Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing 100083, China; College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing 100083, China; Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing 100083, China.
Int J Biol Macromol. 2019 Aug 15;135:768-775. doi: 10.1016/j.ijbiomac.2019.05.173. Epub 2019 May 23.
A codon-optimized cutinase gene (TtCutopt) from Thielavia terrestris was over-expressed in Pichia pastoris. An extracellular activity reached 10,200 U/mL using high cell density fermentation. The optimal pH and temperature of TtCutopt were 7.0 and 50 °C, respectively. It displayed high stability over a wide range of pH from 3.0 to 11.0 and up to 85 °C. Among tested p-nitrophenyl esters and triglycerides, TtCutopt showed the highest activity towards p-nitrophenyl butyrate and tributyrin, with specificity activity of 2322.4 U/mg and 1152.5 U/mg, respectively. It was extremely stable in organic solvents and surfactants. TtCutopt efficiently catalyzed the synthesis of butyl butyrate, hexyl butyrate, butyl hexanoate and hexyl hexanoate with esterification efficiency of >95%. Furthermore, it catalyzed the degradation of >90% of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and dibutyl phthalate to release their corresponding monoalkyl phthalates within 24 h. Thus, high yield, high stability, and esterification efficiency of TtCutopt make it an attractive candidate for ester biosynthesis and biodegradation.
从土曲霉中过表达了密码子优化的角质酶基因（TtCutopt）。通过高密度发酵，胞外酶活达到 10200 U/mL。TtCutopt 的最适 pH 和温度分别为 7.0 和 50°C。它在 pH 3.0 至 11.0 的较宽范围内和 85°C 下具有很高的稳定性。在测试的对硝基苯酚酯和三酸甘油酯中，TtCutopt 对对硝基苯酚丁酸酯和三丁酸甘油酯表现出最高的活性，比活分别为 2322.4 U/mg 和 1152.5 U/mg。它在有机溶剂和表面活性剂中极其稳定。TtCutopt 能有效地催化丁酸丁酯、丁酸己酯、丁酸己酸酯和己酸己酸酯的合成，酯化效率>95%。此外，它能在 24 h 内催化邻苯二甲酸二甲酯、邻苯二甲酸二乙酯、邻苯二甲酸二丙酯和邻苯二甲酸二丁酯>90%的降解，释放出相应的单烷基邻苯二甲酸酯。因此，TtCutopt 的高产量、高稳定性和酯化效率使其成为酯类生物合成和生物降解的有吸引力的候选物。
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