pig liver Esterase (PLE), horse liver esterase (HLE) and porcine pancreatic lipase (PPL) hydrolyze (R,S)-3-cyclohexene-1-carboxylic acid methyl ester to prepare chiral 3-cyclohexene-1-
  • PLE catalyzed reaction to obtain (S)-configuration carboxylic acid conversion rate is 49%, ee>99%
  • HLE catalyzed substrate hydrolysis also generates (S)-configuration carboxylic acid, the conversion rate reaches 48%, The ee value is 97%.
  • Sheng Wu et al. (Applied and Environmental Microbiology, 2016, AEM.01817-16) used Mhg esterase to hydrolyze methyl 3-cyclohexene-1-carboxylate to obtain chiral 3-cyclohexene-1-carboxylic acid.
  • the conversion rate of the product was 53%, and the ee value was 25%.
  • Wang Zhao et al. described a commercial protease resolution method to prepare chiral 3-cyclohexene-1-carboxylic acid in the patent, with a yield of 32.8%, e.e. The value is 99.5%.
  • the present invention provides a strain of Acinetobacter that can produce an esterase that enantioselectively catalyzes the hydrolysis of high-concentration substrates, and provides the use of the strain or the crude enzyme catalyzed (R, S) Method for enantioselective hydrolysis of methyl-3-cyclohexene-1-carboxylate to produce high-concentration product (S)-3-cyclohexene-1-carboxylic acid.
  • the first objective of the present invention is to provide a strain of Acinetobacter, whose taxonomic name is Acinetobacter sp., which has been deposited at the China Common Microbial Culture Collection and Management Center on January 21, 2019, and the deposit address is Beijing No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC No. 17220.
  • the second object of the present invention is to provide the application of said Acinetobacter in the production of (S)-3-cyclohexene-1-carboxylic acid.
  • esterase produced by the fermentation of Acinetobacter is used as a catalyst to catalyze the production of (S)-3-cyclohexene-1-carboxylic acid methyl ester .
  • the catalysis is specifically to catalyze the enantioselective hydrolysis of methyl (R,S)-3-cyclohexene-1-carboxylate in a buffer solution containing a cosolvent, and then from the mixture generated by the hydrolysis reaction Collect (S)-3-cyclohexene-1-carboxylic acid methyl ester and heat and hydrolyze under alkaline conditions to obtain (S)-3-cyclohexene-1-carboxylic acid.
  • the co-solvent is an organic solvent that is miscible with water.
  • the added amount of the co-solvent is 5-35% of the total volume of the reaction liquid.
  • the buffer solution is citrate buffer, phosphate buffer or glycine-NaOH buffer, and the pH of the buffer solution is 5.0-9.5.
  • the alkaline condition is 0.5-1.5M sodium hydroxide solution.
  • the third object of the present invention is to provide an esterase, which is produced by fermentation of said Acinetobacter.
  • the fourth object of the present invention is to provide a microbial inoculum comprising the Acinetobacter.
  • the use of the Acinetobacter of the present invention to produce chiral methyl 3-cyclohexene-1-carboxylate has significant advantages.
  • the resulting product (S)-3-cyclohexene-1-methylcarboxylate has an optical purity of 99% Above, and the catalyst has good stability, can tolerate higher concentrations of substrates and products, and the reaction conditions are mild.
  • the resolution process of the present invention can simply and efficiently obtain (S)-3-cyclohexene-1-carboxylic acid with high optical purity and high concentration, and the method is energy-saving and environmentally friendly, and the high-concentration product is beneficial to product recovery , Is an efficient production method of (S)-3-cyclohexene-1-carboxylic acid, and has a good industrial application prospect.
  • Acinetobacter sp. has been deposited at the China Common Microbial Culture Collection and Management Center on January 21, 2019.
  • the deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 17220.
  • the culture media used in the present invention are:
  • Enrichment medium methyl cyclohexenecarboxylate 1.5, (NH 4 ) 2 SO 4 2, KH 2 PO 4 , MgSO 4 0.5, NaCl 1, DMSO 5% (v/v), pH 7.0, 121°C high temperature sterilization for 20min. Take a small amount of soil sample and suspend it in a test tube containing enrichment medium, culture for 48h at 30°C, 180rpm, draw an appropriate amount of culture solution, add it to the test tube containing the second round of enrichment medium, and continue to culture for 48h under the same conditions .
  • Plate separation medium g/L: tributyrin 10, Tween-80 10, peptone 16, yeast extract 10, NaCl 5, agar 15, KH 2 PO 4 0.5, MgSO 4 0.2, pH 7.0, 121°C High temperature sterilization for 20 minutes.
  • the culture solution is diluted and spread on a plate medium, and a single colony with a transparent circle is the target colony.
  • Fermentation medium g/L: glycerol 15, peptone 5, yeast extract 5, NaCl 1, KH 2 PO 4 0.5, pH 7.0, and autoclave at 121°C for 20 min. After sterilization, cool and inoculate. The inoculation amount is 5% (v/v). Fermentation is carried out at 30°C and 180rpm speed. After 12 hours of cultivation, the wet weight of the bacteria can reach 20g/L.
  • the specific activity of the enzyme (U) is defined It is: the amount of cells required to catalyze 1 ⁇ mol (R,S)-3-cyclohexene-1-methyl formate per minute, the enzyme production of JNU9335 can reach 180U/L, and the specific activity is 9U/g wet cells.
  • the reaction conditions for enantioselectively catalyzed hydrolysis the cell concentration is 20-200g/L, the substrate concentration is 50-500mM, the reaction temperature is 10-60°C, the pH is 5-9.5, and the reaction time is 0.5-36h.
  • the enantiomeric excess (ee) and conversion rate of the substrate were analyzed by gas chromatography.
  • the analysis conditions were as follows: B-DM chiral column (30m ⁇ 0.25mm ⁇ 0.25 ⁇ m); nitrogen was used as carrier gas; injection port temperature was 280 °C; air flow 300mL/min, makeup gas flow 25mL/min; split ratio 50:1, sample injection volume 1.0 ⁇ L.
  • Oven heating program keep the initial temperature at 100°C for 2min, and raise the temperature to 150°C at 2°C/min and keep it for 2min.
  • FID detector temperature 280°C.
  • Table 1 shows that the 9 strains screened from the soil all have high enantioselective catalytic activity, among which JNU9335 has the fastest reaction speed and the highest conversion rate.
  • Table 2 shows that as the substrate concentration increases, the activity of most strains is inhibited by the higher substrate concentration, and the increase in hydrolysate is very small. Only strain JNU9335 can maintain a relatively high substrate concentration (500mM). The high conversion rate and higher concentration of the product indicate that this strain has excellent substrate and product tolerance, so this strain was selected as the best strain for future work.
  • the strain JNU9335 has the following microbiological characteristics:
  • the suitable growth temperature is 20 ⁇ 35°C, and it can survive in a pH 5 ⁇ 9 environment.
  • Cultivated on a 30°C plate for 24 hours can form small colonies, and 36 hours form viscous, moist white colonies with smooth edges and prominent middle. Over time, a transparent circle formed on the tributyrin plate.
  • Fermentation medium g/L: glycerol 15, peptone 5, yeast extract 5, NaCl 1, KH 2 PO 4 0.5, pH 7.0. High temperature sterilization at 121°C for 20 min. After sterilization, cool and inoculate, the inoculation volume is 5% (v/v), ferment at 30°C, 180rpm speed, culture for 12h, collect the bacteria at 8000rpm, 10min, discard the upper medium, and use normal saline Wash once, dry in a vacuum freeze dryer (SCIENTZ-10N) for 24 hours, collect the enzyme powder and store at -20°C.
  • SCIENTZ-10N normal saline Wash once, dry in a vacuum freeze dryer
  • the specific activity (U) of the enzyme is defined as the amount of cells required to catalyze 1 ⁇ mol (R,S)-3-cyclohexene-1-carboxylic acid methyl ester per minute, and the specific activity of the enzyme powder is determined to be 96 U/g.
  • Example 5 The effect of temperature on the enzymatic hydrolysis of methyl cyclohexenate
  • Example 6 Enzymatic hydrolysis of (R,S)-3-cyclohexene-1-carboxylic acid methyl ester by Acinetobacter esterase
  • Table 4 shows that when the esterase catalyzes different concentrations of methyl (R,S)-3-cyclohexene-1-carboxylate, the product concentration increases with the increase of the substrate concentration, up to 215mM, and still retains
  • the higher optical purity of the product indicates that higher concentrations of substrates and products have no significant impact on the activity of the Acinetobacter esterase, and the esterase can tolerate high concentrations of substrates and products. This is the example with the highest concentration of products among the biocatalytic methods for the production of chiral cyclohexenic acid reported so far, and it is also the only report on microbial enzymatic conversion.
  • the method of the present invention has very broad practical industrial application prospects.
  • reaction was stopped after 12h, after filtering to remove the enzyme, adjusting the pH to 9, adding ethyl acetate for extraction 3 times, combining the organic phases and rotary evaporation until no liquid flowed out to obtain methyl (S)-3-cyclohexene-1-carboxylate , Then add (S)-3-cyclohexene-1-carboxylic acid methyl ester to 1M NaOH aqueous solution at 50°C and stir for 6h at reflux, then add 1M HCl aqueous solution to adjust the pH to 5, add equal volume of ethyl acetate and extract 3 times.

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Abstract

提供一株不动杆菌及其在生产手性3-环己烯-1-甲酸中的应用。该不动杆菌的分类学命名为不动杆菌Acinetobacter sp.，于2019年01月21日保藏于中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号CGMCC No.17220。采用该不动杆菌生产手性3-环己烯-1-甲酸甲酯，所得产物(S)-3-环己烯-1-甲酸甲酯的光学纯度在99％以上，而且催化剂的稳定性好，能耐受较高浓度的底物和产物，反应条件温和。使用该拆分工艺能简单、高效地获得高光学纯度和高浓度的(S)-3-环己烯-1-甲酸，且该法节能、环保，高浓度的产物有利于产物的回收，是一种高效的(S)-3-环己烯-1-甲酸的生产方法，具有很好的工业应用前景。

Description

一株不动杆菌及其在生产手性3-环己烯-1-甲酸中的应用 技术领域
本发明涉及一株不动杆菌及其在生产手性3-环己烯-1-甲酸中的应用，属于微生物技术领域。
背景技术
3-环己烯-1-甲酸作为手性化合物是一种重要的化学试剂和有机中间体，广泛应用于医药、化工等多个领域。(S)-3-环己烯-1-甲酸是凝血因子Xa的抑制剂—依度沙班中间体3,4-二氨基环己烷羧酸衍生物的重要起始原料。而(R)-3-环己烯-1-甲酸也可以用于合成多种药物中间体，如抗肿瘤药物(+)-Phyllanthocin的中间体、FK 506(普乐可复)的C24-C34片段、磷酸奥司他韦(特敏福)的初始原料。
目前，仅有少数关于手性3-环己烯-1-甲酸拆分制备的文献报道，例如Cihangir Tanyeli等(Tetrahedron Asymmetry,2004,15,2057-2060)描述了一种利用商品化的猪肝酯酶(PLE)，马肝酯酶(HLE)和猪胰脂肪酶(PPL)水解(R,S)-3-环己烯-1-甲酸甲酯制备手性3-环己烯-1-甲酸，PLE催化反应得到(S)-构型的羧酸转化率为49％，e.e.>99％，HLE催化底物水解后也生成(S)-构型的羧酸，转化率达到48％，e.e.值为97％，与此相反，PPL产生(R)-构型羧酸的e.e.值为91％，转化率为49％。徐春秀等(现代药物与临床,2013,28(2):126-128)在2013年报道了通过化学拆分法以手性苯乙胺为手性拆分剂，(R,S)-3-环己烯-1-甲酸在丙酮中形成非对映异构体，并利用它们的溶解度差别来拆分为(R)-(+)-3-环己烯-1-甲酸(收率28.3％)和(S)-(-)-3-环己烯-1-甲酸(收率28.7％)，光学纯度均大于99％。Sheng Wu等人(Applied and Environmental Microbiology,2016,AEM.01817-16)利用Mhg酯酶对3-环己烯-1-甲酸甲酯进行水解得到手性3-环 己烯-1-甲酸，底物的转化率为53％，e.e.值为25％。王钊等人在专利中描述了一种商品化的蛋白酶拆分制备手性3-环己烯-1-甲酸的方法，收率为32.8％，e.e。值为99.5％。
然而这些研究存在一定的局限性，如：商品酶价格昂贵，且催化的水解反应底物浓度较低，利用微生物酶催化过程又存在立体选择性不高的问题，化学法的手性拆分试剂价格非常昂贵。因此，目前仍然缺乏可高立体选择性催化3-环己烯-1-甲酸甲酯制备手性3-环己烯-1-甲酸的微生物酶。
发明内容
为解决上述技术问题，本发明提供一株能够生产对映选择性地催化水解高浓度底物的产酯酶的不动杆菌，并提供利用该菌株或其产生的粗酶催化(R,S)-3-环己烯-1-甲酸甲酯对映选择性水解生产高浓度产物(S)-3-环己烯-1-甲酸的方法。
本发明的第一个目的是提供一株不动杆菌，其分类学命名为不动杆菌Acinetobacter sp.，已于2019年01月21日保藏于中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号CGMCC No.17220。
本发明的第二个目的是提供所述的不动杆菌在生产(S)-3-环己烯-1-甲酸的中应用。
进一步地，应用所述的不动杆菌发酵生产的酯酶为催化剂，催化(R,S)-3-环己烯-1-甲酸甲酯生产(S)-3-环己烯-1-甲酸。
进一步地，所述催化具体是在含有助溶剂的缓冲溶液中，催化(R,S)-3-环己烯-1-甲酸甲酯的对映选择性水解，然后从水解反应生成的混合物中收集(S)-3-环己烯-1-甲酸甲酯，在碱性条件下加热水解得到(S)-3-环己烯-1-甲酸。
进一步地，所述的助溶剂为与水互溶的有机溶剂。
进一步地，所述的助溶剂的添加量为反应液总体积的5～35％。
进一步地，所述的缓冲溶液为柠檬酸盐缓冲液、磷酸盐缓冲液或甘氨酸-NaOH缓冲液，所述缓冲溶液的pH为5.0～9.5。
进一步地，所述的碱性条件为0.5～1.5M氢氧化钠溶液。
本发明的第三个目的是提供一种酯酶，由所述的不动杆菌发酵生产得到。
本发明的第四个目的是提供一种微生物菌剂，包含所述的不动杆菌。
本发明的有益效果是：
采用本发明的不动杆菌生产手性3-环己烯-1-甲酸甲酯，具有显著的优点，所得产物(S)-3-环己烯-1-甲酸甲酯的光学纯度在99％以上，而且催化剂的稳定性好，能耐受较高浓度的底物和产物，反应条件温和。使用本发明的拆分工艺能简单、高效地获得高光学纯度和高浓度的(S)-3-环己烯-1-甲酸，且该法节能、环保，高浓度的产物有利于产物的回收，是一种高效的(S)-3-环己烯-1-甲酸的生产方法，具有很好的工业应用前景。
生物材料保藏
不动杆菌Acinetobacter sp.，已于2019年01月21日保藏于中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号CGMCC No.17220。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
本发明用到的培养基有：
富集培养基(g/L)：环己烯羧酸甲酯1.5，(NH 4) 2SO 4 2，KH 2PO 4，MgSO 40.5，NaCl 1，DMSO 5％(v/v)，pH 7.0，121℃高温灭菌20min。取少量土样悬浮于装有富集培养基的试管中，30℃，180rpm下培养48h，吸取适量培养液，加入到装有第二轮富集培养基的试管中，同样的条件继续培养48h。
平板分离培养基(g/L)：三丁酸甘油酯10，Tween-80 10，蛋白胨16，酵母膏10，NaCl 5，琼脂15，KH 2PO 4 0.5，MgSO 4 0.2，pH 7.0，121℃高温灭菌20min。将培养液进行梯度稀释涂布平板培养基，产透明圈的单菌落即为目的菌落。
发酵培养基(g/L)：甘油15，蛋白胨5，酵母膏5，NaCl 1，KH 2PO 4 0.5， pH 7.0，121℃高温灭菌20min。灭菌后冷却、接种，接种量5％(v/v)，在30℃，180rpm转速条件下进行发酵，培养12h后菌体湿重可达20g/L，该酶的比活力(U)定义为：每分钟催化1μmol(R,S)-3-环己烯-1-甲酸甲酯所需要的细胞量，测得JNU9335产酶可达180U/L，比活力为9U/g湿细胞。
对映选择性地催化水解的反应条件：细胞浓度为20～200g/L，底物浓度为50～500mM，反应温度为10～60℃，pH 5～9.5，反应时间为0.5～36h。
底物的对映体过量值(e.e.)和转化率采用气相色谱分析，分析条件如下：B-DM手性柱(30m×0.25mm×0.25μm)；以氮气作为载气；进样口温度280℃；空气流量300mL/min，尾吹气流量25mL/min；分流比50:1，进样量1.0μL。柱箱升温程序：初始温度100℃保持2min，2℃/min升温至150℃，保持2min。FID检测器温度：280℃。
实施例1：不动杆菌的筛选
从江苏、陕西、山东、河南、江西等地区的300多份土壤中进行筛选，筛选的具体步骤如下：
从不同的环境中采集土壤样品，利用环己烯甲酸甲酯为唯一碳源进行三轮富集培养，筛选酯酶产生菌。通过反复筛选，分离得到7株具有很高的对映选择性催化活性的菌株，进一步对7株候选菌的性能进行分析筛选：
7株候选菌株对(R,S)-3-环己烯-1-甲酸甲酯的对映选择性水解性能：
取1.0g各菌株的湿细胞悬浮于10mL的磷酸盐缓冲溶液(100mM，pH 7.0)中，(R,S)-3-环己烯-1-甲酸甲酯的浓度为50mM，助溶剂DMSO的添加量为5％，反应混合物在30℃，180rpm的恒温摇床上反应，在表1中所示的时间取样，产物和底物经乙酸乙酯萃取，无水Na 2SO 4干燥后，再通过手性气相色谱分析底物的转化率和对映体过量值(e.e. s)。各菌株的催化性能如表1所示。
表1 候选菌株的催化性能比较
Figure PCTCN2019120238-appb-000001
表1说明从土壤中筛选的这9株菌均具有很高的对映选择性催化活性，其中JNU9335反应速度最快，转化率最高。
7株候选菌株对不同浓度的(R,S)-3-环己烯-1-甲酸甲酯的耐受能力：
取1g各菌株的湿细胞悬浮于10mL的磷酸盐缓冲溶液(100mM，pH 7.0)中，分别加入不同浓度的(R,S)-3-环己烯-1-甲酸甲酯，反应混合物在30℃，180rpm的恒温摇床上反应24h。反应后取样，产物和底物经乙酸乙酯萃取，无水Na 2SO 4干燥后，再通过手性气相色谱分析底物的转化率。各菌株对不同浓度底物的耐受情况如表2所示。
表2 候选菌株对不同浓度的底物耐受情况的对比
Figure PCTCN2019120238-appb-000002
表2说明随着底物浓度的增加，多数菌株的活性受到较高底物浓度的抑制，水解产物增加很少，而只有菌株JNU9335在较高的底物浓度下(500mM)， 仍能维持较高的转化率，得到较高浓度的产物，说明该菌株具有优异的底物和产物耐受性，所以该菌株被筛选作为最优菌株用于以后的工作。
通过上述筛选得到一株产酶稳定的高活力和高选择性的酯酶产生菌JNU9335，该菌株已于2019年01月21日保藏于中国普通微生物菌种保藏管理中心，保藏编号CGMCC No.17220。
实施例2：不动杆菌Acinetobacter sp.JNU9335的形态及生理学鉴定
菌株JNU9335具有如下的微生物学特征：
1.形态大小
杆状，染色均匀，0.5～0.9×1.5～3μm，不产芽孢，无鞭毛，革兰氏染色阴性。
2.适宜生长环境
适宜的生长温度为20～35℃，可以在pH 5～9环境中生存。
3.平板培养菌落特性
在30℃平板上培养24h可形成小的菌落，36h形成粘稠、湿润的白色菌落，边缘光滑，中间突出。随着时间的延长，在三丁酸甘油酯平板上形成透明圈。
实施例3：不动杆菌Acinetobacter sp.JNU9335的分子生物学鉴定
提取菌株JNU9335的染色体DNA，使用引物(27F:5'-AGAGTTTGATCCTGGCTCAG-3'；1492R:5'-TACCTTGTTACGACTT-3')酶促扩增16S核糖体DNA(16S rDNA)，热循环仪PCR程序为：在95℃变性5min，在95℃40s，在55℃下1min在72℃下2min的30个循环，最后一步在72℃下10min。16S rDNA测序结果分析，鉴定为不动杆菌Acinetobacter sp.。
实施例4：不动杆菌Acinetobacter sp.JNU9335的发酵培养
发酵培养基(g/L)：甘油15，蛋白胨5，酵母膏5，NaCl 1，KH 2PO 4 0.5，pH 7.0。121℃高温灭菌20min。灭菌后冷却、接种，接种量5％(v/v)，在30℃，180rpm转速条件下进行发酵，培养12h，在8000rpm，10min条件下收集菌体，弃尽上层培养基，用生理盐水洗涤一次，在真空冷冻干燥机(SCIENTZ-10N)中干燥24h，收集酶粉于-20℃保存。该酶的比活力(U)定义为：每分钟催化1μmol (R,S)-3-环己烯-1-甲酸甲酯所需要的细胞量，测得酶粉的比活力为96U/g。
实施例5：温度对环己烯甲酸甲酯的酶促水解的影响
将70mg的(R,S)-3-环己烯-1-甲酸甲酯，0.5mL二甲基亚砜、25mg冻干的粗酶粉加入到10mL磷酸盐缓冲液(100mM，pH 7.0)中，混合均匀后分别置于20，30，40和50℃，180rpm的恒温摇床上反应振荡6h。反应后取样，产物和底物经乙酸乙酯萃取，无水Na 2SO 4干燥后，再通过手性气相色谱分析底物的转化率和对映体过量值(e.e. s)。如表3所示，温度在20～40℃时，该酯酶具有很高的转化率和稳定的e.e. s，说明该温度范围内，该酯酶是比较稳定的，活性没有受到很大的损失。温度超过50℃时，该酯酶的转化能力大大下降，这可能是高温导致该酯酶空间构型发生变化，从而导致酶的活性下降。
表3 温度对不动杆菌酯酶催化(R,S)-3-环己烯-1-甲酸甲酯的酶促水解的影响
Figure PCTCN2019120238-appb-000003
实施例6：不动杆菌酯酶对不同浓度(R,S)-3-环己烯-1-甲酸甲酯的酶促水解
将浓度为100，200和500mM的(R,S)-3-环己烯-1-甲酸甲酯和0.5mL二甲基亚砜加入到10mL磷酸盐缓冲液(100mM，pH 7.0)中，再分别加入相应量的酶，反应混合物置于30℃，180rpm的恒温摇床上振荡反应。反应后取样，产物和底物经乙酸乙酯萃取，无水Na 2SO 4干燥后，再通过手性气相色谱分析底物的转化率和对映体过量值(e.e. s)。结果如表4所示。
表4 不动杆菌酯酶对不同浓度的(R,S)-3-环己烯-1-甲酸甲酯的酶促水解
Figure PCTCN2019120238-appb-000004
表4显示，该酯酶催化不同浓度的(R,S)-3-环己烯-1-甲酸甲酯时，产物浓度随着底物浓度的增加而增加，最高可达215mM，且仍保留较高的产物光学纯度，说明较高浓度的底物和产物对该不动杆菌酯酶的活性没有造成显著的影响，该酯酶可以耐受高浓度的底物和产物。这是至今报道的生物催化法生产手性环己烯甲酸的方法中，生成产物浓度最高的例子，也是唯一一篇微生物酶法转化的报道。本发明的方法具有很广阔的实际工业应用前景。
实施例7：(S)-环己烯-1-甲酸的克级制备
将3.50g(R,S)-3-环己烯-1-甲酸甲酯与2.5mL二甲基亚砜和47.5mL磷酸盐缓冲液(200mM，pH 7.0)混合均匀后，加入1g酶粉。反应在恒温30℃的250mL圆底烧瓶中进行，400rpm下机械搅拌。通过手性气相色谱监测底物的转化率和产物的对映体过量值。12h后停止反应，过滤除酶后，调节pH至9，加入乙酸乙酯萃取3次，合并有机相后旋转蒸发至无液体流出，获得(S)-3-环己烯-1-甲酸甲酯，然后将(S)-3-环己烯-1-甲酸甲酯加入1M NaOH水溶液50℃加热回流搅拌反应6h，再加入1M HCl水溶液调节pH至5，加入等体积乙酸乙酯萃取3次，合并有机层，用无水Na 2SO 4干燥，过滤，旋转蒸发得到(S)-3-环己烯-1-甲酸，所得产物为具有特殊气味的液体，分离后总得率为40％，光学纯度为99％e.e.。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一株不动杆菌，其特征在于，其分类学命名为不动杆菌Acinetobacter sp.，已于2019年01月21日保藏于中国普通微生物菌种保藏管理中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏编号CGMCC No.17220。
  2. 权利要求1所述的不动杆菌在生产(S)-3-环己烯-1-甲酸的中应用。
  3. 根据权利要求2所述的应用，其特征在于，应用所述的不动杆菌发酵生产的酯酶为催化剂，催化(R,S)-3-环己烯-1-甲酸甲酯生产(S)-3-环己烯-1-甲酸。
  4. 根据权利要求3所述的应用，其特征在于，所述催化具体是在含有助溶剂的缓冲溶液中，催化(R,S)-3-环己烯-1-甲酸甲酯的对映选择性水解，然后从水解反应生成的混合物中收集(S)-3-环己烯-1-甲酸甲酯，在碱性条件下加热水解得到(S)-3-环己烯-1-甲酸。
  5. 根据权利要求4所述的应用，其特征在于，所述的助溶剂为与水互溶的有机溶剂。
  6. 根据权利要求4所述的应用，其特征在于，所述的助溶剂的添加量为反应液总体积的5～35％。
  7. 根据权利要求4所述的应用，其特征在于，所述的缓冲溶液为柠檬酸盐缓冲液、磷酸盐缓冲液或甘氨酸-NaOH缓冲液，所述缓冲溶液的pH为5.0～9.5的。
  8. 根据权利要求4所述的应用，其特征在于，所述的碱性条件为0.5～1.5M氢氧化钠溶液。
  9. 一种酯酶，其特征在于，由权利要求1所述的不动杆菌发酵生产得到。
  10. 一种微生物菌剂，其特征在于，包含权利要求1所述的不动杆菌。
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CN111778229B (zh) 2020-07-23 2022-02-22 江南大学 环己烯甲酸酯水解酶及其突变体、编码基因、表达载体、重组菌与应用
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CN115583900A (zh) * 2022-04-29 2023-01-10 张邦成都生物医药科技有限公司 一种高纯度依度沙班中间体的制备方法
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CN116396950B (zh) * 2023-04-27 2023-10-20 江南大学 羧酯酶突变体及其在(r)-3-环己烯-1-甲酸合成中的应用
CN119876086B (zh) * 2025-01-08 2026-07-14 浙江工业大学 一种脂肪酶突变体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1408848A (zh) * 2002-09-17 2003-04-09 华东理工大学 不动杆菌及采用该菌种拆分制备手性环戊烯酮的方法
US20040138429A1 (en) * 2002-07-30 2004-07-15 Council Of Scientific And Industrial Research Bioemulsifier production by Acinetobacter strains isolated from healthy human skin
US20090238811A1 (en) * 2002-09-09 2009-09-24 Mcdaniel C Steven Enzymatic Antimicrobial and Antifouling Coatings and Polymeric Materials
CN104845922A (zh) * 2015-06-15 2015-08-19 中国海洋大学 一种不动杆菌及其在磷脂酰丝氨酸合成中的应用
CN106119303A (zh) * 2016-07-01 2016-11-16 浙江工业大学 一种蛋白酶拆分制备手性3‑环己烯‑1‑甲酸的方法
CN110272839A (zh) * 2019-05-08 2019-09-24 江南大学 一株不动杆菌及其在生产手性3-环己烯-1-甲酸中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101139559A (zh) * 2006-09-05 2008-03-12 许雷 一株高效降解菊酯类及有机磷农药的新菌株及其用途
CA3035878C (en) * 2007-03-28 2023-01-03 REG Life Sciences, LLC Enhanced production of fatty acid derivatives
MX354883B (es) * 2012-04-13 2018-03-23 Reg Life Sciences Llc Produccion microbiana de alcanolamidas y amidoaminas y usos de las mismas.

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040138429A1 (en) * 2002-07-30 2004-07-15 Council Of Scientific And Industrial Research Bioemulsifier production by Acinetobacter strains isolated from healthy human skin
US20090238811A1 (en) * 2002-09-09 2009-09-24 Mcdaniel C Steven Enzymatic Antimicrobial and Antifouling Coatings and Polymeric Materials
CN1408848A (zh) * 2002-09-17 2003-04-09 华东理工大学 不动杆菌及采用该菌种拆分制备手性环戊烯酮的方法
CN104845922A (zh) * 2015-06-15 2015-08-19 中国海洋大学 一种不动杆菌及其在磷脂酰丝氨酸合成中的应用
CN106119303A (zh) * 2016-07-01 2016-11-16 浙江工业大学 一种蛋白酶拆分制备手性3‑环己烯‑1‑甲酸的方法
CN110272839A (zh) * 2019-05-08 2019-09-24 江南大学 一株不动杆菌及其在生产手性3-环己烯-1-甲酸中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAO-DI MA, XU-DONG KONG, HUI-LEI YU, ZHI-JUN ZHANG, SHUAI DOU, YAN-PENG XU, YAN NI, AND JIAN-HE XU: "Increased Catalyst Productivity in α‑Hydroxy Acids Resolution by Esterase Mutation and Substrate Modification", ACS CATALYSIS, vol. 4, no. 3, 17 February 2014 (2014-02-17), pages 1026 - 1031, XP055751877, ISSN: 2155-5435, DOI: 10.1021/cs401183e *
KIM, H.E. ET AL.: "Gene Cloning, Sequencing, and Expression of an Esterase from Acinetobacter lwoffii I6C-1", CURRENT MICROBIOLOGY, vol. 46, 31 December 2003 (2003-12-31), DOI: 20200218140556A *
ZHAO YUQIAO, WU HUIYUAN, XU JIANHE ,ZHOU LINTING: "Isolation and application of acinetobacter SP. YQ231 highly producing enantioselective esterases", CHINESE JOURNAL OF APPLIED AND ENVIRONMENTAL BIOLOGY, vol. 8, no. 6, 25 December 2002 (2002-12-25), pages 640 - 643, XP055751881 *

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