枯草芽孢杆菌β-甘露聚糖酶在毕赤酵母中的分泌表达  

Secreting Expression of Bacillus subtilis β-Mannanase in Pichia pastoris

作　　者：马威[1] 沈志娜[2] 陈轶群[1] 李志民[1] 曹云鹤[1] 

机构地区：[1]中国农业大学动物营养学国家重点实验室,北京100193 [2]哈尔滨师范大学生命科学院,黑龙江哈尔滨150025

出　　处：《生物技术通讯》2010年第2期171-174,共4页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2007AA100601);教育部新世纪优秀人才支持计划(NCET-07-0807);动物营养学国家重点实验室自主研究课题(2004DA125184(团)0806)

摘　　要：目的:研制高效分泌表达枯草芽孢杆菌β-甘露聚糖酶的毕赤酵母基因工程菌株。方法与结果:将优化设计的枯草芽孢杆菌MA139β-甘露聚糖酶基因用EcoRⅠ/XbaⅠ双酶切,克隆到诱导型表达载体pPICzαA中α因子信号肽编码序列的下游,转化大肠杆菌筛选重组质粒,转化毕赤酵母X-33感受态细胞,经Zeocin筛选,获得重组表达菌株X-33/mann。将重组菌株在10L全自动发酵罐中进行高密度发酵培养,甲醇诱导72h发酵活力达到2100U/mL。重组甘露聚糖酶的最适催化温度为40℃,最适催化pH值为6.0。结论:枯草芽孢杆菌β-甘露聚糖酶在毕赤酵母中获得了高效分泌表达,具有开发作为饲料添加剂的潜能。Objective：To construct an engineering Pichia pastoris with secretory over-expression of β-mannanase from Bacillus subtilis.Methods Results：A codon-optimized β-mannanase gene from B.subtilis MA139 cleaved by EcoRⅠ and XbaⅠ was fused downstream of an α-factor in the expression plasmid of pPICzαA,and transformed into E.coli.The resulting plasmid linearized with SacⅠ was transformed into P.pastoris X-33.The recombinant strain with high-level expression of β-mannanase was cultured in 10 liter fermenter.The β-mannanase activity of the supernatant reached 2100 U /mL after the yeast was induced by methanol for 72 h.The recombinant βmannanase showed optimum of activity at 40℃ and pH6.0.Conclusion：The B.subtilis β-mannanase was high-level expressed by P.pastoris.The biochemical characteristics of the β-mannanase suggest the enzyme has a prospective application in feed industry as an additive.

关 键 词：Β-甘露聚糖酶 枯草芽孢杆菌 毕赤酵母 分泌表达 

分 类 号：Q785[生物学—分子生物学]