β-甘露聚糖酶基因克隆与在大肠杆菌中表达

The Cloning and Expression of β-Mannanase Gene in E. coli

为了通过基因工程方法在大肠杆菌中发酵生产β-甘露聚糖酶,采用多重比较几种来源的β-甘露聚糖酶氨基酸序列,获得了该酶的保守结构域,并按此设计简并引物.以能水解魔芋葡甘聚糖的枯草杆菌属野生筛选菌种A33为材料,通过简并引物PCR法从A33基因组中扩增出β-甘露聚糖酶基因核心区段.经过克隆、测序及BLASTN比对分析,证实该DNA区段推导的编码蛋白具有β-甘露聚糖酶的保守结构域,属于该酶家族中的一员.将该片段构建到大肠杆菌表达载体pRSET-A并转入大肠杆菌表达系统BL21DE3(pLysS),经过诱导获得了此酶的高效表达.

β-Mannanase is a general induced enzyme that can hydrolyze hemicellulose with the activity of cellulase. Degenerate primers are designed according to β-mannanase conservative sequence of several sources. A selected strain A33 of Bacillus which can hydrolysis the glucomannan of Amorphophallus was used as material to isolate the gene of β-mannanase by PCR. An identical molecule is amplified and cloned from A33 genomic DNA. Sequence analysis shows this DNA fragment encodes a putative protein containing the conserved domain of β-mannanase and it belongs to the family of mannanase. The amplified fragment is recombinated into the high efficiency expressive vector of pRSET-A and a recombinant plasmid pRSET-A-Mann is constructed. β-Mannanase is expressed in the E. coli strain BL21DE（pLysS） efficiently after inducing.