来源于欧文氏杆菌CXJZ95-198的β-甘露聚糖酶基因高效表达体系构建

doi:10.11924/j.issn.1000-6850.casb14110094

中国农学通报 ›› 2015, Vol. 31 ›› Issue (10): 240-245.doi: 10.11924/j.issn.1000-6850.casb14110094

所属专题：生物技术

来源于欧文氏杆菌CXJZ95-198的β-甘露聚糖酶基因高效表达体系构建

成莉凤,冯湘沅,段盛文,郑科,刘正初

(中国农业科学院麻类研究所,长沙 410205）

收稿日期:2014-11-17 修回日期:2015-03-06 接受日期:2015-01-23 出版日期:2015-05-05 发布日期:2015-05-05
通讯作者: 刘正初
基金资助:
国家高技术发展计划“复合酶用于麻类生物脱胶的工艺研究”(2012AA022209D)；国家麻类产业技术体系建设专项“脱胶技术与工艺”(CARS-19-E24)；国家微生物资源平台专项经费课题“麻类加工微生物资源整理整合”(NIMR-2013-1-4)；长沙市科技计划重点项目“麻类加工高效生物制剂产业化关键技术研究”(K1205005-11-2)；中国农业科学院科技创新工程“农产品加工微生物遗传改良与应用”(ASTIP-IBFC09)。

Construction of High Efficient Expression System of the β-mannanase Gene from Erwinia carotovora CXJZ95-198

Cheng Lifeng, Feng Xiangyuan, Duan Shengwen, Zheng Ke, Liu Zhengchu

(Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205)

Received:2014-11-17 Revised:2015-03-06 Accepted:2015-01-23 Online:2015-05-05 Published:2015-05-05

摘要/Abstract

摘要： 旨在构建β-甘露聚糖酶基因的高效表达体系。从原基因工程菌株slp-man-pET28a/JM提取重组质粒slp-man-pET28a，转化Escherichia coli BL21(DE3)。用水解圈大小，β-甘露聚糖酶活力高低和天然半纤维素底物降解能力等综合指标衡量新基因工程菌株的β-甘露聚糖酶表达效果。新基因工程菌株slp-man-pET28a/BL在选择平板上能产生明显的水解圈，其分泌的β-甘露聚糖酶活性最高达 645.5 U/mL，是原基因工程菌株slp-man-pET28a/JM分泌的1.28倍，是出发菌株Erwinia carotovora CXJZ95-198分泌的1.97倍；新基因工程菌株在苎麻原料发酵液的COD净增加值达5.13 g/L左右，分别为原基因工程菌株和出发菌株净增值的1.5和1.2倍；其还原糖生成量为0.721 mg/mL，比原基因工程菌株和出发菌株分别提高了26.7%和10.1%。新基因工程菌株的β-甘露聚糖酶分泌量大幅度提高，可为麻类生物脱胶β-甘露聚糖酶制剂制备提供科学依据。

关键词: 辽西地区, 辽西地区, 地面温度, 气候特征, 气候突变

Abstract: High efficient expression system of the β-mannanase gene was built. Recombinant plasmid slp-man-pET28a was extracted from the genetic engineering strain slp-man-pET28a/JM, and then transformed to E. coli BL21 (DE3). β-mannanase of the new genetic engineering strain was tested by comparing hydrolysis circle, determination of β-mannanase activity and degradation effect of the natural hemi-cellulose. New genetic engineering strain slp-man-pET28a/BL produced significant hydrolysis circles in selective medium, and secreted β-mannanase up to 645.5 U/mL, as 1.28 times and 1.97 times as that of genetic engineering strain slp-man-pET28a/JM and original strains E. carotovora CXJZ95-198, respectively. COD value of the fermented liquid in ramie raw material by the new genetic engineering strain increased by 5.13 g/L, as 1.5 times and 1.2 times as that of slp-man-pET28a/JM and E. carotovora CXJZ95-198, respectively. Reducing sugar of the fermented liquid in ramie raw material by new genetic engineering strain was 0.721 mg/mL, higher than that of slp-man-pET28a/JM strain and E. carotovora CXJZ95-198 strain by 26.7% and 10.1%, respectively. β-mannanase activity is increased greatly by the construction of high efficient expression system of the new β-mannanase gene, and it may provide scientific basis for preparation used for bast fiber bio-degumming.

成莉凤,冯湘沅,段盛文,郑科,刘正初. 来源于欧文氏杆菌CXJZ95-198的β-甘露聚糖酶基因高效表达体系构建[J]. 中国农学通报, 2015, 31(10): 240-245.

Cheng Lifeng,Feng Xiangyuan,Duan Shengwen,Zheng Ke and Liu Zhengchu. Construction of High Efficient Expression System of the β-mannanase Gene from Erwinia carotovora CXJZ95-198[J]. Chinese Agricultural Science Bulletin, 2015, 31(10): 240-245.

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来源于欧文氏杆菌CXJZ95-198的β-甘露聚糖酶基因高效表达体系构建

Construction of High Efficient Expression System of the β-mannanase Gene from Erwinia carotovora CXJZ95-198

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