用酶作催化剂合成乙酸异戊酯

研究了用固定化脂肪酶(Rhizomucor miehei酯酶RMIM和Candida antarctica Novozym 435)催化合成乙酸异戊酯，考察了酶的类型和用量、反应时间、温度及摇动速度对酯化反应的影响。     

凹凸棒土固定脂肪酶及其在催化菜籽油转酯反应中的应用

采用吸附法对来源于,Staphylococcus aureus JH的脂肪酶进行了固定化.以凹凸棒土作为载体系统研究了固定化条件对固定化效率及固定化酶转酯活力的影响.结果表明最适加酶量、缓冲液pH和吸附时间分别为0.35 g/g、8.0和3 h,在上述优化条件下固定化酶的转酯活力为465.5 U/g,而所用的游离酶只有较低转酯活性.利用该固定化酶催化菜籽油转酯反应生产生物柴油时,叔丁醇为适宜的反应介质,其最适添加昔为0.8 mL/g;适宜的酶鼍、加水晕和反应温度分别为40.0 U/g、油质量的的1.2%和40℃.按甲醇/油摩尔为3.5的比例在优化反应条件下,反应12 h后甲酯产率达96.0%;固定化脂肪酶具有较好的操作稳定性,反应160批次时,相对酶活力为78.4%.     

非水相脂肪酶催化阿魏酸双甘酯的合成

在有机相和无溶剂反应体系中进行不同来源的脂肪酶催化阿魏酸与油酸甘油酯转酯反应的比较研究。通过筛选发现固定化脂肪酶Novozym435因其独特的催化专一性减少了副产物,提高了目标产物的产率。同时研究了不同反应介质对转酯反应的影响,结果表明在无溶剂反应体系中,Novozym435脂肪酶催化阿魏酸乙酯与油酸甘油酯的转酯反应目标产物得率较高,与甲苯作为反应介质的体系相比酶的操作稳定性得到了明显提高。     

脂肪酶固定化研究和应用

采用硅藻土作载体,进行了脂肪酶的固定化。利用固定化酶选择性催化1-苯乙醇与乙酸乙烯酯的转酯化反应,得到R-乙酸苯乙酯,进行1-苯乙醇的拆分。实验考察了不同吸附方法固定化酶的效果,确定效果最好的固定方法为载体涂布法,并对该法的固定化条件进行了优化。制备的固定化酶的转酯比活比游离酶提高了14．3倍。固定化没有改变酶的选择性．对映体过剩值仍大于98％。初步探讨了固定化酶和游离酶的反应过程。     

离子液体在脂肪酶催化合成生物柴油中的应用发展趋势

从固定化和非水介质两方面介绍了脂肪酶催化合成生物柴油的现状。简述了离子液体作为绿色溶剂的特性及对脂肪酶催化反应的影响,明确使用离子液体可提高酶的稳定性,维持酶的催化活性。综述了离子液体作为潜在的反应介质应用于脂肪酶催化高产生物柴油的前景。     

溶胶-凝胶法固定化Candida sp.99-125脂肪酶

利用正硅酸甲酯(TMOS)和丙基三甲氧基硅烷(PTMS)为复合硅源,以PEG(MW=20000)为稳定剂,以HCl为催化剂,经过溶胶-凝胶过程包埋假丝酵母99-125脂肪酶. 研究得到最适的固定化条件为：PTMS与TMOS的摩尔比4: 1, R值(水与硅源的摩尔比)20, 给酶量(酶占硅源的质量百分数)3.71%, PEG与酶的质量比(1~1.5):1, 硅源水解时间35 min. 在该条件下,固定化脂肪酶的最高酯化活力是游离酶最高酯化活力的2.02倍. 固定化脂肪酶在100℃保温2 h后酶活仍维持为59.1%,固定化酶催化特定酯化反应,经过8批连续反应96 h后酶活维持不变.     

树脂吸附法固定Candida rugosa脂肪酶

Candida rugosa脂肪酶具有优良的催化性能,对其进行固定化可以很方便地实现酶的回收和再利用。采用南开大学化工厂生产的4种阴离子交换树脂和4种大孔吸附树脂为载体,对来源于Candida rugosa的脂肪酶进行了吸附固定化,结果表明,以大孔吸附树脂AB-8为载体的固定酶比活性最高。固定化酶制备过程中缓冲液的最适宜pH值为7.2,最佳固定化时间为1 h,载体和酶的最佳质量配比为10∶1。与游离酶相比,固定化后酶活损失大约30%,但稳定性平均约提高60%。     

酶定向固定化方法及应用的研究进展

传统固定化方法常会导致酶活性大幅度下降,回收率较低,而酶定向固定由于固定化后可完全暴露其活性部位,因而可以保持较高的酶活回收率。本文主要综述了定向固定化酶的两种方法,分别为共价定向固定和非共价定向固定。其中非共价定向固定化主要是抗体与抗原、亲和素/链霉亲和素和生物素以及组氨酸标签与Co2+/Ni2+之间的亲和作用;共价定向固定化主要是通过半胱氨酸残基上的巯基与载体作用。简述了其在生物传感器、分子识别、酶生物燃料电池及酶纯化方面的应用。最后指出今后的主要研究方向为探索新的定向固定化标签以降低对酶活性部位的影响,应用新的载体以提高固定化酶酶活回收率,优化固定化过程及简化固定化步骤等。     

磁性纳米粒子的制备及脂肪酶的固定化

建立了以纳米级磁性粒子为载体固定化脂肪酶的方法,优化了脂肪酶的固定化条件,考察了固定化酶的性质. 制备的磁性载体平均粒径20 nm,具有超顺磁性,分散和再分散效果好. 固定化酶的最适吸附时间为60 min,酶用量:载体量为1:1,固定化酶的酶活达到718 U/g. 结果表明,经纳米磁性粒子固定化后,脂肪酶得到活化,固定化酶比活为游离酶的1.8倍. 同时,固定化脂肪酶的pH稳定性显著提高.     

黏土吸附法固定化脂肪酶的初步研究

为研究天然黏土为载体固定化脂肪酶的可行性,采用羟基化、硅烷化处理,对黏土进行改性,并以此为载体吸附固定化脂肪酶,探讨黏土固定化脂肪酶的条件对酶活及蛋白吸附量的影响,优化固定化脂肪酶条件。研究结果表明:黏土经羟基化、硅烷化改性处理后能显著提高固定化酶活和蛋白固定量,其中硅烷化改性最优;载体固定脂肪酶最优条件为:加酶量50 mg/g,载体粒径180—250μm,pH值为4.0,固定化温度25℃,固定化时间2.0 h;与游离酶相比,固定化酶显示出更广的pH值适应性。黏土固定化脂肪酶重复使用10批次后,仍能保留76.85%的初始活力。以天然黏土为载体固定化脂肪酶,具有较好的实际可应用性及操作稳定性,在较低pH值条件下应用具有一定优势。     

Immobilization of lipase onto metal–organic frameworks for enantioselective hydrolysis and transesterification

Enzyme immobilization enhances the catalytic activity and stability of the enzyme, and also improves reusability. Metal–organic frameworks (MOFs), which possess diversified structures and porosity, have been used as excellent carriers for enzyme immobilization. Pseudomonas fluorescens lipase (PFL) has been successfully immobilized onto MOFs by covalent cross-linking to obtain a series of immobilized lipase (PFL@MOFs). PFL@MOFs are used for catalytic enantioselective hydrolysis of 2-(4-hydroxyphenyl) propionic acid ethyl ester enantiomers (2-HPPAEE) in aqueous medium and transesterification of 4-methoxymandelic acid enantiomers (4-MMA) in organic medium. The experimental results indicated that PFL@Uio-66(Zr) exhibits excellent enzymatic catalysis performances and high enantioselectives. In addition, to improve catalytic activity and reusability, PFL is modified by the polyethylene glycol (PEG) to prepare PEG-modified lipase (PFL-PEG), then PFL-PEG is immobilized onto Uio-66(Zr) to prepare PFL-PEG@Uio-66(Zr), demonstrating better reusability and catalytic activity compared with PFL@Uio-66(Zr).     

一种选择性拆分布洛芬对映体的固定化脂肪酶

为了提高扩展青霉TS414脂肪酶(PEL)对布洛芬的拆分效率,建立了适于非水相中选择性拆分(R,S)-布洛芬的固定化方法. 结果表明,固定化介质的类型、冻干pH和外加水量等因素对固定化PEL酶促拆分(R,S)-布洛芬有较大影响. 在冻干pH为9.0、外加水量为0、以大孔吸附树脂AB-8为固定化载体的体系中,40℃反应30 h后,拆分反应的转化率可达47%,对映体过量值eeP可达98.75%. AB-8固定化后,PEL在有机相反应体系中的分散性得到了明显改善,大幅度提高了酶促拆分反应的效率;大孔吸附树脂AB-8固定化PEL具有较高的操作稳定性,连续10批拆分反应的平均转化率在47%以上,eeP值均稳定在98%以上.     

Immobilization of hydrophobic lipase derivatives on to organic polymer beads

A simple and effective method of lipase immobilization is described. Lipase from Candida rugosa was first modified with several hydrophobic modifiers before being adsorbed on to organic polymer beads. The soluble hydrophobic lipase derivatives adsorbed more strongly on to the various polymers as compared with the native lipase. The optimal adsorption temperature of the native and modified lipases on all the polymers was 40°C. The optimal pH of adsorption was between 6 and 7. Lipase immobilized in this manner produced high catalytic recoveries which were affected by the type of modifiers, degree of modification and type of supports used. Monomethoxypolyethylene glycol (1900) activated with p-nitrophenyl chloroformate was found to be the best modifier of the enzyme at 95% modification, for adsorption to the polymers. Increasing the degree of modification of the enzyme increased the activity which was immobilized. Generally, both native and hydrophobic lipase derivatives showed higher specific activities when immobilized on polar polymers compared with non-polar polymers.     

Preparation of an Immobilized Lipase‐Palladium Artificial Metalloenzyme as Catalyst in the Heck Reaction: Role of the Solid Phase

A p‐nitrophenylphosphonate palladium pincer was synthesized and selectively inserted by irreversible attachment on the catalytic serine of different commercial lipases with good to excellent yields in most cases. Among all, lipase from Candida antarctica B (CAL‐B) was the best modified enzyme. The artificial metalloenzyme CAL‐B‐palladium (Pd) catalyst was subsequently immobilized on different supports and by different orienting strategies. The catalytic properties of the immobilized hybrid catalysts were then evaluated in two sets of Heck cross‐coupling reactions under different conditions. In the first reaction between iodobenzene and ethyl acrylate, the covalent immobilized CAL‐B‐Pd catalyst resulted to be the best one exhibiting quantitative production of the Heck product at 70 °C in dimethylformamide (DMF) with 25% water and particularly in pure DMF, where the soluble Pd pincer was completely inactive. A post‐immobilization engineering of catalyst surface by its hydrophobization enhanced the activity. The selectivity properties of the best hybrid catalyst were then assessed in the asymmetric Heck cross‐coupling reaction between iodobenzene and 2,3‐dihydrofuran retrieving excellent results in terms of stereo‐ and enantioselectivity.

     

Microwave‐assisted immobilization of lipase from Candida rugosa on Eupergit® supports

BACKGROUND: Immobilization of lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from Candida rugosa on Eupergit^® C and Eupergit^® C 250L was performed under microwave irradiation in order to reduce immobilization time. Lipase loading, hydrolytic activity, esterification activity and operational stability in organic solvent of immobilized lipase preparation were determined. RESULTS: The microwave‐assisted procedure resulted in a 29% lower lipase loadings, compared with immobilized lipase obtained without microwaves. In hydrolytic activity assay, lipase immobilized under microwaves exhibited a 23% higher specific activity. Slight activation of lipase by microwave‐assisted immobilization was observed, since specific activity was around 5% higher than for free lipase. Lipase of highest activity was obtained after 2 min immobilization on Eupergit^® C. The same preparation exhibited high esterification activity in organic medium and a half life of 212 h was determined in multiple use assay. CONCLUSION: The application of microwave irradiation leads to reduction of immobilization time from 2 days to only 2 min. The immobilized lipase obtained has prospects for further application due to its high retained activity and stability. Copyright © 2009 Society of Chemical Industry     

Catalytic Behaviour of Lipase Free and Immobilized in Biphasic Membrane Reactor with Different Low Water-Soluble Substrates

The catalytic activity and reaction rate of lipase have been studied using the biocatalyst free in an organic/aqueous emulsion and immobilized in a biphasic organic/aqueous membrane reactor. The first reaction system was realized in a stirred tank reactor. The other was obtained by immobilizing the enzyme in the sponge layer of an asymmetric capillary membrane and recirculating the two phases along the two separate circuits of the membrane module. The performance of the reactors has been studied using two different low water-soluble substrates: triglycerides present in commercial olive oil and (R,S)-cyanomethyl-[2-(4-isobutylphenyl)propionate] (CNE). The effects of substrate viscosity and flow dynamics conditions, such as organic phase flow rate, on the biphasic membrane reactor performance have been evaluated on the basis of observed reaction rate and catalytic activity of free and immobilized lipase for both substrates. It has been observed that free lipase showed higher catalytic activity with olive oil, while immobilized lipase showed higher catalytic activity with CNE which has a lower viscosity than olive oil. The increase of organic phase flow rate negatively affected the reactor performance, with a minor effect when using CNE rather than olive oil. The influence of temperature on the biocatalyst performance with the two substrates has also been investigated. The optimal temperature value of lipase was different for the two substrates: 28°C with CNE and 40°C with olive oil. © 1997 SCI.     

Biodiesel production using immobilized enzyme-current state and future perspective

近年来由于化石燃料的全球性短缺、原油价格的过度上涨和环境问题的加剧致使生物柴油的生产迅速增长。生物柴油生产的关键反应是化学或生物催化的转酯反应,酶催化的转酯反应与化学催化相比有相对节能、副产物甘油易回收及适合高含量游离脂肪酸油脂作为底物等明显优势。本文综述了固定化脂肪酶生产生物柴油的现状及最新进展,包括生物柴油的原料、脂肪酶的来源、酶的固定化技术、甲（乙）醇及甘油对脂肪酶的失活作用,展望了固定化脂肪酶生产生物柴油的未来前景。     

Carbonaceous–siliceous composite materials as immobilization support for lipase from Alcaligenes sp.: Application to the synthesis of antioxidants

Two carbonaceous–siliceous composite materials, produced by hydrothermal and carbonization processes, were evaluated as immobilization support for lipase from Alcaligenes sp. These materials exhibited similar chemical characteristics but their carbon content and porous characteristics were different, which explain the catalytic behavior and stability of the biocatalysts immobilized on them. Higher activity and immobilization selectivity was achieved with the microporous material that had higher carbon content. The lipase immobilized on the mesoporous material had a higher thermal stability at 55 °C, pH 7.0 or at 40 °C in tert-butanol, simulating the reaction conditions required for organic synthesis. Both biocatalysts were tested in the synthesis of palmitoyl ascorbate and they were compared with the commercial biocatalyst QLC. The synthesis conversions with the lipase immobilized in mesoporous materials and with the biocatalyst QLC were similar (50%), but only the former could be reused. These are promising biocatalysts for industrial applications.