生物柴油是由动植物油脂转化得到的环保型可再生能源。生物酶法合成生物柴油具有反应条件温和，无污染物排放等优点，是近年来的研究热点。目前生物酶法合成生物柴油没有实现工业化，其主要瓶颈在于反应过程中酶易失活，使用寿命太短，从而导致生产成本过高。针对传统酶法工艺的瓶颈问题，本论文提出了采用新型酰基受体乙酸甲酯与油脂酯交换反应制备生物柴油的新方法，并系统研究了脂肪酶在催化油脂转化为生物柴油反应体系中的催化特性。 首先考察了以甲醇作为酰基受体时酶促油脂转化为生物柴油过程中脂肪酶的催化活性和稳定性。在该反应体系中，甲醇难溶于油脂，局部甲醇浓度过高导致酶易失活，副产物甘油易吸附在固定化酶表面，影响酶的活性和稳定性。采用分步加入甲醇法并用有机溶剂冲洗酶上吸附的甘油，可在一定程度上减缓酶的失活，但回用过程中酶活性仍出现明显下降，回用10次酶活性下降10%。 在进一步研究中，针对传统酶法工艺中脂肪酶易失活，酶使用寿命短等问题，本论文提出了采用新型酰基受体乙酸甲酯和油脂酯交换反应合成生物柴油的新方法。在优化的反应条件下，生物柴油得率可达90%；酶的使用寿命显著延长，回用近180批次酶活性未见下降。以乙酸甲酯作为反应酰基受体，脂肪酶可以有效转化廉价的废油脂（如高酸价废油）进行生物柴油的制备，而且在操作过程中脂肪酶表现出较好的操作稳定性；而在常规醇解工艺中，高酸价废油中的游离脂肪酸与甲醇酯化生成大量的水，酶容易聚集成团，活性迅速下降。 论文作者采用油脂、脂肪酸甲酯或疏水性有机溶剂对脂肪酶浸泡预处理，可有效提高酶催化活性。论文研究了油脂中关键组分磷脂和游离脂肪酸对酶活性的影响，发现磷脂对不同脂肪酶的影响不同；油脂中含游离脂肪酸时，脂肪酸比中性油脂更易被转化为生物柴油；加入固体缓冲剂可进一步提高酶促高酸价油脂与乙酸甲酯酯交换反应速率。 最后论文还对酶促油脂与乙酸甲酯酯交换反应动力学进行了探讨。脂肪酶催化甘油三酯与乙酸甲酯的酯交换反应包含三步连续可逆反应，反应动力学符合乙酸甲酯抑制的乒乓BiBi机制。本文分别建立了中性油脂、含磷脂油脂和高酸价油脂的酯交换反应动力学模型，考察了油脂成分对酶促反应动力学的影响。
Biodiesel fuel (fatty acid methyl esters) originating from vegetable oils and animal fats has received considerable attention in recent years as a renewable, biodegradable, and non-toxic fuel. The production of biodiesel fuel by enzymatic method has attracted great interests because of its waste-free process. However, enzymatic methods have not been industrialized because of the high price and the instability of enzymes. A novel route for biodiesel production was developed in this thesis in which methyl acetate was used as acyl acceptor. The catalytic performances of the lipases were investigated systematically.In the conventional enzymatic process for biodiesel production, short chain alcohol (methanol) was used as acyl acceptor. However, methanol denatured the lipase to some extent and glycerol, as one of the products was easily adsorbed on the surface of lipase resulting in serious negative effect on the reaction. In order to slow the inactivation of the lipase, some attempts such as stepwise methanolysis and glycerol removal by solvent washing were adopted in this study, however, the enzymatic activity decreased inevitably during the repeated uses. In order to overcome the bottlenecks of conventional enzymatic process, a novel route for biodiesel production was developed in which methyl acetate was used instead of methanol as acyl acceptor. Methyl acetate didn’t inactivate the lipase and no glycerol was produced in the process, so it was very convenient to recycle the lipase without any additional treatment. Under the optimized conditions, the biodiesel yield reached 90%；the immobilized lipase could be repeatedly used for more than 170 times with neglectable decrease in enzymatic activity. Furthermore, different oils or fats were successfully converted to corresponding fatty acid methyl esters by this novel route. Even in the transformation of some cheap waste oils containing large amount of free fatty acids, the lipase could still maintain high activity during the repeated uses; while during the conventional methanolysis, the esterification of free fatty acids and methanol produced large amount of water, which would cause the aggregation of lipase and the enzymatic activity decreased seriously.Immersing with oils, biodiesel or hydrophobic solvents could enhance enzymatic activity dramatically. Components of oils showed several effects on enzymatic activity: phosphilipids inhibited Novozym435 to some extent, while could be transformed by LipozymeIM TL and LipozymeIM RM; free fatty acids were easier to be converted to methyl esters than neutral lipids; the addition of Tris or HEPES/HEPES•Na buffer could effectively enhance enzymatic activity during the lipase-catalyzed transformation of waste oil with high acid value.A kinetic model based on mass balance of three second-order reversible reactions was proposed since it was observed that three consecutive and reversible reactions occurred during the interesterification of triglycerides and methyl acetate. The lipase-catalyzed interesterification was found to obey Ping-Pong Bi-Bi mechanism with substrate inhibition of methyl acetate. In the further study, the reaction kinetics models of the interesterification of neutral glycerides, triglycerides containing phospholipids and oils containing free fatty acids were developed, respectively.
