Preparation of S-(+)-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.     

Enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester in dispersed aqueous reaction system induced by chiral cyclodextrin

The enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester to the optically active (S)-ketoprofen was carried out in a dispersed aqueous lipase reaction system induced by the inclusion of chiral cyclodextrins for complexation of the substrate. Hydroxypropyl-beta-cyclodextrin was the most effective chiral selector and disperser giving an enantiomeric excess and conversion yield of 0.99 and 0.49, respectively.     

(S)-3-Pentyn-2-ol production through microbial enzyme-catalyzed, highly enantioselective hydrolysis of racemic 3-pentyn-2-ol esters

Air-dried cells of Hansenula nonfermentans AKU 4332 catalyzed the production of (S)-3-pentyn-2-ol from (RS)-3- pentyn-2-ol acetate ester at 10% (v/v). The product was formed at 96.6% e.e. with a molar yield of 45% in 24 h. © Rapid Science Ltd. 1998     

Isolation and characterization of Acinetobacter sp. ES-1 excreting a lipase with high enantioselectivity for (S)-ketoprofen ethyl ester

A new Acinetobacter sp. ES-1, grown on triolein, tryptone and Triton X-100, excreted a lipase that hydrolyzed 10m M (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10Uml^-1 and, at 30°C and pH7 over 48h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.     

(S)-酮基布洛芬拆分用脂肪酶基因的克隆及表达

本实验室筛选出一株具有不对称拆分消旋酮基布洛芬氯乙酯的菌株NK13为材料,经鉴定为巨大芽孢杆菌Bacillus megaterium。通过构建其基因文库,从中筛选得到阳性克隆重组子pUC-NK1。测序分析表明,该重组子质粒中包含一长度为633bp的脂肪酶基因的完整开放阅读框,核苷酸同源性对比证明该脂肪酶基因属首次发现(GenBank Accession No.EU381317),将此基因克隆到原核表达载体pET21b(+)中构建重组表达质粒pET-NKest1,转化Escherichia coli BL21,经Isopropyl-β-D-Thiogalactoside(IPTG)诱导在宿主菌中得到表达,经SDS-PAGE电泳检测证明该脂肪酶成熟蛋白分子量约为20kDa。薄层层析与HPLC检测结果显示,表达菌株转化外消旋酮基布洛芬氯乙酯得到(S)-酮基布洛芬过量(e.e.%),由野生菌NK13的5.84%提高到75.28%,提高约15倍,说明该脂肪酶具有优先拆分得到(S)-酮基布洛芬的特性。     

一种不对称水解（R,S）-2,6-二甲基苯基氨基丙酸甲酯的新酯酶基因的克隆表达和酶学性质研究

本文将来自反硝化无色杆菌Achromobacterdenitrificans1104的酯酶基因EHest,转化大肠杆菌中,成功表达了具有不对称水解农药甲霜灵的中间体(R,S)-2,6-二甲基苯基氨基丙酸甲酯( MAP )活性的酯酶EHesterase。用重组酯酶EHesterase催化MAP 的水解,底物浓度50 g/L,反应1h的转化率29.5%,产物( R-酸)的eep 是85.1%。该酶的最适反应pH和温度分别为9.0和50℃,在50℃以下和pH5~9之间具有较好的稳定性。该酶水解MAP 的米氏动力学参数Vm、Km 分别是0.733 g/(L·min)和7.49 g/L。加入10%DMSO对酶EHesterase的立体选择性和催化速度有一定的促进作用。 Cu2+、Fe3+对酶活有明显抑制作用。该酶水解MAP 的活性与水解p-对硝基苯乙酸酯的活性数量级相当,是水解橄榄油活性的333倍。     

拆分获得(R)-酮基布洛芬酯酶基因的筛选、克隆与表达

以自筛选出的具有一定不对称拆分外消旋酮基布洛芬氯乙酯能力的野生菌Bacillus megaterium NK13为材料,通过构建其基因文库,筛选得到一个阳性克隆重组子pUC18-NK-HYD3。分析测序结果发现外源片段中包含一段完整的741 bp的开放阅读框,其编码的蛋白中含有酯酶的GXSXG保守序列。经在NCBI的BLAST系统中比对,证明该酯酶基因属于首次发现(GenBank Accession Number: GU143552)。将酯酶基因克隆到载体pET21b(+)中,转化E. coli BL21(DE3),经IPTG诱导后在宿主菌得到表达。SDS-PAGE电泳检测证明该酯酶蛋白分子量约为28 kDa。TLC和HPLC检测结果显示,该酯酶优先水解(R)-型底物,在重组菌菌液体系,转化率为15%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)最高,达62.74％;改用重组菌湿菌体的PBS体系后,在转化率为10%~50%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)一直保持在73%~76%之间。     

脂肪酶产生菌的筛选及不对称水解合成S-(+)-萘普生

从１６个土样中分离获得一株脂肪酶产生菌Ｅ－５３＃,该菌可优先水解Ｓ－（＋）－萘普生甲酯为Ｓ－（＋）－萘普生,ｅｅ值可超过８７％。该菌经初步鉴定为芽孢杆菌,最佳产酶培养基为葡萄糖０．５％、蛋白胨０．５％、酵母膏０．２％。０．５％的橄榄油,能诱导脂肪酶的大量产生。     

Enantioselective lipase-catalyzed ester hydrolysis: Effects on rates and enantioselectivity from a variation of the ester structure

In order to make a preliminary study of substituent effects on the rate and enantioselectivity obtained in esterolytic reactions catalyzed by a lipase from Candida rugosa, a series of racemic esters, derived from some α-alkyl and α-halo phenylacetic acids, were prepared. The reactions were studied at pH 6.0 and 50°C under which conditions uncatalyzed hydrolysis was relatively slow. Reaction samples were studied at different points of time by means of analytical chiral reversed-phase liquid chromatography, which permitted the simultaneous determination of product enantiomeric excess and of the degree of total ester hydrolysis. These data were then used to calculate initial rates as well as enantioselectivity. An increase of the steric bulk of the α-substituent was found to highly decrease the rate of the reaction. On the other hand, rates were higher for the p-nitrophenyl esters than for the corresponding 2-chloroethyl esters. Consistently, the enantioselectivity was found to be higher for the latter type of ester. The esters of the α-halo (bromo and chloro) phenylacetic acids gave mandelic acid as the final product. This was caused by a rapid solvolysis of the α-halo phenylacetic acid initially formed. © 1993 Wiley-Liss, Inc.     

Improving lipase-catalyzed enantioselective ammonolysis of phenylglycine methyl ester in organic solvent by in situ racemization

Lipase-catalyzed enantioselective ammonolysis of phenylglycine methyl ester was processed by in situ racemization with ammonium carbamate as the acyl acceptor. Using 1 mM benzaldehyde or 0.6 mM chloropyridoxal as the racemizing catalyst, 80% substrate conversion with an enantiomeric excess of the product of 95% were achieved at 20 °C after 7 h reaction.     

Lipase-catalyzed enantioselective esterification of (S)-naproxen hydroxyalkyl ester in organic media

A lipase-catalyzed, enantioselective esterification process in organic solvents was developed for the synthesis of (S)-naproxen hydroxyalkyl ester. With the selection of lipase (Candida rugosa lipase) and reaction medium (isooctane and cyclohexane), a high enantiomeric ratio of <100 for the enzyme was obtained. 1,4-Butanediol was the best acyl acceptor. The carbon chain length of the alcohol had a major effect on the enzyme activity and enantioselectivity of lipase-catalyzed esterification.     

Immobilization of Candida rugosa lipase on glass beads for enantioselective hydrolysis of racemic Naproxen methyl ester

Candida rugosa lipase (CRL) was immobilized on glutaraldehyde-activated aminopropyl glass beads by using covalent binding method or sol-gel encapsulation procedure and improved considerably by fluoride-catalyzed hydrolysis of mixtures of RSi(OCH₃)₃ and Si(OCH₃)₄. The catalytic properties of the immobilized lipases were evaluated into model reactions, i.e. the hydrolysis of p-nitrophenylpalmitate (p-NPP). It has been observed that the percent activity yield of the encapsulated lipase was 166.9, which is 5.5 times higher than that of the covalently immobilized lipase. The enantioselective hydrolysis of racemic Naproxen methyl ester by immobilized lipase was studied in aqueous buffer solution/isooctane reaction system and it was noticed that particularly, the glass beads based encapsulated lipases had higher conversion and enantioselectivity compared to covalently immobilized lipase. In short, the study confirms an excellent enantioselectivity (E > 400) for the encapsulated lipase with an ee value of 98% for S-Naproxen.     

恶臭假单胞菌CGMCC 1388对映选择性生物降解制备(S)-扁桃酸及其衍生物的研究

从土壤中分离得到一株（R）-扁桃酸选择性降解菌,经鉴定为恶臭假单胞菌,保存于中国普通微生物保藏中心,编号为CGMCC1388。考察了扁桃酸及其降解产物对扁桃酸脱氢酶活力的影响。研究表明,在培养基中添加少量扁桃酸、苯甲酰甲酸或苯甲酸均可显著提高其产量,以扁桃酸的诱导效果最佳。3种诱导物的最适添加质量浓度分别为4、4和2 g/L。当以消旋扁桃酸为反应底物时,该菌可高选择性降解（R）-扁桃酸,回收得到的（S）-扁桃酸对映体过量值（e.e.）高于99%,反应的对映选择率（E）达130。用静息细胞催化扁桃酸降解的最适温度和pH分别为30 ℃和6.0,最适底物浓度为60 mmol/L,以双倒数法求得Km为47 mmol/L。考察了该菌对扁桃酸苯环取代衍生物的生物转化,并以高产率制备获得高对映纯度的（S）-对羟基扁桃酸和（S）-对氯扁桃酸。     

Resolution of 2-cyano-2-methylalkanoic acids via porcine pancreatic lipase-catalyzed enantioselective ester hydrolysis: effect of the alcohol moiety of the substrate ester on enantioselectivity

2-Cyano-2-methylalkanoic acids were resolved via porcine pancreatic lipase-catalyzed enantioselective ester hydrolysis. The importance of the alcohol moiety of the substrate ester on enantioselectivity was confirmed: the E value was increased up to 9-fold by using the n-butyl ester instead of the conventional methyl ester. The maximum E value was 180.     

不对称水解(R,S)-2,6-二甲基苯基氨基丙酸甲酯新菌种的分离鉴定及酯酶基因的克隆、表达

【目的】筛选鉴定1株可以选择性水解农药甲霜灵的中间体(R,S)-2,6-二甲基苯基氨基丙酸甲酯(MAP)的菌株,并克隆、表达该菌株中的酯酶基因。【方法】以MAP为唯一碳源,对活性污泥样品中的微生物进行富集培养,采用罗丹明B平板显色法进行初筛,通过摇瓶复筛得到了1株对MAP具有最高对映体选择性和水解活力的新菌株,根据其形态、生理生化特征及16S rRNA序列分析,确立该菌株的系统发育学地位。构建该菌株的基因文库,筛选获得含目的基因的克隆子,通过序列分析和引物扩增得到酯酶基因,将基因与表达载体pET28a(+)连接后,转化大肠杆菌BL21Gold(DE3)plysS,构建重组菌。【结果】该菌属于革兰氏阴性菌,结合16S rRNA基因、形态特征和生理生化实验结果,鉴定该菌为反硝化无色杆菌。通过基因文库法,找到了该菌中的酯酶基因EHest,并成功构建了重组大肠杆菌EHest-p ET28a(+)-BL21Gold(DE3)plys S,表达了来自Achromobacter denitrificans 1104且具有不对称水解MAP活性的酯酶EHesterase,大小约27 kDa,表达酶活是原始菌株的27.1倍。用EHesterase催化MAP水解,底物浓度50 g/L,反应1 h,底物转化率为29.5%,产物(酸)的ee_p为85.1%,对映体选择性为R型。该酶的最适反应pH和温度分别为pH 9.0和50°C。它水解MAP的活性分别是水解橄榄油和乙酸乙酯活性的333倍和667倍。【结论】筛选到1株具有不对称水解MAP能力的新菌株Achromobacter denitrificans 1104。     

产碱杆菌ECU0401催化拆分扁桃酸及其衍生物

从土壤中分离的1株产碱杆菌Alcaligenes sp.ECU0401具有扁桃酸脱氢酶活性,可以以扁桃酸、苯甲酰甲酸或苯甲酸为唯一C源生长,并且具有较高的脱氢酶活力。以外消旋扁桃酸为C源,采用分批补料策略培养（或反应）99h,扁桃酸累计投入量为30.4g/L,（S）-（＋）-扁桃酸被完全降解,（R）-（-）-扁桃酸回收产率为32.8%,对映体过量值（e.e.）〉99.9%。利用静息细胞作为催化剂不对称降解外消旋扁桃酸的氯代衍生物,制备获得光学活性的（R）-（-）-邻氯扁桃酸、（S）-（＋）-间氯扁桃酸和（S）-（＋）-对氯扁桃酸,光学纯度均超过99.9%e.e.。     

来源于西梅的(R)-醇腈酶促立体选择性转氰合成(R)-酮醇腈

在水/有机溶剂双相反应体系中,研究了来源于西梅的(R)-醇腈酶催化酮与丙酮醇腈合成(R)-酮醇腈的立体选择性转氰反应．系统探讨了不同酶源、酶粉颗粒大小、底物浓度、两底物配比、酶浓度和底物结构对转氰反应的影响．结果发现西梅醇腈酶能高效催化三甲基硅酮与丙酮醇腈的立体选择性转氰．酶粉颗粒大小以直径0.3～0.45 mm为优,底物浓度以21 mmol/L左右为佳,底物丙酮醇腈与三甲基硅酮摩尔浓度比以2∶1为宜,酶浓度以60.9 g/L左右为好．西梅醇腈酶对3, 3-二甲基-2-丁酮几乎没有催化活性,而对其硅结构类似物三甲基硅酮却具有非常高的立体选择性和催化活性,在上述优化反应条件下反应24 h的底物转化率和产物光学纯度均高达99%以上,表明底物中的硅原子对西梅醇腈酶的催化活性有非常显著的促进作用．     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Effect of solvent on enantioselective esterification of naproxen by lipase with trimethylsilyl methanol

Improvement of stereoselective resolution of racemic Naproxen, 2-(6-methoxy-2-naphthyl)propionic acid, was attempted with esterifcation reaction by Candida cylindracea lipase. By carefully selecting the organic medium, a 72-time enhancement of yield of the desired S-ester was achieved. The optimal reaction temperature was approximately 53 degrees C, and an alcohol concentration between 20 mM and 40 mM in an 80% (v/v) isooctane and 20% (v/v) toluene mixture was found. (c) 1994 John Wiley & Sons, Inc.     

芦笋抗S—（2—氨乙基）—L—半胱氨酸（AEC）变异体的筛选

S-(2-氨乙基)-L-半胱氨酸(AEC)可抑制芦笋愈伤组织的生长,此抑制作用可被赖氨酸或甲硫氨酸部分解除。用0.5mmol/L的AEC进行筛选,得到抗性愈伤组织AR10并再生植株。AR10愈伤组织经一年多的继代培养,在离开选择剂组培继代两代后仍保持对AEC的抗性。抗性系愈伤组织还表现出对2mmol/L的半胱氨酸具交叉抗性,对1mmol/L的赖氨酸加苏氨酸表现部分交叉抗性。AR10再生植株一部分保持对AEC的抗性,而一部分则无抗性。对抗性愈伤组织及其再生植株的氨基酸分析表明,愈伤组织内游离赖氨酸、苏氨酸、甲硫氨酸都有增加,而在再生植株内却发现半胱氨酸和赖氨酸的特异性增加,分别是对照植株的5.4和4.6倍。