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曹磊,刘滔滔,张宏涛,谢敏,杜丽琴,梁智群,韦宇拓.纤维微菌海藻糖合成酶基因克隆表达及酶学特性鉴定[J].广西科学,2016,23(1):19-24. [点击复制]
- CAO Lei,LIU Taotao,ZHANG Hongtao,XIE Min,DU Liqin,LIANG Zhiqun,WEI Yutuo.Clonging,Expressionan and Characterization of a Novel Gene Encoding Trehalose Synthase from Cellulosimicrobium cellulans[J].Guangxi Sciences,2016,23(1):19-24. [点击复制]
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| 纤维微菌海藻糖合成酶基因克隆表达及酶学特性鉴定 |
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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
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| (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) |
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| 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5.58.5保存24h,相对酶活力仍保留80%以上。Cu2+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。 |
| 关键词: 海藻糖合成酶 纤维微菌 反向PCR 酶学性质 |
| DOI：10.13656/j.cnki.gxkx.20160315.001 |
| 投稿时间：2015-11-16修订日期：2016-02-26 |
| 基金项目:国家自然科学基金项目(31160311)和广西自然科学基金项目(2012GXNSFAA053051)资助。 |
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| Clonging,Expressionan and Characterization of a Novel Gene Encoding Trehalose Synthase from Cellulosimicrobium cellulans |
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CAO Lei1,2, LIU Taotao1,2, ZHANG Hongtao1,2, XIE Min1,2, DU Liqin1,2, LIANG Zhiqun1,2, WEI Yutuo1,2
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| (1.College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;2.State Key Laboratory for Conservation and Utilizaiton of Subtropical Agro-bioresources, Nanning, Guangxi, 530005, China) |
| Abstract: |
| [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 from Cellulosimicrobium 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 from Cellulosimicrobium 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 5.58.5for 24 hours.Cu2+ 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. |
| Key words: trehalose synthase Cellulosimicrobium cellulans inverse-PCR enzymatic characteristics |
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