手性技术与生物催化

简要介绍了手性,手性技术与生物催化的基本概念。手性,是指一个有机分子具有不对称性,形成两种空间排布方式不同的对映异构体。手性技术即生产手性化合物的技术,手性化合物的制备方法主要有手性源、外消旋体拆分、不对称合成等几种。生物催化,即利用酶或微生物等生物材料催化进行某种化学反应,被认为是手性化合物生产取得突破的关健技术。文章还介绍了生物催化外消旋体拆分、生物催化不对称合成等几种生产手性化合物的应用实例。     

Serratia marcescens H30脂肪酶的重组可溶表达、固定化及其酶学性质

为了提高Serratia marcescens H30脂肪酶的可溶表达水平,分别将目的基因与p GEX-4T-1、p ET28a和p ET32a构建重组表达载体,转入大肠杆菌BL21(DE3),通过优化诱导过程,发现可溶性酶的最高活性可达25 000 U/L。再经Ni2+亲和柱纯化、LH-EP固定化后,固定化酶的比酶活为214 U/g(以1 g湿质量计),酶活回收率为51%。固定化后重组脂肪酶的最适温度由30℃提高到35℃,最适p H从7.0偏移至8.0左右,并且稳定性也有所增加。该固定化重组脂肪酶同样能够拆分消旋体反式-4-甲氧苯基缩水甘油酸甲酯,光学选择性没有改变。反应14 h,转化率为48.5%,底物的e.e.值为99.2%,表明该固定化脂肪酶能有效拆分消旋体反式-4-甲氧苯基缩水甘油酸甲酯,为工业生物催化制备地尔硫卓提供了可能。     

微生物酶转化合成手性药物的研究进展

通过微生物酶催化不对称合成反应或拆分外消旋体合成医药手性中间体具有独特的优势。结合作者自身近年来在该技术领域的实践对相关课题作了介绍,总结了微生物酶催化不对称反应和拆分反应得到手性药物的研究进展。     

微环境对脂肪酶催化拆分外消旋2－辛醇的影响

微环境对脂肪酶催化拆分外消旋２－辛醇的影响　　　　　　　杨红,曹淑桂,韩四平,黄仲丽,杨同书（吉林大学酶工程国家重点实验室,长春１３００２３）手性２－辛醇不仅是制备液晶材料不可缺少的重要手性原料,也是合成具有光学活性的医药和农药的重要手性中间体．本文...     

固定化脂肪酶催化制备L-薄荷醇

用大孔树脂NKA固定高选择性的脂肪酶,催化有机相中转酯化反应,从而拆分八异构体消旋薄荷醇来制备L-薄荷醇。研究pH、载体与酶比例对固定化酶制备的影响及固定化酶的反应稳定性;考察温度、转酯化过程醇酯比例、及底物醇异构组成变化对拆分效果的影响。结果表明:固定化酶的最适pH为8,载体与酶的比例为5∶1时,所得固定化酶的反应稳定性比游离酶的反应稳定性提高了约50%;转酯化反应的最优温度为40℃,醇酯比例为1.5∶1时最佳,改进八异构体消旋薄荷醇组分比例后,非对映体选择率dep达到了95.1%。     

拆分获得(S)-酮基布洛芬脂肪酶基因在枯草芽孢杆菌中的克隆与表达

【目的】筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因,构建其表达分泌型工程菌,并进一步提高该脂肪酶的立体选择性。【方法】以自筛选出的一株具有不对称拆分消旋酮基布洛芬氯乙酯能力的菌株NK13为材料,通过构建其基因组文库,筛选具有不对称拆分消旋酮基布洛芬氯乙酯能力的脂肪酶基因。通过构建该脂肪酶基因的分泌型诱导表达载体pHY300-plk-sacR-gene,将其转入枯草芽孢杆菌WB600,获得基因重组菌WB600(pHY300-plk-sacR-gene)。用SDS-PAGE检测其表达和转化情况,采用非变性聚丙烯酰胺凝胶电泳的方法纯化脂肪酶;并利用TLC和HPLC检测该酶的立体选择专一性。【结果】得到了具有专一性拆分获得(S)-酮基布洛芬能力、长度为633bp的脂肪酶基因(GenBank登录号为:EU381317)。该脂肪酶在枯草芽孢杆菌WB600中得到了分泌表达。TLC和HPLC检测结果显示,纯化的脂肪酶对底物转化40h时转化率为30%,生成(S)-酮基布洛芬的e.e.%值最高,达60.02%,与未加Tween-80的枯草芽孢杆菌转化子体系相同。而在含Tween-80的环境下,枯草芽孢杆菌表达重组菌对底物转化36h时转化率约为45%,生成(S)-酮基布洛芬的e.e.%值最高,达93.64%,是野生菌NK13的16倍。【结论】从NK13号菌株中筛选得到的新的脂肪酶具有很高的不对称拆分获得(S)-酮基布洛芬的能力,实现了NK13菌中633bp脂肪酶基因在枯草芽孢杆菌中的分泌表达,研究证明Tween-80能提高该脂肪酶的拆分专一性。     

有机相中脂肪酶催化不对称酯合成反应动力学的研究

有机相中脂肪酶催化不对称酯合成反应动力学的研究杨红,高修功,郭妮妮,曹淑桂,杨同书（吉林大学酶工程国家重点实验室,长春１３００２３）在环已烷中应用酵母脂肪酶催化外消旋２－辛醇和辛酸的不对称酯合成反应,研究了该反应的动力学机制,测定了表现动力学常数．利...     

洋葱伯克霍尔德菌脂肪酶：一种应用前景广阔的生物催化剂

洋葱伯克霍尔德菌脂肪酶对有机溶剂（醇）、热、氧化剂、表面活性剂、去污剂、蛋白酶等具有良好的抗性,在有机合成、对映体拆分、非水相催化等领域应用十分广泛。综述了洋葱伯克霍尔德菌脂肪酶的发酵生产、分离纯化、基因克隆与表达、固定化与生物印迹、蛋白质结构解析及应用研究等,并展望了其未来发展方向,以期为该工业酶的研发与广泛应用提供参考。     

酶法拆分手性化合物HPBE

R-HPBE(2-羟基4苯基丁酸乙酯)是一种重要的医药中间体,可以通过脂肪酶催化水解外消旋体得到。介绍了此催化过程的机理、工艺、产物检测等,并通过酶在疏水载体上的界面吸附对酶进行固定化,以提高酶活及对映选择性。     

有机介质中脂肪酶催化反应在有机合成中的应用

综述了近年来有机介质中脂肪酶催化反应在酯合成,酯交换,内酯合成,多肽合成,高聚物合成及立体异构体拆分等有机合成领域的应用。对脂肪酶催化不对称反应合成光学纯化合物进行了较详细的介绍。     

Application of lipases in kinetic resolution of racemates

Lipases have been well established as valuable catalysts in organic synthesis. This review article focuses on some of the recent developments in the rapidly growing field of lipase-catalyzed kinetic resolution of racemates as a versatile method for the separation of enantiomers. The literature search dates back to the last five years and covers some comprehensive examples. The main emphasis is on the use of lipases in organic solvents.     

Production, purification, characterization, and applications of lipases

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyze the hydrolysis and the synthesis of esters formed from glycerol and long-chain fatty acids. Lipases occur widely in nature, but only microbial lipases are commercially significant. The many applications of lipases include speciality organic syntheses, hydrolysis of fats and oils, modification of fats, flavor enhancement in food processing, resolution of racemic mixtures, and chemical analyses. This article discusses the production, recovery, and use of microbial lipases. Issues of enzyme kinetics, thermostability, and bioactivity are addressed. Production of recombinant lipases is detailed. Immobilized preparations of lipases are discussed. In view of the increasing understanding of lipases and their many applications in high-value syntheses and as bulk enzymes, these enzymes are having an increasing impact on bioprocessing.     

Are Lipases Still Important Biocatalysts? A Study of Scientific Publications and Patents for Technological Forecasting

The great potential of lipases is known since 1930 when the work of J. B. S. Haldane was published. After eighty-five years of studies and developments, are lipases still important biocatalysts? For answering this question the present work investigated the technological development of four important industrial sectors where lipases are applied: production of detergent formulations; organic synthesis, focusing on kinetic resolution, production of biodiesel, and production of food and feed products. The analysis was made based on research publications and patent applications, working as scientific and technological indicators, respectively. Their evolution, interaction, the major players of each sector and the main subject matters disclosed in patent documents were discussed. Applying the concept of technology life cycle, S-curves were built by plotting cumulative patent data over time to monitor the attractiveness of each technology for investment. The results lead to a conclusion that the use of lipases as biocatalysts is still a relevant topic for the industrial sector, but developments are still needed for lipase biocatalysis to reach its full potential, which are expected to be achieved within the third, and present, wave of biocatalysis.     

Hydrolysis of oils by using immobilized lipase enzyme: A review

This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.     

Novel microbial lipases: catalytic activity in reactions in organic media

2,000 microbial strains were isolated from soil samples and tested to determine their lipolytic activity by employing screening techniques on solid and in liquid media. Culture broths were initially tested with 1,2-O-dilauryl-rac-glycero-3-glutaric acid-resorufinyl ester, a chromogenic substrate specific for lipases. Fourteen lipase-producing microorganisms were selected and their taxonomic identification was carried out. Hydrolysis of tributyrin or olive oil and the esterification of oleic acid with heptanol were selected to preliminary evaluate the catalytic activity of these lipases. All the selected lipases catalysed this esterification reaction with good yields. Resolution of (R,S)-2-(4-isobutylphenyl) propionic acid, (R,S)-1-phenylethanol, (R,S) 1-phenylethylamine and of (R) or (S) glycidol were performed to evaluate the stereoselectivity of these novel enzymes as biocatalysts in reactions in organic media. Lipases from the fungi Fusarium oxysporum and Ovadendron sulphureo-ochraceum gave the best yields and enantioselectivities in the resolution of racemic ibuprofen and 1-phenylethanol. Several lipases displayed a high stereoselectivity in the resolution of chiral amines by an alcoxycarbonylation reaction.     

Three New Lipases from Actinomycetes and Their Use in Organic Reactions

Three novel lipase-producing microorganisms have been isolated from 526 actinomycete strains by employing screening techniques on solid media. Time-course and scale-up of enzyme production were analyzed. The lipases, produced by microorganisms belonging to the Streptomyces genus, were tested in several reactions in organic medium using unnatural substrates. The lyophilized crude lipases are stable at least for 1 month at 4°C (100% recovered activity). The lipase activity per milliliter of cell culture broth was higher than described in the literature for other lipases from actinomycetes. The three selected lipases displayed better activity than commercial lipase from Candida rugosa in the resolution of chiral secondary alcohols. The lipase from S. halstedii also displayed very good activity in the synthesis of carbamates.     

Enzymatic resolution of racemic ibuprofen by surfactant-coated lipases in organic media

Summary Surfactant-coated lipases have been utilized as a biocatalyst for the resolution of racemic ibuprofen. S-(+)-ibuprofen was selectively transferred to the ester form by Mucor javanicus or Candida rugosa lipase. The enzymatic activity of upases in organic media was remarkably enhanced by coating with a nonionic surfactant. The reaction rates of the coated lipases were increased around 100-fold that of the powder lipases.     

Hydrophobic adsorption in ionic medium improves the catalytic properties of lipases applied in the triacylglycerol hydrolysis by synergism

It is known that lipases may have their catalytic properties improved by the action of some salts or by the adsorption on hydrophobic supports. However, what we present in this work is more than that: we evaluate the combination of these two factors of hyperactivation of lipases from Acremonium-like ROG 2.1.9, a study that has not been done so far. This work proves that a synergistic effect occurs when the lipases are immobilized on hydrophobic supports at the presence of sodium chloride and are applied in triacylglycerol hydrolysis. This assay made it possible to achieve the highest hyperactivation of 500 % with the lipases immobilized on Phenyl-Sepharose and applied with 0.1 M of sodium chloride. Besides this positive effect on enzyme activity, the use of these two factors led to the thermal stability increasing of the immobilized lipases. For this derivative, the recovered activity was approximately 85 % after 6 h incubated at 55 °C and 1.0 M of the sodium chloride against 50 % of the same derivative without this salt. Furthermore, others assays were performed to prove the evidences about the synergistic effect, showing a promising method to improve the catalytic properties of the lipases from Acremonium-like ROG 2.1.9.     

Preparation and catalytic performance of lipases encapsulated in sol-gel materials

Three kinds of lipases (from Candida antarctica, Pseudomonas cepacia, and Pseudomonas fluorescens) were encapsulated in inorganic matrices by the sol-gel method in order to synthesize chiral compounds by kinetic resolution. Sol-gel lipases prepared with vinyltriethoxysilane had higher hydrolysis activity for 2-octyl acetate than those with other silane precursors: tetramethoxysilane, methyltrimethoxysilane, and propyltrimethoxysilane.     

Lipases: Useful biocatalysts for the preparation of pharmaceuticals

Biocatalysis offers a clean and ecological way to perform chemical processes, in mild reaction conditions and with high degree of selectivity. The use of enzymes, specially lipases, in organic solvents proves an excellent methodology for the preparation of single-isomer chiral drugs. This review covers some general aspects and representative examples of the use of lipases for the enantioselective or regioselective preparation of alcohol and amine intermediates in the synthesis of pharmaceuticals.