内切葡聚糖酶与木聚糖酶在毕赤酵母中的共分泌表达.pdf

研究论文
内切葡聚糖酶与木聚糖嗨在毕赤酵母中的共分泌表达
刘高磊,胡蝶2,邬敏辰”,殷欣2,汪俊卿2
(1.江南大学药学院,江苏无锡214122:2.江南大学生物工程学院,江苏无锡214122;3.江南大学无锡医
学院,江苏无锡214122
摘要:为实现内切耥聚耱酶和木聚糖酶在毕赤酵母中的共分泌表达,进而降低酶制剂的生产成
本,构建含木聚糖酶基因的重组表达載体 PPICZAA- Aoxynlla,经SacI线性化后,电转化至含
内切葡聚糖酶基因 A uceli2A的重组毕赤酵母CSC7中,获得双重重组毕赤酵母CSCX8。经甲醇
诱导表达后,CSCX8发酵上清液中内切葡聚糖酶和木聚糖酶的活性分别为47.77U/mL和
192.71IU/mL,为单独表达菌株GSC7和CSXS5的85%和80%。酶学性质分析显示,内切葡聚糖
酶的最适pH为4.0,在pH3.0-8.5稳定;最适温度为50℃,在60℃以下稳定。木聚糖酶的最适
pH为5.5,在pH3.0~10.0稳定;最适温度为55℃,在50℃以下稳定。CSCX8遗传稳定性测试结
果表明,内切葡聚糖酶和木聚糖酶在毕赤酵母中实现了稳定的共表达。
关键词:内切聚糖酶;木聚糖酶;毕赤酵母;共分泌表达
中图分类号:Q814文献标志码:A文章编号:1673-1689(2015)03-0253-07
Co-expression of Endoglucanase and Xylanase in Pichia pastoris
LIU Gaolei, HU Die, WU Minchen", YIN Xin?, WANG Junging?
(1. School of Pharmaceutics, Jiangnan University, Wuxi 214122, China; 2. School of Biotechnology, Jiangnan
University, Wuxi 214122, China; 3. Wuxi Medical School, Jiangnan University, Wuxi 214122, China)
Abstract: Both endoglucanase and xylanase are additives in feed for mono-gastric animal. Co
expression of endoglucanase and xylanase in P. pastoris is a useful way to reduce the production
cost in feedstuff industry. An endoglucanase-encoding gene(A ucel12a) from A spergillus usamii
was expressed in P. pastoris, and the engineering strain with the highest endoglucanase activity was.
labeled G$C7. Then, a recombinant vector, PPLCZAA-AOXYN11A, was constructed and linearized
Sac I, followed by transforming it into GSC7. Ultimately, the endo lucanase and xylanase genes were
successfully co-expressed in P. pastoris. One strain harbored genes Axyn11a and Aucel12a, with
the highest xylanase and endoglucanase activities, was labeled GSCX8. The enzymatic properties of
the recombinant A/A and AOXYN11A, expressed in GSCX8, were characterized. Meanwhile, to
analyze the genetic stability of GSCX8, the xylanase and endo lucanase activities of different strains
收稿日期:2014-04-17
基金项目:国家自然科学基金项目(31101229;31271811)。
本通信作者:邬敏辰(1962一),男,江苏无锡人,理学博士,教授,博士研究生导师,主要从事酶T程与基因工程研究。
E-mail:biowme@126.com
含出5すわ技メ影2015年第34卷第3期253
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