【摘要】 为提高Tremetes viscolor漆酶Lcc的表达量,本研究主要针对漆酶转录调节区域进行定向进化。利用易错PCR,选择不同浓度的Mn2+对调节区域进行突变,并以酿酒酵母为宿主细胞进行异源表达,构建S. c/p YES2-Lcc调节子突变库。选用2,2-联氮基双(3-乙基苯并噻唑啉-6-磺酸)二胺盐(ABTS)为底物的96孔板测定漆酶酶活高通量筛选方法对突变库中960株突变体进行筛选,并对酶活高的突变体进行摇瓶复筛,最终获得三株遗传稳定性良好的酶活较高地突变体M1、M2、M3,其粗酶液酶活分别是野生型菌株粗酶液酶活的1. 73倍、1. 46倍、1. 37倍,突变菌株M1粗酶液酶活最高可达35. 4 U/L。更多还原

【Abstract】 To improve the expression of the Tremetes ciscolor laccase Lcc,the transcription regulatory regions of Lcc were engineered by directed evolution. The mutation library was generated using error-prone PCR with different concentrations of Mn2 +and the Saccharomyces cerevisiae was used as host cell for heterologous expression laccase Lcc. Totally 960 mutants were picked into 96-well plates for cultivation and screened using 2-nitrogen-bis( 3-ethylbenzothiazoline-6-sulfonic acid)diamine salt( ABTS) assay. The mutants with enzyme activity higher than wild type were selected and then rescreened by shaking flask cultivation. Finally,three mutants( M1,M2,M3) with higher total enzyme activity were selected. The crude enzyme activities of these three mutants are 1. 73 times,1. 46 times and 1. 37 times of that of the wild-type strain,respectively. The crude enzyme activity of the best mutant strain M1 is up to 35. 4 U/L.更多还原