中文摘要：

【目的】探讨复合酶协同催化体系在含水量较高的体系中催化油脂制备生物柴油的工艺条件。【方法】通过基因工程手段在毕赤酵母中分别高效分泌表达南极假丝酵母脂肪酶（CALB）和米根霉脂肪酶（ROL）,构建CALB和ROL复合酶协同催化体系制备生物柴油,利用单因素实验优化工艺条件,以甲酯化得率作为复合酶协同催化体系效能的评价标准。【结果】优化工艺条件为：CALB？ROL最佳复合酶配比为7？3,每克大豆油中加入16 U的复合脂肪酶,甲醇与大豆油摩尔比为4？1,并按0 h时2？1醇油摩尔比,12 h和24 h时以1？1醇油摩尔比分批加入甲醇,含水量为30%-60%之间,40°C反应29-34 h,甲酯得率达到93%。【结论】该复合酶协同催化体系对环境友好,与常规酶法制备生物柴油工艺相比对酶的使用量和催化时间减少幅度都在50%以上,本复合酶协同催化体系能有效降低生物柴油制备成本,具有较好的工业化应用前景。

英文摘要：

[Objective] To optimize the process for transesterification reaction in biodiesel production by the synergistic biocatalyst in high water content system. [Methods] The synergistic biocatalyst of Candida antarctica lipase B（CALB） and Rhizopus oryzae lipase（ROL） were expressed and secreted in Pichia pastoris using genetic engineering techniques. The single factor experiment was used to optimize the process conditions, and the methyl ester（ME） yield was used to evaluate the transesterification ability of synergistical biocatalyst. [Results] The maximum ME yield of 93% could be achieved using 16 U/g oil combined with mixed lipase（CALB：ROL=7：3） for the methanolysis. The molar ratio of methanol to oil is 4：1（initial methanol/oil molar ratio 2：1 and 1：1 at 12 h and 24 h, respectively）, water content 30%-60%（W/W）（based on oil weight）, temperature 40 °C for 29-34 h. [Conclusion] This synergistic catalytic system is environmental friendly. Furthermore, both the consumption of lipase and reaction time were decreased more than 50% compared with the conventional enzymatic preparation of biodiesel. Therefore, this study provides an effective way to decrease the cost of biodiesel preparation and also has great potential in biodiesel industry.