纤维微菌海藻糖合成酶基因克隆表达及酶学特性鉴定  

Clonging,Expressionan and Characterization of a Novel Gene Encoding Trehalose Synthase from Cellulosimicrobium cellulans

作　　者：曹磊[1,2] 刘滔滔[1,2] 张宏涛[1,2] 谢敏[1,2] 杜丽琴[1,2] 梁智群[1,2] 韦宇拓[1,2] 

机构地区：[1]广西大学生命科学与技术学院,广西南宁530005 [2]亚热带农业生物资源保护与利用国家重点实验室,广西南宁530005

出　　处：《广西科学》2016年第1期19-24,共6页Guangxi Sciences

基　　金：国家自然科学基金项目(31160311);广西自然科学基金项目(2012GXNSFAA053051)资助

摘　　要：【目的】开发适用于海藻糖生产的新型海藻糖合成酶。【方法】通过反向PCR技术,从一株纤维微菌的基因组DNA中获知海藻糖合成酶基因完整ORF序列,进而克隆得到纤维微菌海藻糖合成酶基因(CCTreS),将其与表达载体pSE380构建重组质粒后转入大肠杆菌BL-21(DE3)中诱导表达,通过镍柱亲和层析纯化得到纯酶并进行酶学性质测定。【结果】从纤维微菌中克隆并在大肠杆菌中成功表达海藻糖合成酶基因(CCTreS)。经纯化获得的重组酶(CCTreS)在以麦芽糖为底物生成海藻糖时,最适反应pH值为7.0,最适反应温度为45℃,40℃保温1h仍具有80%以上的相对酶活力,在pH值5.5~8.5保存24h,相对酶活力仍保留80%以上。Cu^(2+)对其有明显抑制作用。【结论】该重组酶具有很好的热稳定性和pH稳定性,具有一定的研究价值和潜在的工业应用价值。【Objective】A new trehalose synthase was developed for application in industrial production of trehalose.【Methods】The complete ORF sequense of trehalose synthase gene was obtained fromCellulosimicrobium cellulans by inverse-PCR,and then it was amlified by PCR.The recombinant plasmid pSE380-CCTreS was constructed and transformed into Escherichia coli BL-21(DE3).The expressed protein was purified using nickel-nitrilotriacetic acid agarose resin.At last,the enzymatic characteristics of recombinant enzyme were studied.【Results】The trehalose synthase gene(CCTreS)was cloned fromCellulosimicrobium cellulans and successfully expressed in Escherichia coli.The optimum activity for the purified recombinant enzyme to convert maltose into trehalose was at 45℃ and pH 7.0.CCTreS had a good stability and could be reserved above 80% relative activity when itwas placed in the environment at 40℃ for an hour or the environment in pH 5.5~8.5for 24 hours.Cu^(2+)strongly inhibited the enzyme activity.【Conclusion】The recombinant enzyme has good thermal stability and pH stability,which provide certain research value and potential industrial application value.

关 键 词：海藻糖合成酶 纤维微菌 反向PCR酶学性质 

分 类 号：Q785[生物学—分子生物学] Q786