地衣芽孢杆菌β-1,3-1,4葡聚糖酶基因的克隆和表达  

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

作　　者：吕文平[1] 许梓荣[1] 杜文理[1] 李卫芬[1] 孙建义[1] 

机构地区：[1]浙江大学饲料科学研究所教育部动物分子营养重点实验室,杭州310029

出　　处：《农业生物技术学报》2004年第4期446-449,共4页Journal of Agricultural Biotechnology

基　　金：高等学校骨干教师资助计划资助;国家自然科学基金(No.30000118)。

摘　　要：提取地衣芽孢杆菌(Bacilluslicheniformis)基因组,通过PCR克隆了该菌的β-1,3-1,4-葡聚糖酶基因全长。该基因全长818 bp,ORF为732 bp,编码243个氨基酸,计算分子量为27.35 kD,等电点为8.31。经Blast分析,该序列与短小芽孢杆菌(B.pumilus )同源性最高(99%),而与基因库中地衣芽孢杆菌的同源性为94%,该基因已被GenBank接受(AY225317)。用BamHⅠ和XhoⅠ双酶切目的片断和表达载体pET-30a(+)后相连接,构建重组表达载体pET-lic,并导入大肠杆菌(Escherichiacoli )BL21菌株中表达。酶学特性表明:SDS-PAGE电泳在27 kD左右有表达蛋白带,该工程菌总酶活达67.34 U/mL,是出发菌的60倍,最适温度在50 ℃左右,最适pH在5～6之间。该工程菌可作为材料构建耐热性好和酶活高的杂合基因工程菌。A gene encodingβ-glucanase from Bacillus licheniformis was cloned and expressed in Escherichia coli.. The whole length of the gene was 818 bp, including an ORF of 732 bp and encodeing 243 amino acids. The enzyme showed a molecular size of 27.35 kD and a pI of 8.31. The nucleotide sequence of the gene shared high degree of sequence similarity to that of B.pumilus and B.licheniformis accessed in GenBank, and their similarities were 99% and 94%respectively. The gene has been registered in Genank(Accession number is AY225317). The gene from recombinant cloning plasmid was subcloned into Bam HⅠand XhoⅠsite of expression plasmid pET-lic in E. coli . The recombinant expression plasmid was transformed into E. coli strain BL21. The result of SDS-PAGE showed that about 27 kD protein of theβ-1,3-1,4-glucanase was expressed in E.coli strain BL21. The enzyme activity of E.coli expressed strain was 67.34 U/mL, which was about 60-fold higher than that of original strain. The pH and temperature at the highest activity was found were pH 5~6 and 50 ℃ respectively. It’s a good material for gene engineering to construct highly active and thermophilic enzymatic gene.

关 键 词：地衣芽孢杆菌 葡聚糖酶基因 β-1 克隆和表达 SDS-PAGE电泳 GENBANK 短小芽孢杆菌 重组表达载体 PCR克隆 Blast 基因工程菌 大肠杆菌 最适温度 最适PH 同源性 基因组 氨基酸 分子量 等电点 基因库 双酶切 特性表 耐热性 

分 类 号：Q939.124[生物学—微生物学] S565.4[农业科学—作物学]