两拷贝β-甘露聚糖酶基因在毕赤酵母中的表达  

Expression of Two Copies of β-mannase Gene in Pichia pastoris

作　　者：聂金梅[1] 李阳源[1] 刘金山[1] 

机构地区：[1]广东溢多利生物科技股份有限公司,广东珠海519060

出　　处：《生物技术》2014年第2期24-28,共5页Biotechnology

摘　　要：目的:旨在获得高效分泌表达的两拷贝β-甘露聚糖酶(β-mannase)毕赤酵母工程菌株。方法:以作者研究室保藏的黑曲霉β-mannase X33菌株(X33-β-mannase)为出发菌株,体外构建两拷贝重组质粒pPIC-mannase,用电击法转化至X33感受态细胞,用抗生素Zeocin筛选阳性转化子,用BMGY培养基培养,BMMY诱导其表达,利用DNS法测定酶活。结果:实验已成功构建了两拷贝β-mannase毕赤酵母工程菌株,50 L发酵罐培养7 d后,β-mannase产酶水平达到21 300 U/ml。Objective： To obtain recombinant strain of two copies of β - mannase which expressed the β - mannase protein at high level. Method： The parent strain producing β - mannase named as β - mannase - X33, two copies of recombinant plasmid of pPIC - man- nase was constructed in vitro, then the recombinant plasmid was transformed to Pichia pastoris X33 by electroporation, positive transforma- nts was screened by Zeocin in high concentration. Cultured with BMGY medium and its expression was induced by BMMY medium, en- zyme activity was measured using DNS method. Result： β - mannase activity of two copies of recombinant strain β - mannase gene was 21300 u/ml by fermentation for 7days. Conclusion： An efficient and stable expression of two copies of β - mannase gene in Pichia engi- neering strains was obtained successfully.

关 键 词：Β-甘露聚糖酶 感受态细胞 毕赤酵母 

分 类 号：Q786[生物学—分子生物学]