产木聚糖酶毕赤酵母工程菌株构建及表达条件优化研究  

Study on the construction and expression of the engineered strain of Pichia pastoris for xylanase production

作　　者：周晨妍[1] 刘振华[1] 王丹丹[1,2] 李同彪 高启禹[1] 

机构地区：[1]新乡医学院生命科学技术学院,合成生物学改造工程与应用实验室,河南新乡453000 [2]新乡医学院三全学院,河南新乡453000

出　　处：《食品工业科技》2016年第13期162-167,共6页Science and Technology of Food Industry

基　　金：河南省科技攻关计划项目(162102210118);河南省教育厅科学技术研究重点项目(13A180861;14A180018);河南省高等学校青年骨干教师资助计划项目(2011GGJS-125);新乡医学院科研项目培育基金(2013ZD113)

摘　　要：将黑曲霉XZ-3S木聚糖酶Xyn43A成熟肽基因插入表达载体p PIC9K,重组质粒SalⅠ线性化后分别电击转化2种毕赤酵母GS115和KM71,转化液经MD平板、G418浓度梯度平板和摇瓶复筛,获得两株重组菌GS115/Xyn43A(Mut^+)和KM71/Xyn43A(Mut^s)。其中GS115/Xyn43A菌株最优表达条件为:甲醇浓度2.0%,接种时间24 h,诱导时间108 h,诱导温度30℃、诱导培养基初始p H6.3;KM71/Xyn43A菌株最优表达条件为:甲醇浓度1.75%,接种时间26 h,诱导时间132 h,诱导温度30℃、诱导培养基初始p H6.5。在最优表达条件下两重组菌GS115/Xyn43A和KM71/Xyn43A比酶活力分别可达139.36、143.29 U/mg。A gene encoding xylanase Xyn43A from Aspergfllus niger XZ-3S was cloned into the Pichia pastoris expression vector, pPIC9K.The recombinant plasmid was linearized with Sa/ I and then transformed into Pichia pastoris GSI]5 and KMT] by electroporation, respectively. After screening by MD medium, G418 concentration plate and shake bottle selection,two recombinant strains GSl15/Xyn43A and KM7]/Xyn43A were obtained.The best optimization schemes were obtained by single factor experiment and L9 （34） experiment. The optimal expression conditions of GSli5/Xyn43A were methanol concentration of 2.0%, inoculation time of 24 h, induction time of 108 h, induction temperature of 30℃,and initial pH6.3.The optimal expression conditions of KM71/Xyn43A were methanol concentration of 1.75%, inoculation time of of 26 h, induction time of 132 h, induction temperature of 30℃,and initial pH6.5. Under the optimum conditions, the xylanase activity of the two recombinant strains were 139.36 U/mg and 143.29 U/mg,respectively.

关 键 词：黑曲霉 毕赤酵母 木聚糖酶 诱导表达 

分 类 号：TS201.3[轻工技术与工程—食品科学]