一株微生物菌株催化水解拆分外消旋6-羟基-8-氯辛酸乙酯

从土壤中筛选到一株能够拆分外消旋的硫辛酸中间体6-羟基-8-氯辛酸乙酯的微生物菌株T4,并对其拆分反应条件进行了优化,确定了最适反应温度为30℃,最适pH为7．0,最适摇床转速为170r／min,确定了有效表面活性剂为CTAB,在此优化条件下反应8h,得到（R）-6-羟基-8-氯辛酸乙酯的对映体过量值（e．e．）为92．8％,产率为26．3％。该研究为（R）-硫辛酸的制备提供了一条可行的途径。     

Bacillus megaterium WZ009催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯

以外消旋4-氯-3-羟基丁酸乙酯为唯一C源的富集培养筛选得到一株菌株WZ009,经16S rDNA测序鉴定为巨大芽胞杆菌(Bacillus megaterium)。B.megaterium WZ009静息细胞可以立体选择性催化(S)-4-氯-3-羟基丁酸乙酯水解和脱氯反应得到光学纯的(R)-4-氯-3-羟基丁酸乙酯(e.e.≥99%)和(S)-3-羟基-γ-丁内酯(e.e.≥95%)。笔者对B.megaterium WZ009不对称催化反应影响因素(温度、pH、中和剂、底物浓度、时间进程以及细胞重复利用)进行优化研究,确定了该反应体系最优条件:底物浓度200 mmol/L,中和剂氨水,pH 7.2,40℃反应12 h,转化率达到50.6%,底物对映体过量值为99.6%。该生物催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯过程具有良好的工业化应用前景。     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从11株微生物中筛选出4株具有不对称还原2′-氯-苯乙酮能力的酵母,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对2′-氯-苯乙酮还原的最佳反应时间为24h;最佳pH 8.0;最佳反应温度为25℃;最佳共底物为5%（体积比）乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为10.75mg/mL（细胞干重/反应体积）的酿酒酵母B5可将647mmol/L的2′-氯-苯乙酮100%地转化为R-2′-氯-1-苯乙醇,其对映体选择性为100%。酿酒酵母B5可重复利用的特点可提高产物的产量。     

新颖深海微生物酯酶EstC11的酶学性质及其在手性拆分乙酸苏合香酯中的应用

【背景】手性乙酸苏合香酯是重要的手性香料产品,在食品及精细化工等领域都有重要的应用。酶催化不对称合成手性乙酸苏合香酯产品具有极好的工业应用前景。【目的】研究酯酶EstC11的基本酶学性质及其在制备手性乙酸苏合香酯中的应用。【方法】对来自西太平洋深海热液口芽孢杆菌Bacillus sp.CX01中的新颖微生物酯酶基因EstC11进行克隆、表达及酶学性质鉴定。通过对p H、温度、有机溶剂等反应条件的优化提高酯酶手性拆分乙酸苏合香酯的光学纯度。【结果】酯酶EstC11的最适反应p H为8.5,最适温度为25°C,一些金属离子和有机溶剂对酯酶EstC11的水解活性具有不同程度的抑制作用。通过对反应条件的优化,在最适反应条件下(p H 9.0 50 mmol/L Tris-HCl,20°C,50 mmol/L底物浓度)反应3 h后,(R)-乙酸苏合香酯的光学纯度达98%,得率为39%。【结论】通过对酯酶拆分条件的优化,手性拆分乙酸苏合香酯生成(R)-乙酸苏合香酯的光学纯度明显提高,为酯酶EstC11在工业化上的应用奠定了基础。     

（S）-（-）-β-羟基苯丙酸乙酯的微生物法合成

采用酿酒酵母CGMCC No．2266菌体,不对称还原β-羰基苯丙酸乙酯制备光学纯（S）-（-）-β-羟基苯丙酸乙酯。结果表明：采用初始pH为8．0的液体发酵培养基培养的CGMCCNo．2266菌体经过50℃预热处理30min后用于生物转化获得的（S）-（-）-G-羟基苯丙酸乙酯对映体过剩值可以达到100％ee。确定了合成（S）-（-）-β-羟基苯丙酸乙酯的较佳转化条件为pH7．0,温度30℃,转化时间24h,底物浓度为3．63mmol／L,菌体用量为86g/L（干重／反应体积）。以10％葡萄糖为辅助底物,产率比不加辅助底物时提高了75．4％。在最佳转化条件下反应转化率及（S）-（-）-β-羟基苯丙酸乙酯对映体过剩值可分别达到98．4％和100％ee。     

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。     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从 11株微生物中筛选出 4株具有不对称还原 2′ 氯 苯乙酮能力的酵母 ,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对 2′ 氯 苯乙酮还原的最佳反应时间为 2 4h ;最佳pH 8 0 ;最佳反应温度为2 5℃ ;最佳共底物为 5 % (体积比 )乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为 10 75mg mL(细胞干重 反应体积 )的酿酒酵母B5可将 6 47mmol L的 2′ 氯 苯乙酮10 0 %地转化为R 2′ 氯 1 苯乙醇 ,其对映体选择性为 10 0 %。酿酒酵母B5可重复利用的特点可提高产物的产量。     

羰基还原酶不对称还原（R）-6-氰基-5-羟基-3-羰基己酸叔丁酯

选择R-羰基还原酶和葡萄糖脱氢酶双酶,协同催化（R）-6-氰基5-羟基-3-羰基己酸叔丁酯不对称还原制备阿托伐他汀关键手性合成子6-氰基-（3R,5R）-二羟基已酸叔丁酯。转化条件优化结果显示：在不添加外源性辅酶NADP（H）、菌体用量15．0g/L、147．0g/L（R）-6-氰基-5-羟基-3-羰基己酸叔丁酯、128．2g／L葡萄糖,30℃、pH6．5条件下反应6h后,底物转化率达到100％,产物d．e．值大于99．5％。     

固定化细胞拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的研究

【目的】筛选鉴定一株产酯酶用于选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,利用该菌株固定化细胞催化拆分外消旋底物。【方法】通过富集培养、罗丹明B平板初筛及复筛培养获得一株选择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的菌株,通过对其形态、生理生化特征及16S r DNA序列分析,确立该菌株系统发育地位。优化了利用硅藻土-戊二醛吸附交联法对该菌体细胞固定化的条件,研究固定化细胞催化性质及操作稳定性。【结果】该菌为革兰氏阴性菌,鉴定其为甲基球状菌属(Methylopila)。固定化体系最优条件:聚乙烯亚胺0.15%(V/V),戊二醛0.2%(V/V),硅藻土6 g/L,菌体质量浓度100 g/L。与游离细胞相比,固定化细胞最适p H由8.0变为8.5,最适温度由35°C变为40°C,p H稳定性和温度稳定性都有所提高。Cu~(2+)、Mn~(2+)、Ca~(2+)能促进酶活,Zn~(2+)、Fe~(2+)抑制酶活。固定化细胞的有机溶剂耐受性较游离细胞有所提高。动力学分析细胞固定化后Km值变大,底物亲和力降低。利用固定化细胞水解(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯,底物浓度200 g/L,反应20 h,保留构型为S型,得率47.8%,对映体过量值ees为99.4%,重复使用12次后仍保留初始酶活的80%以上。【结论】开发了利用Methylopila sp.cxzy-L013固定化细胞择性拆分(R,S)-α-乙基-2-氧-1-吡咯烷乙酸甲酯的工艺,该工艺具有良好的工业应用前景。     

重组青霉素G酰化酶拆分制备（S）-邻氯苯甘氨酸

对产青霉素G酰化酶的重组枯草芽胞杆菌发酵产酶条件进行优化,确定优化后的发酵条件：可溶性淀粉10g/L、蛋白胨12g/L、酵母粉3g/L、NaCl10g,／L;pH7．5、培养温度37℃、装液量80mL（500mL三角瓶）、培养28h,青霉素G酰化酶的表达水平由最初的7．34U／mL提高至18．23U／mL。以表达青霉素G酰化酶的枯草芽胞杆菌发酵液为酶源,在水相中对映选择性催化N-苯乙酰-（R,S）-邻氯苯甘氨酸制备（S）-邻氯苯甘氨酸,当底物浓度为100mol／L时转化4h,转化率达44．2％。对底物浓度为80mmoL／L反应液中的（S）-邻氯苯甘氨酸进行分离,达到理论收率的94．29％（以N-苯乙酰-（R,S）-邻氯苯甘氨酸的0．5倍摩尔量为理论产率）,e．e．值大于99．9％。170℃条件下,N-苯乙酰-（R）-邻氯苯甘氨酸与苯乙酸共熔消旋为N-苯乙酰-（R,S）-邻氯苯甘氨酸可用于循环拆分。     

Sequential Kinetic Resolution by two Enantioselective Enzymes

Enhancement of the enantioselectivity by simultaneous use of two enzymes in a sequential kinetic resolution process is presented. The model system consisted of carboxylesterase NP catalyzed hydrolysis of racemic methyl 2-chloropropionate, followed by dehalogenation of the enantiomerically enriched 2-chloropropionate by DL-dehalogenase into lactate. Optimal results are shown to be attained when the conversion rates of both faster reacting enantiomers are the same. An optimization parameter D for sequential resolutions is introduced. The kinetics of both reaction steps were investigated separately by progress curve analysis, and the enantioselectivity of the enzymes was determined. From a quantitative kinetic model we could formulate the sequential resolution, which yielded the predicted improvements of product enantiomeric excess.     

Sequential Kinetic Resolution by two Enantioselective Enzymes

Enhancement of the enantioselectivity by simultaneous use of two enzymes in a sequential kinetic resolution process is presented. The model system consisted of carboxylesterase NP catalyzed hydrolysis of racemic methyl 2-chloropropionate, followed by dehalogenation of the enantiomerically enriched 2-chloropropionate by DL-dehalogenase into lactate. Optimal results are shown to be attained when the conversion rates of both faster reacting enantiomers are the same. An optimization parameter D for sequential resolutions is introduced. The kinetics of both reaction steps were investigated separately by progress curve analysis, and the enantioselectivity of the enzymes was determined. From a quantitative kinetic model we could formulate the sequential resolution, which yielded the predicted improvements of product enantiomeric excess.     

Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.     

新颖微生物低温酯酶EstP8的酶学性质研究与在手性催化中的应用

从假单胞菌Pseudonocardia antitumoralis HUP007基因组中克隆了一个1 041bp的酯酶基因EstP8,编码的蛋白具有377个氨基酸残基。在E.coli BL21（DE3）中实现酯酶EstP8的高效异源表达和纯化。EstP8为脂肪酶家族Ⅳ中的一员,具有HGGG保守序列。EstP8最适底物为对硝基苯酚乙酸酯（p-NPO）,最适温度和pH分别为50℃和8.0。EstP8催化p-NPO水解反应的活性、V_max和K_m分别达到105.19U/mg、89.4μM/min、1.144mM。EstP8在pH7.0~8.0范围内具有良好的pH稳定性;在4℃时,酯酶相对活力为41.78%,在10~40℃内具有很好温度稳定性。EstP8对大部分金属离子有很好的耐受性,低浓度的Cu²⁺、Mn²⁺、Zn²⁺对该酶的活性有激活作用。辛烷、庚烷、甲苯、丙酮、DMF等有机溶剂对EstP8的活性同样具有激活作用。酯酶EstP8还可以通过水解拆分高效地制备手性（R）-1-苯基乙醇;添加有机溶剂可以很好地促进该酯酶的光学选择性和产率,在共溶剂甲苯的存在下,所制备的（R）-1-苯基乙醇的e.e.和产率可达91%和18%;在共溶剂DMSO的存在下,所制备的（S）-乙酸苏合香酯的e.e.和产率可达98%和60%。酯酶EstP8在手性生物催化等诸多工业领域有很好的应用潜力。     

Synthesis and enantioselective rearrangement of (Z)-4-triphenylmethoxy-2,3-epoxybutan-1-ol enantiomers

Efficient enzyme catalyzed kinetic resolutions of a synthetically useful chiral building block, (Z)-4-triphenylmethoxy-2,3-epoxybutan-1-ol, are reported. The highest selectivities were achieved by Lipozyme TL IM and Amano Lipase PS enzymes in the presence of vinyl acetate. Enantiomeric enrichment of the optically active acetate isomer was accomplished by selective crystallization of the racemic part of the enantiomeric mixture. Enzyme catalyzed hydrolysis of the acetate also provided an optically pure epoxybutanol derivative. O-Benzylation of (+)-(Z)-1-hydroxy-4-triphenylmethoxy-2,3-epoxybutane followed by super base promoted diastereo- and enantio-selective rearrangement resulted in (+)-(2R,3R,1'R)-3-[1-hydroxy-2-(triphenylmethoxy)ethyl]-2-phenyloxetane in >98% ee and de. Configurations of the new optically active products were determined by chemical correlation.     

Enzyme catalyzed hydrolysis of the diesters of cis- and trans-cyclohexanedicarboxylic acids

Summary Pig liver esterase (EC 3.1.1.1) catalyzed hydrolysis of the dimetrhy ester of meso-cis-1,2-cyclohexanedicarboxylic acid yielded the optically pure (1S,2R)-monoester. The corresponding diethyl ester yielded racemic monoester.The diethyl ester of racemic trans-1,2-cyclohexanedicarboxylic acid was kinetically resolved by partial hydrolysis with subtilisin (EC 3.4.21.14) or pig liver esterase. The (1R,2R)-monoester had an enantiomeric excess of 45% and was obtained in an enantiomerically pure form through recrystallisation. The remaining (1S,2S)-diester exhibited an enantiomeric excess of 83%. The nature of the ester function (methyl, ethyl, and propyl esters) had a great influence on the enantiomeric excess obtained and on the kinetic parameters.     

Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.     

Enzymatic Resolution of Racemates Contaminated by Racemic Product

Enzyme-catalyzed kinetic resolution is sometimes performed starting with substrate already containing small amounts of the racemic product. Then the determination of the enantiomeric ratio may be seriously disturbed when this parameter is calculated from the degree of conversion and the enantiomeric excess of either the substrate or the product (Chen et al., 1982, 1987) or when it is calculated directly from the enantiomeric excess of substrate and product (Rakels et al., 1993).

This paper presents modifications of these methods in order to correctly determine the enantiomeric ratio as well as the amount of racemic product in the substrate. The theoretical predictions were verified for the hydrolysis of racemic ethyl 2-chloropropionate, catalyzed by carboxylesterase NP. Despite the presence of racemic product in the substrate, accurate and reliable values for the enantiomeric ratio were obtained by using the modified methods.     

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)-mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 43.6 of (R,S)-ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM-87 from the producer) to 16.7 mmol/h g and 867 of (R,S)-2-methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)-2-hydroxycarboxylic acid esters, giving improvements of the enzyme performance from V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)-2-methoxyethyl 3-chloromandelate (16) for the immobilized enzyme, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 8.27 of (R,S)-ethyl 4-chloromandelate (10) for free SNSM-87 to 33.5 mmol/h g and 123 of (R,S)-methyl 4-chloromandelate (14) for the immobilized enzyme, as well as V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 7.94 of (R,S)-ethyl 3-phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)-2-methoxyethyl 3-phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach.     

Efficient kinetic resolution of secondary alcohols using an organic solvent-tolerant esterase in non-aqueous medium

Objective

To identify an esterase-mediated kinetic resolution of secondary alcohols in non-aqueous medium.

Results

An esterase, EST4, from a marine mud metagenomic library, showed high activity and enantioselectivity for the kinetic resolution of secondary alcohols in non-aqueous medium. Using 1-phenylethanol as the model alcohol, the effects of organic solvents, acyl donors, molar ratio, temperatures and biocatalyst loading on the kinetic resolution catalyzed by the EST4 whole-cell biocatalyst were investigated and optimized. The optimized methodology was effective on resolving 16 various racemic secondary alcohols in neat n-hexane, providing excellent enantiomeric excess (up to 99.9 % ee). Moreover, EST4 exhibited a strong tolerance for high substrate concentration (up to 1 M), and the optical purity of the desired secondary alcohols was kept above 99 % ee.

Conclusion

The esterase EST4 is a promising biocatalyst for the enantioselective synthesis of various alcohols and esters with interesting practical applications.