黑曲霉XZ-3S木聚糖酶基因xynZF-2在毕赤酵母中的高效表达  

Efficient expression of the xylanase gene xyn ZF-2 from Aspergillus niger XZ-3S in Pichia pastoris

作　　者：王丹丹[1,2] 周晨妍[1] 李同彪 张振群[1] 王亚杰[1] 梁真真[1] 张青[1] 路丹荣 

机构地区：[1]新乡医学院生命科学技术学院,河南新乡453003 [2]新乡医学院三全学院,河南新乡453003

出　　处：《食品工业科技》2015年第7期200-203,280,共5页Science and Technology of Food Industry

基　　金：河南省教育厅自然科学研究计划项目资助(2011A180026);河南省教育厅科学技术研究重点项目(13A180861);河南省科技厅科技攻关项目(112102210299);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木聚糖酶基因xyn ZF-2（成熟肽碱基）插入表达载体p PIC9K中,SalⅠ线性化后电击转化毕赤酵母GS115,经MD平板、G418梯度和摇瓶复筛筛选出多克隆阳性转化子。选择28h的种子,每隔24h补加1.5%的甲醇,发酵96h后,比酶活可达13210U/mg;SDS-PAGE分析表明,该蛋白相对分子质量为23.0ku;重组酶最适作用温度为45℃,最适作用p H为5.0,在温度35--40℃、p H5--7条件下有良好的稳定性,Ca2＋对酶的激活作用最明显。In this study, a recombinant plasmid pPIC9K-xynZF-2was constructed by inserting gene xynZF-2 mature peptide） from Aspergillus niger into Pichia pastoris secretary vector pPIC9K. The pPIC9K- xynZF- 2 finearized by Sal I was transformed into Pichia pastoris GS115 by electroporation.The positive recombinant strain was identified by MD medium,G4]8 and shake bottle selection.The SDS-PAGE analysis showed that the protein molecular weight was about 23.0ku. Under the condition of the vaccination time 28h, 1.5% methanol every 24h added,and the methanol induced time 96h,the enzyme production can be achieved 13210U/mg.The optimal temperature and pH of the enzyme activity was 45~C and 5.0, respectively.The enzyme activity was stable at the conditions of temperature 35-40℃C and pH 5-7.Ca2＋ had an active effect on the enzyme obviously.

关 键 词：黑曲霉 木聚糖酶 毕赤酵母GS115 诱导表达 

分 类 号：TS201.3[轻工技术与工程—食品科学]