碱性芽孢杆菌木聚糖酶基因的克隆及表达

本实验从广西红树林土壤中筛选出一株产木聚糖酶的耐碱菌HSL38，经过16S rDNA鉴定，其与碱性芽孢杆菌Bacillus halodurans C-125(GenBank：NC_002570.2)的同源性达到99%。参考Bacillus halodurans C-125的β-1,4-木聚糖酶基因xynA设计引物，扩增出HSL38中的β-1,4-木聚糖酶基因xyl-BH-G10，其与xynA的同源性达到99%，编码396个氨基酸残基(45.27kD)，属于糖基水解酶GH10家族。将xyl-BH-G10基因与表达载体pET-30a-c(+)连接，构建重组载体，转化大肠杆菌E.coli BL21(DE3)，IPTG诱导表达重组蛋白。结果表明，在最佳诱导条件下，木聚糖酶基因xyl-BH-G10在大肠杆菌细胞内高效表达，酶活力达到55.28U/mL，是原菌株HSL38发酵液酶活力的4倍。

Cloning and Expression of Xylanase Gene from Alkaliphilic Bacillus halodurans

In this study, an alkaliphilic strain (HSL38) producing xylanase was isolated from mangrove soil, and identified as Bacillus halodurans C-125 (GenBank: NC_002570.2) based on 16S rDNA analysis, which revealed a similarity of 99%. Specific primers based on theβ-1,4-xylanase gene xynA of Bacillus halodurans C-125 were designed, and theβ-1,4-xylanase gene (xyl-BH-G10) from HSL38 strain was amplified. Sequence analysis showed 99% similarity between the xynA gene and the amplified gene encoding a precursor of 396 amino acid residues (45.27 kD), which belonged to glycoside hydrolase family 10 GH10. A recombinant expression plasmid was constructed by linking xyl-BH-G10 to the expression plasmid pET-30a-c(+), and then transformed into E. coli BL21(DE3). The expression of recombinant protein was achieved under IPTG induction. These investigations indicated that theβ-1,4-xylanase gene xyl-BH-G10 was highly expressed in E. coli BL21 (DE3) cells under the optimal induction conditions, and an xylanase activity of 55.28 U/mL was obtained, which exhibited a 4-fold enhancement when compared to the fermentation of original HSL38 strain.