离子液体中酶催化反应的研究进展

讨论了近年来离子液体作为酶催化反应介质的应用发展。说明了不同类别的酶在离子液体或水合离子液体两相体系中具有催化活性，尤其是脂肪酶，在无水的离子液体介质反应体系中不仅能保持很好的活性，其手性选择性和操作稳定性往往优于传统介质中的表现。指出了离子液体的环境相容性使其成为绿色生物反应工程新的应用溶剂。对不适于在传统溶剂体系中进行的生物催化再生和产物回收研究，离子液体溶剂已经逐渐表现出其优势。     

反胶束技术在生物工程中的研究进展

本文综述了反胶束萃取的基础理论研究,反胶束作为酶催化反应介质的优点,酶在反胶束中的催化活性和稳定性以及反胶束技术的应用。     

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。     

修饰固定化青霉素酰化酶在非水相中的催化

为提高酶的催化水解活力和稳定性,将青霉素酰化酶组装于介孔泡沫二氧化硅(MCFs)中,并应用于水/有机混合体系催化水解。分别考察了有机介质种类和体积分数、葡聚糖(Dex10k)修饰对固定化酶活力的影响,研究了不同条件下固定化酶的稳定性。实验结果显示:体积分数20%石油醚中,添加Dex10k的介孔泡沫硅固定化酶比活力达209.5U/mg,是缓冲液中MCFs固定化酶活力的196.2%。20%石油醚中,经25次连续操作,固定化酶保持初始活力的71.5%。结果表明:石油醚等烷烃形成的水/有机体系是适合青霉素酰化酶催化的二相体系,且添加Dex10k能提高固定化酶在二相体系中的催化活力及稳定性。     

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。     

甲醇对NS81006催化大豆油制备生物柴油的影响研究

游离脂肪酶催化法以其酶催化剂生产成本较低,反应速率较快的优点,成为生物柴油制备的新的关注方向.游离脂肪酶NS81006可以有效催化大豆油甲醇解制备乍物柴油,但其催化活性在反应后期下降,甲醇可能对NS81006催化活性及回用稳定性造成影响.研究了甲醇对NS81006酶活的影响以及不同甲醇添加策略对NS81006催化效果的影响.研究结果表明,反应体系中过多甲醇的存在对NS81006酶活产生负而影响;通过减少甲醇添加量和缩短甲醇与NS81006作用时间可有效降低酶活损失,提高NS81006回用稳定性;在优化策略下,NS81006连续使用6个批次,生物柴油得率保持90%以上.     

生物柴油制备过程中磷脂及水分含量对脂肪酶的影响

用叔丁醇作为反应介质,利用固定化脂肪酶催化油脂原料甲醇解反应制备生物柴油,消除了甲醇和甘油对酶的负面影响,酶的使用寿命显著延长,进一步系统研究了以叔丁醇作为反应介质,磷脂及水含量对脂肪酶催化特性的影响。当体系中中含水量达2%以上或磷脂含量达0.1%油重时将对固定化脂肪酶LipozymeIM TL 和Novozym 435的催化活性造成明显的不利影响。同时还发现把脂肪酶LipozymeIM TL 和 1%Novozym 435混合起来能大大提高脂肪酶的耐水、耐磷脂能力。     

木质素降解产物用于漆酶-介质体系的结构理论研究

为从天然产物中寻找廉价、低毒、高效的漆酶新介质,利用分子模型和分子对接技术在分子水平研究木质素降解产物在漆酶-介质体系中与漆酶活性位点的结合模式与反应能力,以探明其结构特征。结果表明,10种木质素降解产物小分子介质酚羟基邻位取代基可保持与漆酶作用过程中介质构象的稳定,邻位取代基的给电子能力可增强介质自由基中间体的稳定性,介质酚羟基对位取代基的吸电子性有利于加强与Phe265间的π-π堆积作用,但对位取代基吸电子性过强反而不利于提高介质反应活性及苯氧自由基稳定性。该研究为新介质的发现及介质结构的改造与修饰奠定了理论基础与研究方向。     

伴生介质漆酶的生产优化及其酶学特性研究

为了缓解人工合成漆酶介质价格昂贵和环境污染的问题,利用血红密孔菌NFZH-1制备伴生介质漆酶,以实现漆酶介质体系(LMS)的工业化应用。通过优化菌株NFZH-1液态培养基和发酵条件,提高了伴生介质漆酶产量,并揭示了其酶学特性。结果表明:血红密孔菌NFZH-1为伴生介质漆酶高产菌株,经产酶优化,漆酶活力提高了91%,达26.1 U/m L。伴生介质漆酶在50～85℃和p H2.5～4.5范围内具有较高的漆酶活力,在<60℃和p H5.0～7.0范围内具有较好的漆酶稳定性,属耐热型漆酶。高产、稳定的伴生介质漆酶对LMS的工业化应用具有重要意义。     

非水介质中PA修饰对辣根过氧化物酶性质的影响

研究了二甲基甲酰胺（DMF）和乙腈（AcN）有机溶剂水混合体系中邻苯二甲酸酐（PA）修饰对辣根过氧化物酶（HRP）催化性质的影响。研究结果表明,PA修饰HRP可以提高该酶在有机溶剂中的稳定性,且有机溶剂的浓度越高PA修饰对HRP稳定性的提高越明显,在50％的DMF和ACN有机溶剂中PA修饰HRP的催化活性分别比HRP高25％和19％。在10％有机溶剂中的动力学数据表明PA修饰HRP可以提高该酶在有机溶剂中对酚类化合物的亲和性和专一性。     

Preparation of glucose oxidase immobilized in different carriers using radiation polymerization

Glucose oxidase (EC 1.1.3.4) was immobilized on different polymeric materials using different immobilization techniques (entrapping by γ‐irradiation, and covalent binding using epichlorohydrin). Studies were carried out to increase the thermal stability of glucose oxidase (GOD) for different applications. The activity and stability of the resulting biopolymers have been compared with those of free GOD. The effect of different polyvinyl alcohol/polyacrylamide (PVA/PAAm) compositions of the copolymer carrier on the enzymatic activity of the immobilized GOD was studied. The maximum enzymatic activity was obtained with the composition ratio of PVA/PAAm of 60:40. The behaviour of the free and immobilized enzyme was analysed as a function of pH. A broadening in the pH profile (5.5–8) was observed for immobilized preparations. The activity and stability of the resulting biopolymers produced by immobilization of GOD onto different carriers have been compared, in both aqueous and organic media, with those of the free GOD. The enzyme's tolerance toward both heat and organic solvent was enhanced by immobilization onto polymers. The addition of different concentrations of organic solvents (10–50%, v/v) to the enzyme at higher temperature (60 °C) was found to stabilize the enzyme molecule. The strongest stabilizing effect on the enzymatic activity was achieved at a concentration of 10%. Copyright © 2005 Society of Chemical Industry     

Biocatalytic ester synthesis in ionic liquid media

Ionic liquids have shown potential as green reaction media compared with organic solvents, mainly due to their lack of vapour pressure. In non‐aqueous enzymology, ionic liquids are opening up new fields. The advantages of using ionic liquids over the use of organic solvents as reaction medium for biocatalysis include enhancement of enzyme activity, stability and selectivity. In this work, the enzymatic synthesis of esters in ionic liquids has been extensively reviewed. Numerous examples of the application of ionic liquids as reaction medium for the enzymatic production of esters have been included. The effect of the nature of the ionic liquid on activity, selectivity and stability of enzymes which catalyze esters synthesis has been carefully analysed. Innovative reaction methodologies for the biosynthesis of esters, including ionic liquid/supercritical carbon dioxide biphasic systems and the integrated reaction/separation processes using supported liquid membranes based on ionic liquids have been revised. Copyright © 2010 Society of Chemical Industry     

非水介质酶促反应中水的作用

对非水介质酶促反应体系中水的作用以及相关因素进行了初步总结 ,着重讨论了水在体系中的分配及影响因素、水的分配平衡速度、水活度的测定及控制方法和水作为反应物的动力学影响。     

Candida sp. 99-125脂肪酶及其在化学品合成中的应用

传统的酯化或转酯化产品的合成通常需要高温、强酸、强碱等相对苛刻的条件,脂肪酶由于其生物催化过程具有高效、高选择性、条件温和和环境友好等特点,在化学品的合成中越来越受到人们的关注。本课题组开发了一种可以用于酯类合成的新脂肪酶,并且实现了该酶的工业化生产。来源于Candida sp. 99-125的脂肪酶在非水相中对酯化和转酯化反应具有高效的催化活性和稳定性。本文介绍了该脂肪酶的发酵生产及其在中长链脂肪酸酯、二元酸酯、维生素A棕榈酸酯、手性化合物以及生物柴油等多种化学品的合成中的应用。     

整合型酿酒酵母菌株强化发酵和特性比较

为了提高木糖异构酶基因在重组酿酒酵母体内的稳定性,并比较不同真菌来源的木糖异构酶在木糖或者葡萄糖-木糖培养基的发酵利用特性,分别构建来自Piromyces sp.E2和Orpinomyces sp.的木糖异构酶基因的整合表达载体,利用同源重组将其整合进入呼吸缺陷型菌株的18S rDNA非转录区,结果测得Orpinomyces的木糖异构酶酶活活力为0.72 U/mg,比Piromyces的木糖异构酶酶活高2.8倍。在木糖培养基中发酵获得乙醇的得率分别为0.40 g/g和0.48 g/g。且整合入Orpinomyces的木糖异构酶基因菌株能获得最高酶活和乙醇得率。     

Multivariate Analysis of the Stability of Spray-Dried Eupenicillium javanicum Peptidases

Peptidases occupy a central position in the enzyme market because of their importance in many areas, such as for physiological processes, foods, and detergents, as well as in the pharmaceutical, leather, and biotechnology industries. Microbial production is among the major sources of peptidases because it presents many advantages when compared with other methods. In this study, the metallopeptidases produced by the fungus Eupenicillium javanicum under a solid-state fermentation bioprocess were spray-dried. The enzymatic extract was dried using drying adjuvants, and optimal conditions for preserving enzymatic activity were studied following a Box-Behnken experimental design. The spray process factors studied were the air-drying temperature, enzyme feed flow rate, and the proportion of enzyme/additive. The responses analyzed were the dry extract yield and enzymatic activity after spray drying. Additionally, the stability of the dry extracts was assessed during 180 days at 4°C and 25°C. The results revealed extract yields of up to 66.12% and good enzymatic activity for intermediate values of temperature and adjuvant proportions. Furthermore, the dried enzymatic extracts showed potential for future commercial applications because of their stability at 25°C for 180 days.     

INFLUENCE OF ENZYMATIC HYDROLYSIS ON THE FUNCTIONAL PROPERTIES OF SESAME CAKE PROTEIN

This study investigated the effect of enzymatic hydrolysis on the functional properties of sesame cake protein. Protein hydrolyzates from sesame cake protein were obtained by enzymatic hydrolysis using Alcalase at 50°C and pH 8.5. Water-holding capacity, oil-holding capacity, foam capacity and stability, and emulsifying activity and stability of the hydrolyzates were determined. All protein hydrolyzates showed better functional properties than the original protein. Foaming and emulsifying stability decreased as the degree of hydrolysis increased. The water-holding capacity, foaming activity, and emulsifying activity of the hydrolyzates increased with the increase in levels of hydrolysis. Enzymatic modification was responsible for the changes in protein functionality. These improved functional properties make sesame cake protein hydrolyzates a useful product in foods such as bread, cake, ice cream, meat products, desserts, and salad dressing.     

Study on the activity and stability of urease immobilized onto nanoporous alumina membranes

Nanoporous alumina membranes were employed as substrate materials for urease immobilization. Anodic porous alumina was prepared by the two-step anodization of high purity aluminum. By controlling anodization conditions, the nanoporous structure with desired dimension was obtained. Urease immobilization onto nanoporous alumina membranes was performed by four different protocols. Effect of pore diameter, pore length and immobilization methods on the activity and stability of immobilized enzyme was discussed in detail. The results show that the enzymes immobilized onto porous alumina with big pore diameter possess high activity and poor stability as compared to small pore diameter. The effect of pore length is complicated, the activity of enzyme increases with the increasing pore length for big pore size; while for correspondingly small pore size, enzymatic activity slightly depends on pore length. The immobilization methods have a slight effect on enzymatic activity, whereas enzyme immobilization by chitosan coating and reticulation with glutaraldehyde exhibits a good long-term stability as compared to that only via physical adsorption.     

小球藻在蔗渣酶解液中的异养生长及其脂肪酸生成

在高温液态水处理的甘蔗渣酶解过程中添加Tween80可使聚糖转化率提高11.4%。根据蔗渣酶解液中糖的种类及含量,用葡萄糖、木糖和纤维二糖标准品模拟蔗渣酶解液组成配制成相应的混合糖培养基,同时配制仅含葡萄糖的培养基,在有、无Tween80和BG11(Blue-Green 11)的条件下,考察小球藻在不同培养基中的异养生长及脂肪酸生成。结果显示Tween80对小球藻的生长具有抑制作用,纤维二糖也会影响小球藻的生长;小球藻在添加BG11的葡萄糖培养基中的生物量最高,为1.97 g·L^-1,在添加BG11的蔗渣酶解液中的生物量高出未添加BG11的2倍,在含有Tween80和BG11的蔗渣酶解液中的总脂肪酸含量最高,达到6.90%,在所有培养基中产生的脂肪酸以C_16:0、C_18:1、C_18:3、C_20:1和C_20:4为主;培养基组成优化可进一步提高微藻生物量和油脂产量。     

Lipase-Catalyzed Esterification

Lipases are versatile catalysts. In addition to their natural reaction of fat hydrolysis, lipases catalyze a plethora of other reactions such as esterification, amidation, and transesterification of esters as well as organic carbonates. Moreover, lipases accept a wide variety of substrates while maintaining their regioselectivity and stereoselectivity. Lipases are highly stable even under adverse conditions such as organic solvents, high temperatures, and so forth. Applications of lipases include production of food additives, chiral intermediates, and pharmaceutical products. Among these, synthesis of various chiral intermediates in pharmaceutical industry and cocoa butter substitutes is being commercially exploited currently.

Lipase-catalyzed esterification and transesterification in anhydrous media (e.g., organic solvents and supercritical fluids) has been an area of major research activity in the past decade or so. Absence of water eliminates the competing hydrolysis reaction. Moreover, substrate specificity, regioselectivity, and stereoselectivity of the enzyme can be controlled by varying the reaction medium. Although organic solvents, which are generally used for lipase-catalyzed reactions, are nearly anhydrous; they contain water in trace quantities. This water content can be controlled over a range and has a profound effect on the activity of lipases. Water not only affects the enzyme but also acts as a competing nucleophile. Enzyme activity has been correlated with thermodynamic activity of water in the medium rather than with the concentration of water. Because lipases are not soluble in most organic solvents, the method of preparation of the enzyme has a strong influence on the enzymatic activity. The major factors are the pH of the aqueous solution in which the enzyme last existed, additives used during preparation, and method of removal of water (e.g., freeze-drying, evaporation, extraction of enzyme into solvent, etc.). Immobilization of lipases allows easy recovery and reuse of the enzyme. Various immobilization techniques have been studied for lipases and some of them have been shown to enhance the activity and stability of the enzyme. Enzyme stability is an important parameter determining the commercial feasibility of the enzymatic process. Various factors, such as temperature, reaction medium, water concentration, as well as the method of preparation, affect the stability of the lipases.

This review deals with fundamental as well as practical aspects of lipase catalysis. A discussion has been presented on various factors affecting lipase activity and stability. Moreover, a brief account of current and potential applications of lipases has been given.