芽孢杆菌β-甘露聚糖酶基因的克隆及表达  

The Cloning and Expression of Bacillus sp.MX β-mannanase Gene

作　　者：马鑫[1] 赵仲麟[1,2] 李亮[1] 陈明[1] 林敏[1] 平淑珍[1] 

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]中国农业大学生物学院,北京100193

出　　处：《生物技术通报》2009年第2期77-81,共5页Biotechnology Bulletin

基　　金：国家863计划重大专项“新型植酸酶分子设计技术”(2006AA020202);国家863计划重点项目“特殊环境微生物资源的开发利用技术”(2007AA021304)

摘　　要：从新疆极端干燥环境土壤样品中筛选到具有高β-甘露聚糖酶活性的芽孢杆菌(Bacillussp.MX)。运用PCR技术从该菌基因组中克隆得到β-甘露聚糖酶基因,连接到表达载体pET-28a上,在大肠杆菌BL21中高效表达的基因产物经亲和层析纯化,SDS-PAGE凝胶电泳分析显示该蛋白的相对分子质量为41kD。酶学性质分析表明该酶在25～95℃,pH3.0～9.6范围内均具有酶活。最适作用温度55℃和pH值5.0,酶比活力为4572U/mg。在最适pH5.0,高温85℃和95℃分别处理10min后,该酶相对酶活力仍保持51%和34%,显示β-甘露聚糖酶具有较好的耐酸性和热稳定性。A strain of Bacillus sp. MX chat have a high activity of β-mannanase was isolated fi＇om the soil sample of extremely dry environment in Xin.jiang province. The complete gene sequece of β-mannanase was amplified by PCR and inserted into the pLasmid pET-28a. The target protein was expressed efficiendy after transformed into E. coli BL21 and induced with IPTG. The molecular mass of the purified protein was determined to be approximately 41 kD by SDS-PAGE. The analysis of enzymatic properties showed that the specific activity of β-mannanase was 4 572 U/rag,and the optimal reaction temperature were 53℃ and pH5.0,respectively. The enzyme activity of the purified protein remained 51% and 34% of the initial activity at pH 4.0,85℃ and 95℃ treated for 10 min,respectively,suggesting that the β-mannanase from Bacillus sp MX presented good acid resistance and thermal stability.

关 键 词：芽孢杆菌 Β-甘露聚糖酶 表达 酶活 

分 类 号：Q78[生物学—分子生物学] S816[农业科学—饲料科学]