β-甘露聚糖酶基因和木聚糖酶基因在毕赤母中的共表达.pdf

研究论文
β-甘露聚糖醇基因和木聚糖酶基因
在毕赤酵母中的共表达
李剑芳,赵顺阁,邬敏辰2,张慧敏,魏喜換
(1.江南大学食品学院,江苏无鐫214122:2.江南大学医约学院,江苏无锡214122)
摘要:为实现β-甘露聚糖酶基因和木聚糖酶基因在毕赤酵母中的共表达,作者将经SalI线性化
的pPlC9K-xyn∥电转化至工程菌CSl15/ inman5A中,经C418浓度梯度筛选后获得能同时高产
-甘露聚糖酶和木聚糖酶的双重重组子GS115/ amana-xymnⅡ。SDS-PAGE显示目的蛋白的相
对分子质量分別约为5200024100摇瓶发酵筛选出产酶活性最强的转化株,命名为CSMX2,
随后对该菌株的表达条件进行初步优化,优化后的表达条件为:诱导时间120h,培养基起始pH
值为7.0,甘油添加量为1.5%,甲醇添加量为1.5%。在此培养条件下,产β-甘露聚糖酶和木聚糖
酶的活性分别达到37.1U/mL.和193.6U/mL
关键词:β-甘露聚耱酶;木聚耱酶;共表达;条件优化
中图分类号:S37文献标志码:A文章编号:1673-1689(2012)011-1136-06
Coexpression of B-mannanase Gene and Xylanase Gene in Pichia pastoris
LI ian-fang', ZHAO Shung, WU Min-chen, ZHA NG Hui-min, WE Xi-huan
1. School of Food Seience and Technology, Jiangnan University, Wuxi 214122, China; 2. School of Medicine and
Pharmaceutics, Jiangnan University, Wuxi 214122, China
Abstract: B-mannanase and xylanase are extensively used in food industry. Synergistic action
between the two enzymes is obersved to effectively improve digestibility of the hemicelluloses of
food material. In order to coexpress -mannanase gene and xylanase gene in Pichia pastoris
pplc9k-ayn l was linearized with Sal I and transformed into GS115/A man5A by electroporation
GS115/A nman 5A -xm with high production of -mannanase and xylanase was screened by using
G418. SDS-PAGE demonstrated two protein bands with apparent molecular weight of about 52 000
and 24 100. One G$115/Anman5a-xyn I transformant producing the highest activities of two
enzymes in shake flasks was selected and numbered as GSMX2. Subsequently the expression
conditions for enzymes production by using GSMX2 were optimized, the optimum expression
conditions listee as follows initial PH 7.0,glycerol concentration 1.5%, methanol concentration
收稿日期:2012-04-06
基金项目:国家自然科学基金项目(3127181)201年高校博士研究生科研创新计划项目( TUDCF10056
作者简介:李剑芳(1965一),女,江苏无锡人,工学博士,副教授,主要从事食品生物技术研究。E-mail:lijf@163.com
通信作者:邬敏辰(1962-),男,江苏无锡人,理学博士,教授,博士研究生导师,主要从事酶工程与基因工程研究。E-mail:bioch(@163.com
Journal of Food Science and Biotechnology Vol 31 No11 2012
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