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

作　　者：成莉凤[1] 冯湘沅[1] 段盛文[1] 郑科[1] 刘正初[1] 

机构地区：[1]中国农业科学院麻类研究所,长沙410205

出　　处：《中国农学通报》2015年第10期240-245,共6页Chinese Agricultural Science Bulletin

基　　金：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)

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-p ET28a/JM提取重组质粒slp-man-p ET28a,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-p ET28a/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/m L,是原基因工程菌株slp-man-p ET28a/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、1.2倍;其还原糖生成量为0.721 mg/m L,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。High efficient expression system of the β-mannanase gene was built. Recombinant plasmid slpman-p ET28 a was extracted from the genetic engineering strain slp-man-p ET28a/JM, and then transformed to E. coli BL21（DE3）. β-mannanase of the new genetic engineering strain was tested by comparing hydrolysiscircle, determination of β- mannanase activity and degradation effect of the natural hemi- cellulose. Newgenetic engineering strain slp-man-p ET28a/BL produced significant hydrolysis circles in selective medium,and secreted β-mannanase up to 645.5 U/m L, as 1.28 times and 1.97 times as that of genetic engineeringstrain slp-man-p ET28a/JM and original strains E. carotovora CXJZ95-198, respectively. COD value of thefermented liquid in ramie raw material by the new genetic engineering strain increased by 5.13 g/L, as 1.5times and 1.2 times as that of slp-man-p ET28a/JM and E. carotovora CXJZ95-198, respectively. Reducingsugar of the fermented liquid in ramie raw material by new genetic engineering strain was 0.721 mg/m L, higherthan that of slp- man- p ET28a/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 systemof the new β- mannanase gene, and it may provide scientific basis for preparation used for bast fiber bio-degumming.

关 键 词：麻类脱胶 Β-甘露聚糖酶 表达体系 启动子 

分 类 号：Q939.97[生物学—微生物学]