重组角质酶的制备及合成乙酸乙酯条件优化

为了实现镰孢菌角质酶的高效表达，对重组菌株P. pastoris KM71/pPIC9K-ScCuA在3 L发酵罐进行了发酵工艺优化，获得的最优发酵条件为：诱导温度28 ℃，初始诱导菌浓OD600为100，诱导阶段甲醇体积分数为1%条件下进行诱导产酶。在该条件下发酵138 h时上清酶活可达到最大值419 U/mL，是优化前的2倍。对重组角质酶在有机溶剂中合成乙酸乙酯的酯化条件进行了优化，结果表明，当底物乙酸浓度为 0.1 mol/L，乙醇浓度为0.15 mol/L，温度 50 ℃，在异辛烷中反应16 h，乙酸乙酯可以达到最大转化率 94.12%。
In order to achieve efficient production of the cutinase from Sirococcus conigenus，the optimization of fermentation conditions was investigated in P. pastoris KM71 harboring the plasmid pPIC9K-ScCuA. The optimized fermentation conditions were as follows：the inducing temperature is 28 ℃，the induction phase methanol concentration is maintained at 1% When the initial cell concentration OD600 reached 100. Under the optimum condition，the enzyme activity reached 419 U/mL at cultivation of 138 h，was 2 times than before.Different reaction parameters for the synthesis of ethyl acetate by direct esterification in organic solvents are investigated. The highest yield of 94.12% is obtained at 0.1 mol/L acetic acid，0.15 mol/L alcohol using a reaction temperature of 50 ℃ for 16 h incubation time in iso-octane