Biodiesel Synthesis in a Solvent-Free System by Recombinant Rhizopus oryzae Lipase. Study of the Catalytic Reaction Progress

The recombinant 1,3-positional selective Rhizopus oryzae lipase (rROL) was used to synthesize biodiesel and monoacylglycerols simultaneously. The reaction was carried out in a solvent-free system with the enzyme immobilized on octadecyl-Sepabeads. Using response surface methodology, the methyl ester yield was optimized by means of the study of the effect of water, substrate molar ratio (methanol:olive oil) and methanol stepwise addition. It was concluded that in order to prevent enzyme inactivation by methanol, alcohol should be added slowly; otherwise a large amount of water would be present. Taking the best conditions, a 50.3 % yield was achieved in 3 h, which corresponds to 75.4 % of the acyl groups at the 1,3-position undergoing transesterification. It was also concluded that methyl esters result from the esterification of the free fatty acid hydrolyzed by the enzyme and also from a direct transesterification of oil. In addition, the fatty acid selectivity of rROL was found not to favor one fatty acid in olive oil over another.     

Effect of reaction parameters on conversion of krill (Euphausia superba) oil by immobilized lipase ethanolysis

Monoglyceride and diglyceride were produced by performing ethanolysis of krill oil with immobilized lipase and the influence of various parameters on the enzymatic ethanolysis was assessed. As an immobilized lipase, lipozyme TL-IM (thermonuces lanuginose) was used. Ethanolysis was done in non-pressurized and pressurized system to compare the reaction rate and yield. The optimal condition was found at 2.0 of ethanol mole ratio, temperature of 60 °C, lipases amount of 5 wt% in non-pressurized system. At pressurized system the optimal temperature and pressure was found at 50 °C and 10 MPa. However, at 50 °C monoglyceride was higher in pressurized system than in non-pressurized system.     

含离子液体介质中脂肪酶高效催化对羟基苯甘氨酸甲酯不对称氨解

利用离子液体C6MIm·BF4 (20%,v/v)/叔丁醇混合介质中脂肪酶Novozym 435催化对羟基苯甘氨酸甲酯不对称氨解反应,高效地制备了对映体纯D-对羟基苯甘氨酸酰胺.该反应的最适氨源为氨基甲酸铵,其较佳浓度为120～160 mmol·L-1;最适水活度、反应温度、pH值、对羟基苯甘氨酸甲酯浓度分别为0.55～0.75、35～40℃、7.5～8.0和40 mmol·L-1.在上述条件下,反应初速度和对映体选择比分别为5.88 mmol·L-1·h-1和83 (E值),反应6 h,产率为47.6%,产物e.e.值为92.9%.与有机溶剂叔丁醇相比,脂肪酶Novozym 435在C6MIm·BF4 (20%,v/v)/叔丁醇混合溶剂中的操作稳定性显著提高,反应7批次后,仍能维持90%以上的催化活性,而在叔丁醇反应介质中,仅能维持41%的催化活性.     

Production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor

A laboratory-scale continuous reactor was constructed for production of specific structured triacylglycerols containing essential fatty acids and medium-chain fatty acids (MCFA) in the sn-2 and sn-1,3 positions, respectively. Different parameters in the lipase-catalyzed interesterification were elucidated. The reaction time was the most critical factor. Longer reaction time resulted in higher yield, but was accompanied by increased acyl migration. The concentration of the desired triacylglycerol (TAG) in the interesterification product increased significantly with reaction time, even though there was only a slight increase in the incorporation of MCFA. Increased reactor temperature and content of MCFA in the initial reaction substrate improved the incorporation of MCFA and the yield of the desired TAG in the products. Little increase of acyl migration was observed. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA, or on the resulting composition of TAG products and their free fatty acid content. Therefore, we conclude that the water in the original reaction substrate is sufficient to maintain the enzyme activity in this continuous reactor. Since the substrates were contacted with a large amount of lipase, the reaction time was shorter compared with a batch reactor, resulting in reduced acyl migration. Consequently, the purity of the specific structured TAG produced was improved. Interesterification of various vegetable oils and caprylic acid also demonstrated that the incorporation was affected by the reaction media. Reaction conditions for lipase-catalyzed synthesis of specific structured TAG should be optimized according to the oil in use. Presented in part at Food Science Conference, Copenhagen, Denmark, January 30–31, 1997.     

酶法催化生产生物柴油的研究进展

生物柴油是一种可生物降解的、无毒的、清洁的可再生能源,可以部分代替石油柴油.酶法催化生产生物柴油具有反应条件温和、原料来源广泛、无皂化等副反应发生、产物易于分离纯化和设备要求低等优点.对脂肪酶、油脂原料和酰基受体在酶法催化生产生物柴油中的研究应用进行了介绍,指出了酶法催化生产生物柴油中存在的问题,针对酶法催化生产生物柴油难以实现产业化的原因进行了分析,并提出了相应的建议.     

从适应低温的细菌中提取的低温酶（英）

从覆盖有积雪的土壤中的大麻哈鱼肠的螃蟹肠中分离出了新的细菌苗种，并对它们分泌出的淀粉酶、脂肪酶和蛋白酶的性能进行了研究。发现了一种淀粉酶、一种脂肪酶和三种蛋白酶，它们的最佳活性温度明显地朝低温方向移动，而且活化能有所降低。这些酶在高于最代温度（即３０℃￣４０℃）时迅速失活。结果表明，这些酶具有低温活性。低温活性最好的蛋白酶生产者从大麻哈鱼肠中分离出来的菌种通过１６Ｓｒ核糖核酸分析已验明为黄杆菌属，     

Enhanced activities of lipase pretreated with organic solvents

The catalytic activities of lipases derived from Pseudomonas sp and pretreated with various organic solvents were investigated. The activity of the solvent‐pretreated lipase was greater than that of native lipase in both the esterification reaction in an organic medium and the hydrolysis reaction in an aqueous medium. With esterification calalysed by pretreated lipase, the product, benzyl octanoate, was detected without time‐lag. Conversions at equilibrium state were correlated with the hydrophobicities of the solvents used. In the hydrolysis reaction, most pretreated lipases yielded increased acid production compared with native lipase. A linear correlation was observed between the solvent hydrophobicity and the relative initial reaction rate of the hydrolysis reaction when using pretreated lipases. © 2001 Society of Chemical Industry     

Measuring stereoselectivity in lipase-catalyzed acidolysis reactions by ultra-high resolution 13C nuclear magnetic resonance

Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuterium-labeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution ¹³C nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the ¹³C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.