摘要
为了提高甘露聚糖酶Man23的表达量,降低它的体外降解程度,将其基因连接到质粒pHY-p43上,由强启动子p43启动基因man23的表达,将构建的重组质粒pHY-p43-man23转化至短短芽孢杆菌(Brevibacillus brevis)中,经转化的B.brevis发酵后的上清液中酶活高达22480 U?mL-1,较出发菌株Bacillus subtilisB23所产生的甘露聚糖酶活力提高了26.7%.B.brevis体系的表达产物经SDS-PAGE检测,其图谱比出发菌株表达产物的更为明晰,目的蛋白带更宽,带色更深,说明目的酶的产量得到了大幅提高,表达产物得到了明显纯化.试验表明以p43为启动子,B.brevis为表达质粒的宿主菌,不仅保证了基因产物的分泌和正确折叠,而且实现了基因的高表达和产物的高活力.
To promote the production of mannanase （Man23） and reduce its degradation in vitro, the man23 gene was ligated with a high-expression vector pHY-p43. The recombinant plasmid pHY-p43- man23 was transformed into Brevibacillus brevis. The man23 gene regulated by the strong promoter p43 produced mannanase activity of 22480 U·mL^-1, which was 26.7% higher than original strain Bacillus subtilis B23. Because B. brevis is a protease-defective strain, SDS-PAGE of the extracellular protein from B. brevis showed a much stronger band of the target protein and week or little non-target protein bands. This efficient and economic system can produce correctly folded extracellular mannanase with much higher yield and activity. The expression profile indicated that B. brevis was a perfect host cell for gene engineering.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2008年第4期389-394,共6页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
湖南省科技厅重点资助项目(01NKY1002)
