β-甘露聚糖酶基因的克隆表达及酶学性质

Cloning and Expression of β-mannanase Gene and Study of Enzymatic Properties

以能水解魔芋葡甘聚糖的野生筛选菌种枯草杆菌A 33为材料,通过PCR技术从A 33基因组中扩增出β-甘露聚糖酶基因编码序列.经过克隆、测序及BLA ST比对分析,证实该基因编码β-甘露聚糖酶,属于β-甘露聚糖酶家族中的一员,该基因已注册G enBank.将该基因克隆到原核表达载体pRSET-A中并转入大肠杆菌表达系统BL 21(DE 3)pLysS,经过诱导获得了此酶的高效表达.其表达量达到37.78 U/mL,酶学特性分析表明其作用的最适pH为5.0,最适温度为60℃.

A selected strain of Bacillus subtilis A33 which can hydrolyze the glucomannan of Amorphophallus was used as material to isolate β-mannanase gene by PCR. Sequencing analysis shows that the sequence was encoded β-rnannanase which belongs to the family of mannanase. This sequence was submitted to GenBank. To express it in E. coli it was recombinant into the high efficiency expressive vector pRSET-A and a recombinant plasmid was constructed and transformed into the strain E. coli BL21（DE3）pLysS, β-mannanase gene was expressed efficiently after induction with IPTG.. An expression activity 37. 78U/ml was obtained. The enzymatic analysis of the mannanase reveals that its optimal pH and temperature were respectively 5.0 and 60℃.