短小芽孢杆菌β-1,3-1,4-葡聚糖酶基因克隆、表达及其酶特性研究

Cloning and Expression of β-1,3-1,4-glucanase Gene from Bacillus pumilus.

提取短小芽孢杆菌(Bacilluspumilus)基因组,通过PCR克隆了短小芽孢杆菌β-葡聚糖酶基因全长序列.结果表明:该基因全长754bp,ORF为717bp,编码239个氨基酸,计算分子量为26.98kD,等电点为6.08.经Blast分析,该序列与地衣芽孢杆菌同源性最高(91%),该基因首次在GenBank上登录(登录号AY164456).在克隆载体上亚克隆基因全长,构建重组表达载体pET-pum,导入大肠杆菌BL21中表达.SDS-PAGE电泳表明:在27kD左右有表达带,酶活较低(7.24U/mL),仅为出发菌酶活的16%,其最适温度为50℃左右,最适pH在5左右.

A gene encoding β-glucanase from Bacillus pumilus has been cloned and expressed in Escherichia coli. The whole length of the gene was 754 bp, including an ORF of 717 bp encoding 239 amino acids. The enzyme showed a molecular size of 26.98 kD and a pI of 6.08.The gene showed a high degree of similarity to B.pumilus,amounting to homology of 91%. The gene was firstly registered in GenBank (Accession No: AY164456). The expression plasmid named pET-pum was constructed and transformed into E.coli BL21.The result of SDS-PAGE showed that a protein of about 27 kD was expressed in E.coli. The enzyme activity expressed in E.coli was 7.24 U/mL. The optimal pH and temperature for the this enzyme activity were respectively found to be about 5 and 50 ℃.