嗜热内切-1,4-β-木聚糖酶在枯草杆菌中的表达及酶学性质

Expression of Thermophilic Endo-1,4-β-xylanase in Bacillus subtilis

DOI：10.3969/j.issn.1673-1689.2013.12.016

中文关键词: 枯草芽孢杆菌表达系统 分泌表达 耐高温嗜热内切-1,4-β-木聚糖酶

英文关键词: Bacillus subtilis expression system expression and secretion thermophilic endo-1 4-β-xylanase

基金项目:天津市滨海新区科技小巨人成长计划-科技型企业创新发展(科技创业)项目(2010-BK130070);天津市科技型中小企业技术创新基金项目(11C26211203970);天津市滨海新区重大科技项目(2011-BK120014);天津市科技计划项目(12ZCZDSY01800)

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中文摘要:

首先根据Thermopolyspora flexuosa的内切-1,4-β-木聚糖酶基因设计了引物,PCR扩增成功后进行酶切消化,连接载体质粒pHT43,经大肠杆菌DH5α扩增后,挑选阳性克隆,经菌体PCR及测序验证后,将基因序列测序正确的质粒大量抽提,经过转化导入枯草芽孢杆菌Bacillus subtilius WB800N宿主,培养并经过1 mmol/L IPTG诱导表达后,发酵胞外上清经SDS-PAGE电泳证明重组蛋白质成功获得表达。其相对分子质量为27 kD,最适反应温度为80℃,最适反应pH为6.0。由此得知,分泌表达的嗜热内切-1,4-β-木聚糖酶的枯草芽孢杆菌工程菌株构建成功,这为未来该酶的产业化生产奠定了基础。

英文摘要:

The PCR primers were designed based on the endo-1,4-β-xylanase gene of the Thermopolyspora flexuosa was PCR amplified using designed primer. The amplified product was then digested and ligated into plasmid pHT43. After transformation of Escherichia coli DH5α,the positive clones were selected and the correct plasmids were largely extracted and transformed into Bacillus subtilis WB800N,the expression was induced by 1mM IPTG. SDS-PAGE electrophoresis of the extracellular supernatant showed that the targeted protein was successfully expressed. The apparent molecular weight of the endo-1,4-β-xylanase is 27 kD. The optimal reaction temperature is 80 ℃,whereas the optimal pH is 6. Hence,an engineered B. subtilis strain which could secrete endo-1,4-β-xylanase was successfully constructed.

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