本研究将经密码子优化的皱褶假丝酵母脂肪酶(Candida rugosa lipase) CRL1基因克隆到毕赤酵母分泌型表达载体pHKA，构建得到重组菌株GS115/pHKA-CRL1；经摇瓶发酵并结合甲醇诱导120 h处理，该菌株对底物对硝基苯酚丁酯（pNPB）的水解活力达到39.73 U/mL。通过对AOX1启动子和α分泌信号肽同时进行改造，进一步获得重组菌株GS115/pHKA-AOX1m/αm-CRL1，其脂肪酶活力提高达到63.63 U/mL。发酵液上清液经超滤浓缩、硫酸铵沉淀后，再经脱盐和阴离子交换层析处理，获得纯化的重组CRL1。该纯化工艺的纯化倍数为5.41倍，回收率为33.81%。而纯化重组CRL1的比酶活为984.5 U/mg。重组CRL1的最适温度为40 ℃，最适pH为7.5；经40 ℃保温6 h，仍保留52.99%的相对活力；在偏酸性环境中较为稳定。以摇瓶发酵、真空冷冻干燥后的重组CRL1为催化剂，在无溶剂体系中催化合成维生素E醋酸酯，在最适反应条件下（200 mg D-α-生育酚、1 mL乙酸酐、100 mg CRL1、反应温度60 ℃、转速200 r/min、反应时间9 h），D-α-生育酚的转化率在97.00%以上。

In this study, the optimized gene encoding Candida rugosa lipase CRL1 was synthesized in vitro, cloned into the vector pHKA and expressed in Pichia pastoris, producing a lipase activity of 39.73 U/mL towards p-nitrophenyl butyrate (pNPB) after induction for 120 h in flask fermentation. A new gene encoding GS115/pHKA-AOX1m/αm-CRL1 was obtained by modifying the AOX1 promoter and α-factor signal peptide, which lipase activity achieved 63.63 U/mL. Then the purified CRL1 was isolated from the supernatant of the zymotic fluid, through sequential treatments of ultrafiltration, ammonium sulphate precipitation and anion exchange chromatography. The specific enzyme activity of this purified CRL1 was 984.5 U/mg. The recombined CRL1 was characterized by the optimal temperature and pH at 40 ℃ and 7.5, respectively. Besides, it retained 52.99% of its original activity after incubation at 40 ℃ for 6 h and it was relatively stable in a slightly acidic environment. Furthermore, the crude CRL1 was lyophilized and harvested as an effective biocatalyst to synthesize vitamin E acetate in the solvent-free system. The optimal synthesis was performed with 200 mg D-α-tocopherol, 1 mL acetic anhydride, and 100 mg crude CRL1, at 60 ℃ and a shearing rate of 200 r/min for 6 h, of which more than 97% of D-α-tocopherol was transformed.

国家自然科学基金青年基金项目（30471225）；中央高校基本科研业务费项目（2017MS103）