非水相脂肪酶催化有机硅醇的酯化反应

非水相酶催化是酶工程研究热点之一。本文介绍了来自Ｃ．ｃｙｌｉｎｄｒａｃｅａ的脂肪酶催化有机硅醇和脂肪酸的酯化反应。该酶可催化有机硅醇与脂肪酸的酯化反应,并对不同链长的脂肪酸底物、有机溶剂极性及水含量等进行了初步研究。     

有机溶剂中固定化脂肪酶催化硅醇的酯化反应

固定化Mucor miehei脂肪酶可催化有机硅醇和脂肪酸的酯化反应．对固定化酶用量、脂肪酸链长、不同有机硅醇底物、有机溶剂极性和水含量等影响因素进行了初步研究．     

有机相中酶促有机硅烷醇的转酯

在有机介质中,脂肪酶L-1754可以催化非天然有机硅烷醇与脂肪酸酯的转酯反应.反应介质、反应体系水活度和底物的结构对酶促转酯反应有显著的影响.适宜的反应介质为正辛烷,反应体系最适水活度为0.55.     

有机相中脂肪酶L—1754促有机硅烷醇的酯化

在有机介质中脂肪酶Ｌ－１７５４催化非天然有机硅化合物三甲基硅烷醇酯化反应的速度高于Ｌｉｐｏｚｙｍｅ。Ｌ－１７５４对脂肪酸链长有很强的特异性且不同于Ｌｉｐｏｚｙｍｅ。     

有机相中固定化脂肪酶促有机硅烷醇的转酯

探讨了有机相中固定化脂肪酶（Lipozyme）催化非天然的有机硅院醇与脂肪酸酯转酯反应的可能性;系统地研究了有机溶剂特性、水活度、有机硅烷醇结构、脂肪酸酯碳链长等因素对转酯反应的影响。     

有机相脂肪酶催化酮基布洛芬的立体选择性酯化*

从５种脂肪酸中筛选出了具有较高催化活性和对映体选择性的脂肪酶Ｎｏｖｏｚｙｍ ４３５。进一步探讨了酶浓度、底物结构、底物浓度等因素对脂肪酶拆分酮基布洛芬（Ｋｅｔｏｐｒｏｆｅｎ）的影响。结果表明,以１０ｍＬ除水环己烷为反应介质,酶浓度为 ５ｍｇ／ ｍＬ, Ｎｏｖｏｚｙｍ ４３５催化 ６．７ｍｍｏｌ／ Ｌ Ｋｅｔｏｐｒｏｆｅｎ与２６．８ｍｍｏｌ／ Ｌ丙醇进行酯化反应,反应 ３０ｈ,转化率为 ６８％时,Ｓ－酮基布洛芬对映作过剩值可达 ９２％。     

脂肪酶催化合成生物柴油的研究

生物柴油是用动植物油脂或长链脂肪酸与甲醇等低碳醇合成的脂肪酸甲酯,是一种替代能源。这里探讨了生物法制备生物柴油的过程,采用脂肪酶酯化和酯交换两条工艺路线进行催化合成。深入研究制备过程中,不同脂肪酶、酶的用量和纯度、有机溶剂、低碳醇的抑制作用、吸水剂的作用、反应时间和进程、底物的特异性和底物摩尔比等参数对酯化过程的影响。试验结果表明,采用最佳酯化反应参数和分批加入甲醇并用硅胶作脱水剂的工艺过程,酯化率可以达到92％,经分离纯化后的产品GC分析的纯度可达98％以上,固定化酶的使用半衰期可达到360h。同时对酯交换制备生物柴油过程中,甲醇的用量和甲醇的加入方式对脂肪酶催化过程的影响作了初步研究,优化后的酯交换率可达到83％。     

利用麻风树油驯化筛选高活性脂肪酶产生菌及其催化酯化反应

以1株分解麻风树油的脂肪酶产生菌Pseudomonas sp. LP-1为出发菌株, 通过麻疯树油定向驯化筛选获得1株酶活较高且产酶稳定的菌株P. sp. X-2-45, 其水解酶活为29.79 U/mL, 比原始菌株提高了288%。对P. sp. X-2-45生长与产酶特征、对植物油脂水解能力及在有机相中催化脂肪酸和有机醇间的酯化反应研究发现, 该菌株生长速率和产酶速率明显加快, 培养30 h时生物量和酶活达到最大, 稳定期延长, 培养过程中脂肪酶在培养基中的稳定性提高。以麻疯树油诱导合成的P. sp. X-2-45脂肪酶对麻疯树油的水解能力比原始菌株提高了378%, 说明采用麻风树油定向驯化可提高脂肪酶对相应底物的水解能力。X-2-45脂肪酶可以催化月桂酸与正丁醇、正辛醇、月桂醇和丙三醇之间, 棕榈酸、硬脂酸与甲醇、正辛醇、月桂醇和丙三醇之间, 油酸与甲醇、正丁醇、正辛醇、月桂醇和丙三醇之间发生酯化反应。     

非水介质中脂肪酶催化亚油酸油醇酯合成的研究

利用实验室自制及购买的几种脂肪酶制剂催化的酯化反应来制备亚油酸油醇酯,其中本实验室制备的丝孢酵母脂肪酸酯化效果最好,作为进一步研究的实验用酶。以正己烷为反应溶剂,在微水系统中对影响亚油酸油醉酯合成的各种因素进行了研究,确定酯化反应合成的最适温度为３５℃,０～１００℃反应１０ｈ的酯化率均可达到９０％,最适酯化ｐＨ为８．０,最适底物浓度为０．２５ｍｏｌ／Ｌ,最适水含量为０．０５％,在选用的１１种有机溶剂中,以环己烷的酯化率最高,二甲亚砜最低。     

有机相中吸附态固定化脂肪酶催化月桂酸与2—辛醇酯化反应的催化性能研究

本文研究了有机相中吸附在载体6201,102,分子筛、硅藻土、玻璃微珠等上的脂肪酶催化月桂酸与2—辛醇酯化反应的催化活力,关联出载体吸附水量与其固定化脂肪酶活力的关系。并考察了系统含水量、缓冲溶液的pH值、离子强度、反应温度、底物浓度、溶剂等因素对固定化脂肪酶催化活力的影响。在有机相中各最适条件下,载体的吸附性越强,其吸附的脂肪酶催化酯化反应的活力也越高。     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

Fatty acid esterification using nylon-immobilized lipase

The esterification of a long-chain fatty acid was conducted using a nylon-immobilized lipase from Candida cylindracea in a nearly anhydrous, nonpolar organic medium, hexane. Butyl laurate was produced from lauric acid and n-butanol at a maximum initial reaction rate of 37 mmol/h. g immobilized enzyme when the substrates were present in equimolar amounts at an initial concentration of 0.5 mol/L. Lower rates were obtained using nonstoichiometric amounts of the substrates. The rate of reaction increased with temperature, reaching a maximum between 35 and 45 degrees C and decreasing sharply at higher temperatures. (c) 1995 John Wiley & Sons, Inc.     

Hydrolysis of menhaden oil by a Candida cylindracea lipase immobilized in a hollow-fiber reactor

A lipase from Candida cylindracea immobilized by adsorption on microporous polypropylene fibers was used to selectively hydrolyze the saturated and monounsaturated fatty acid residues of menhaden oil at 40 degrees C and pH 7.0. At a space time of 3.5 h, the shell and tube reactor containing these hollow fibers gives a fractional release of each of the saturated and monounsaturated fatty acid residues (i.e., C14, C16, C16:1, C18:1) of ca. 88% of the corresponding possible asymptotic value. The corresponding coproduct glycerides retained over 90% of the initial residues of both eicosapentaenoic (EPA; C20:5) and docosahexaenoic (DHA; C22:6) acids. The half-life of the immobilized lipase was 170 h when the reactor was operated at the indicated (optimum) conditions. Rate expressions associated with a generic ping-pong bi-bi mechanism were used to fit the experimental data for the lipase catalyzed reaction. Both uni- and multiresponse nonlinear regression methods were employed to determine the kinetic parameters associated with these rate expressions. The best statistical fit of the uniresponse data was obtained for a rate expression, which is formally equivalent to a general Michaelis-Menten mechanism. After reparameterization, this rate expression reduced to a pseudo-first-order model. For the multiresponse analysis, a model that employed a normal distribution of the ratio of Vmax/Km with respect to the chain length of the fatty acid residues provided the best statistical fit of the experimental data.     

Characteristics of Lipase Catalysis During Ester Synthesis in Reversed Micellar Systems

The ability of lipase from Candida cylindracea to catalyze ester synthesis from a long chain fatty acid (palmitic acid) and alcohols of varying chain length, is examined. The enzyme is located in the minimal-water environment of reversed micelles. Lipase activity is a strong function of the mode of encapsulation. Direct solid lipase addition to reversed micelles leads to encapsulation in an inactive state unless the enzyme is contacted with the acyl substrate. The alcohol inhibits activity, with low molecular weight alcohols tending to denature the enzyme. Implications to reversed micelle based biocatalyst preparation are briefly discussed.     

Enzymatic synthesis of esters using an immobilized lipase

Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from 25 to 37 degrees C. Ethylpropionate was formed from ethanol and propionic acid at a rate of 0.017 mol/h g immobilized protein. Different esters were formed at comparable rates and equilibrium conversions could generally be approached in less than 10 h in a batch reaction system. The immobilized lipase catalyst was quite stable and retained about one third of the initial activity after repeated experiments during the course of 72 days. A stirred tank continuous flow reactor was used successfully for the continuous production of esters.     

Substrate selectivity of lipases in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the transesterification of cis/trans-isomers of linoleic acid methyl ester

The substrate selectivity of several microbial lipases has been examined in the esterification of oleic acid, linoleic acid, linolenic acid and their all-trans-isomers and in the alcoholysis of isomeric linoleic acid methyl esters with n-butanol. Lipases from Candida cylindracea and Mucor miehei preferred fatty acids and methyl esters with a (first) cis double bond in 9-position, while Chirazyme L-5, a Candida antarctica lipase A, had a preference for trans-9 unsaturated substrates.     

Diacylglycerol lipase and kinase activities in rat brain microvessels

Diacylglycerols can accumulate transiently in intact cells as a consequence of the degradation of phosphatidylinositol by phospholipase C, but little information is available concerning their metabolic fate in the vascular endothelium. Diacylglycerol lipase and kinase activities were measured in rat brain microvessel preparations. Lipase activity, measured by the release of free fatty acids, was much greater at pH 4.5 than at pH 7. The acid lipase was predominantly particulate and likely originated in lysosomes, whereas the neutral lipase was mainly soluble. The fatty acid at the sn-1 position of the diacylglycerol substrate was hydrolyzed faster than that at the sn-2 position at both pH 4.5 and 7. The 2-monoacylglycerol accumulated at pH 4.5 but not at 7 due to the presence of a monoacylglycerol lipase activity with a neutral pH optimum. The formation of phosphatidic acid (kinase activity) was also measured in microvessels. When lipase and kinase activities were measured simultaneously, the formation of phosphatidic acid from a 1-palmitoyl-2-[1-14C]oleoyl-sn-glycerol substrate was 4-fold greater than the release of fatty acid (oleate) from the sn-2 position. Introduction of arachidonic acid to the sn-2 position of the diacylglycerol substrate increased kinase activity but reduced lipase activity. The release of fatty acids from the sn-2 position of phosphatidic acid could not be detected.     

Cloning and nucleotide sequences of two lipase genes from Candida cylindracea.

Two lipase-encoding genes (LIP1 and LIP2) have been isolated from a SacI genomic library of the yeast Candida cylindracea and their nucleotide sequences have been determined. Comparison with the sequence of a cDNA ruled out the presence of introns in the two genes. Both ORFs encode for mature proteins of 534 residues with putative signal peptides of 15 and 14 amino acids, respectively. When compared with other lipase sequences, the two C. cylindracea lipases showed homology only with the Geotrichum candidum lipase, whereas they shared a significant similarity with several esterases.