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【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。
【Objective】 The objective of this study was to investigate the conditions for preparing biodiesel by catalyzing oil in the synergistic catalytic system of complex enzyme with high water content. 【Method】 The gene of Candida antarctica lipase (CALB) and Rhizopus oryzae lipase (ROL) were secreted and expressed in Pichia pastoris respectively. The synergistic catalytic system of CALB and ROL was used to prepare biodiesel. Factors experimental optimization process conditions, methyl ester yield as a composite enzyme synergistic catalytic system efficiency evaluation criteria. 【Result】 The optimum conditions were as follows: the optimal compounding ratio of CALB? ROL was 7? 3, adding 16 U of complex lipase per gram of soybean oil, the molar ratio of methanol to soybean oil was 4? 1, When the molar ratio of 2 to 1 alcohol, 12 h and 24 h at a molar ratio of 1 to 1 alcohol methanol methanol, water content of 30% -60%, 40 ° C for 29-34 h, methyl ester was Rate of 93%. 【Conclusion】 This complex enzyme synergistic catalytic system is environmentally friendly. Compared with the conventional enzymatic process for the preparation of biodiesel, the amount of enzyme used and the reduction of the catalytic time are both above 50%. The synergistic catalytic system of the complex enzyme can effectively reduce the biodiesel Preparation costs, with good prospects for industrial applications.