深海高温淀粉普鲁兰酶异源表达及酶活分析

焦豫良; 王淑军; 吕明生; 房耀维; 刘姝

深海高温淀粉普鲁兰酶异源表达及酶活分析

淮海工学院海洋学院,江苏连云港 222005

基金项目: 国家自然科学基金项目(40746030);江苏省高校自然科学研究重大项目(09KJA170001)。

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出版历程
- 收稿日期: 2010-12-30

Heterologous expression of a deep-sea thermostable amylopullulanase and enzymatic activity analysis of the fusion protein

College of Food Science and Technology, Huaihai Institute of Technology, Lianyungang 222005, China

摘要

摘要: 深海热液口厌氧古菌Thermococcus siculi HJ21中的高温淀粉普鲁兰酶进行分子进化树系分析,并在大肠杆菌中通过pMal-c2x载体表达并纯化其N端催化结构域。通过融合表达,在N端催化结构域的N端融合有麦芽糖结合蛋白MalE。对该融合蛋白的α-淀粉酶和普鲁兰酶活性进行了实验分析。融合蛋白的两种酶活的最适温度均为100 ℃,淀粉酶和普鲁兰酶活性的最适pH值分别为5和6,比活力分别为6.5和11.5 U/mg。结果表明,高温淀粉普鲁兰酶的α-淀粉酶活性相对较弱,其C端578个氨基酸构成的区域非两种酶活性所必需的结构。本研究中获得的高温淀粉普鲁兰酶融合蛋白在工业酶法制糖中可以进一步和高温α-淀粉酶配合使用。
- 高温淀粉普鲁兰酶 /
- 异源表达 /
- 酶活分析
Abstract: Molecular phylogenetic tree analysis on a thermostable amypopullulanase in an Archaeaon strain Thermococcus siculi HJ21 isolated from a deep-sea hydrothermal vents was performed based on experimental enzymatic analysis and amino acid squences of amylopullulanases deposited in GenBank. The N-terminal catalytic region of the amylopullulanase was heterologously expressed in E. coli through pMal-c2x expression system, resulting a fusion protein in which there is a maltose binding protein fused to the N-terminus of the N-terminal catalytic region of the amylopullulanase. Alpha-amylase and pullulanase activities of the fusion protein were experimentally analyzed. The optimal temperatures of the two activities were both at 100 ℃. The optimal pHs of amylase and pullulanase activities were at 5 and 6 respectively. The specific activities of the amylase and pullulanase activities were 6.5 and 11.5 U/mg respectively. The results showed that α-amylase activity was lower than pullulanase activity and the C-terminal region of the thermostable amylopullulanase was non-necessary for the enzymatic activities. The thermostable amylopullulanase fusion protein obtained in this study could be further used for combination with thermostable amylases in the sugar industry.
- thermostable amylopullulanase /
- heterologous expression /
- enzymatic analysis

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参考文献(16)

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