枯草芽孢杆菌Bs-916草酸脱羧酶基因Bacisubin的克隆、原核表达及其表达产物的酶活性分析  

Bacisubin,an oxalate decarboxylase in Bacillus subtilis Bs-916: gene cloning and catalytic activity assay of its prokaryoticlly expressed protein

作　　者：于俊杰[1] 陈志谊[1] 胡建坤[1] 俞咪娜[1] 聂亚锋[1] 尹小乐[1] 刘永锋[1] 

机构地区：[1]江苏省农业科学院植物保护研究所,江苏南京210014

出　　处：《江苏农业学报》2012年第3期497-502,共6页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省科技支撑计划项目(BE2011356);农业部引进国际先进科学技术项目(2011-G4)

摘　　要：为明确枯草芽孢杆菌生防菌株Bs-916的抑菌蛋白质Bacisubin的编码基因,并初步解析Bacisubin的抑菌机理,根据该蛋白质的部分序列设计简并引物,获得了编码基因的部分序列,参考基因组数据库克隆了Bacisubin基因全序列。将Bacisubin基因克隆至PET28a(+)中,在大肠杆菌BL21(DE3)pLysS中表达获得His标签融合蛋白质,并利用次甲基蓝-重铬酸钾法初步检测了该融合蛋白质的酶催化活性。克隆获得Bacisubin基因开放阅读框序列共1 161 bp,G+C含量为51.3%,可编码386个氨基酸,编码蛋白质与Bs-168菌株的草酸脱羧酶Yvrk蛋白质的氨基酸相似度为87%,与其他多种细菌和真菌的草酸脱羧酶的N端和C端Cupin活性中心区域和Mn2+结合区域高度保守。预测Bacisubin分子量为43 600,等电点为5.18,是较稳定的可溶性草酸脱羧酶,其催化最适pH为6.0左右,最适温度为30℃左右。说明枯草芽孢杆菌Bs-916的抑菌蛋白质Bacisubin为草酸脱羧酶,抑菌作用可能与其降解草酸的能力有关。To clone gene of an anti-fungal protein, Bacisubin, in Bacillus subtilis strain BS-916 which was isolated and developed as an biological control agent, the sequence of Bacisubin gene was amplified using a pair of degenerate primer, which was designed according to the small peptides of Bacisubin, and the open reading frame of Bacisubin gene was coloned consulting gene database of B. subtilis strain 168, subsequently. Bacisubin gene was transformed into PET28a（＋） vector, and was subse- quently expressed in Escherichia coli strain BL21 （DE3）pLysS. The oxalate decarboxylase activity of His-tagged Bacisubin was de- termined by methylene blue-dichromate system. Bacisubin gene was confirmed possessing an open reading frame （G＋C =51.3% ） at length of 1 161 bp, which could encode a protein of 386 amino acids. The deduced Bacisubin protein was predicted 43 600 at molecular weight, 5.18 at isoelectric point, and was stable and soluble. It was 87% identical with the oxalate decarboxylase Yvrkin B. subtilis strain 168, and was highly conserved in Mn2＋ binding areas and N- and C- terminal cupin structures of oxalate decarboxylases in several fungi and bacteria. The sol- uble His-tagged Bacisubin was confirmed having oxalate de- carboxylase activity, and its optimum catalytic condition was approximately at pH 6.0 and 30 ℃.

关 键 词：枯草芽孢杆菌 草酸脱羧酶 基因克隆 酶活性 

分 类 号：Q78[生物学—分子生物学]