耐热木聚糖酶基因在毕赤酵母中的表达及酶学性质

Expression of A Thermostable Xylanase Gene in Pichia pastoris and Its Enzymatic Characterization

DOI：10.3969/j.issn.1673-1689.2013.02.003

中文关键词: 耐热木聚糖酶 毕赤酵母 异源表达 酶学性质

英文关键词: thermostable xylanase Pichia pastoris heterologous expression enzymatic characterization

基金项目:国家自然科学基金项目(31101229);2011年高校博士研究生科研创新计划项目(JUDCF10056)

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中文摘要:

将一种11家族极端耐热木聚糖酶的密码子优化基因Syxyn11克隆到毕赤酵母表达载体pPIC9K中,得到重组质粒pPIC9K-Syxyn11,将其经SalⅠ线性化后转化毕赤酵母(Pichia pastoris)GS115。经G418筛选得到重组工程菌GS115/Syxyn11,用甲醇诱导表达重组木聚糖酶SyXyn11,酶活可达到17.74 U/mL。SDS-PAGE显示,SyXyn11的相对分子质量为31 000。SyXyn11的最适反应温度为85℃,在80℃以下稳定。最适反应pH为6.5,在pH 5.0~7.5范围内稳定。EDTA和大多数金属离子对重组酶的活性影响不大。结果表明Syxyn11成功在P.pastoris GS115中实现表达,而且重组木聚糖酶的耐热性并未改变。

英文摘要:

A codon-optimized gene encoding a hyperthermostable xylanase belongs to the glycoside hydrolase family 11 (abbreviated as Syxyn11) has been cloned into the expression plasmid pPIC9K (named pPIC9K-Syxyn11). The pPIC9K-Syxyn11 was linearized with SalⅠ and integrated into the genome of Pichia pastoris GS115 by electroporation. The recombinant P. pastoris GS115/Syxyn11 was screened by G418 and then was induced with methanol to express SyXyn11. The SyXyn11 activity expressed by P. pastoris transformant reached 17.74 U/mL. The molecular weight of SyXyn11 was estimated to be 31 000 by SDS-PAGE. The SyXyn11 displayed the highest activity at 85 ℃and pH 6.5. It was highly stable at a temperature of 80 ℃ or below,and at a pH range of 5.0~ 7.5. Its activity was not significantly affected by most of metal ions tested and EDTA. This revealed that Syxyn11 was successfully expressed in P. pastoris and remained the high thermostability.

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