毕赤酵母GS115中N-乙酰转移酶在大肠杆菌中的克隆表达与性质研究

Cloning,Expression and Characterization of Enzyme for Mpr1 from P. pastoris GS115 in Recombinant E.coli

为了研究P.pastoris Mpr1的生理特性,在E.coli JM109胞内中成功表达来源于P.pastoris GS115的Mpr1酶,并利用响应面分析法对诱导温度、IPTG诱导浓度、起始诱导OD进行发酵优化,酶活达到(610.3±9.5)m U/m L。酶学性质显示,Mpr1酶的最适反应p H范围为7.0-7.5,最适反应温度是30℃。通过分析重组表达Mpr1菌株和对照菌株的培养过程,显示重组菌株的生长能力显著增强,原因是Mpr1降低了细胞内的ROS水平。

Due to the nature in P. pastoris methanol metabolism, it suffers much more ROS oxidative stress. There is one Mpr1 enzyme in P. pastoris. It plays significant physiological roles in ROS oxidative stress resistance ability and related research is still blank. For a detailed study about the physiological characteristics of P. pastoris Mpr1, Mpr1 from P. pastoris GS115 had been successfully expressed in E.coli JM109.Fermentation optimization of recombinant cell was studied from induction temperature, IPTG induction concentration, Initial induction OD by using Response Surface Analysis, activity reached（610.3±9.5）m U/m L. Enzymatic properties showed that the optimal p H of Mpr1 was about 7.0 to 7.5, the optimum temperature of Mpr1 was 30℃. In order to explore the nature of Mpr1, PQE30-E.coli JM109 strain and PQE30-Mpr1-E.coli JM109 strain were cultured under the same fermentation conditions in this experiment. The results showed that the growth capacity of the recombinant strain was stronger. The reason is that Mpr1 reduces levels of intracellular ROS.