Armillariella tabescens EJLY2098β-甘露聚糖酶的诱导、纯化及酶学性质分析  

Inducement, Purification and Characterization ofβ-mannanase from Armillariella tabescens EJLY2098

作　　者：姚冬生[1] 黄小葵[1] 刘大岭[1] 谢春芳[1] 胡熔[1] 

机构地区：[1]暨南大学生命科学院,广州510632

出　　处：《中国生物工程杂志》2006年第7期57-63,共7页China Biotechnology

基　　金：国家"863"计划资助项目(2002AA213011)国家自然科学基金资助项目(30270043)广东省科技攻关计划资助项目(2005820601004)

摘　　要：Armillariella,tabescens EJLY2098经魔芋精粉诱导,可产β-甘露聚糖酶,再用正交实验优化诱导培养基,结果在培养基为魔芋精粉2％、蛋白胨1％、土豆汁25％、KH2PO4 0．3％、MgSO4·7H2O 0．15％、维生素B1 0．01％时可诱导出较高活性的酶。用DEAE-阴离子交换色谱从培养上清分离纯化β-甘露聚糖酶,出现2个活性组份。活性组份P2的SDS-PAGE分析显示电泳纯的一条带,分子量约78．9kDa。HPTLC分析P2为内切β-甘露聚糖酶;酶反应的最适温度为60℃,最适pH为4．0-6．0。保温30min的半失活温度T1/2为63℃,在pH4．5-6．0之间稳定性较好。Na+和Ba2+对其有激活作用,等电点pI约为4．0-4．1。获得了一株产β-甘露聚糖酶的新菌种,为进一步用基因工程方法克隆并构建具有完整自主知识产权的重组β-甘露聚糖酶基因工程菌提供了重要的基础工作。Armillariella tabescens EJLY2098 was capable of secreting β-mannanase by konjac inducement. A 34 orthogonal design was applied to determine the optimum medium of inducing mannanase by Armillariella tabescens EJLY2098. The results suggested that ArmiUariella tabescens EJLY2098 secreted the high-activity enzyme in the optimum medium, which was composed of 2% konjac, 1% peptone, 25% potato juice,0.3% KH2PO4 ,15% MgSO4 · 7H2O, 0.01% VitBt. Purified by DEAE-anion exchange chromatography, two eluting peaks （ P1 and P2） with the β-mannanase activity were obtained, and one of them （ named β-mannanase P2） was a single band by the SDS-PAGE, and the molecular weight of β-mannanase P2 was 78.9kDa. The isoelectric point of β-mannanase P2 was estimated to be 4.0 ～4.1. The optimum activity for the enzyme was found at 60℃ and pH4.0 ～ 6.0, and the enzyme was stable between pH4.5 ～ 6.0. The activity of β-mannanase P2 were enhanced by Na^＋ and Ba^2＋. This β-mannanase can be used in feed industy, a new fungi secreting β-mannanase was obtained, providing an important base for cloning mannanase gene and constructing recombin microbe expressing β-mannanase .

关 键 词：魔芋精粉 Β-甘露聚糖酶 纯化 Armillariella tabescens EJLY2098 

分 类 号：S685.99[农业科学—观赏园艺] TQ925[农业科学—园艺学]