摘要
提取热纤梭菌(Clostridium.sp)基因组DNA,首次通过PCR克隆了该菌的β-葡聚糖酶基因全长。结果表明:该基因全长1040bp,ORF为1003bp,编码334个氨基酸,计算分子质量为37.8kD,等电点为7.67。经Blast分析,该序列与热纤梭菌同源性最高(99%),而与基因库中嗜热梭菌的同源性为94%,该基因已被GenBank接受(AY225318)。用BamHⅠ和XhoⅠ双酶切目的片段和表达载体pET-30a(+)后相连接,构建重组表达载体pET-clo,并导入BL21细菌中表达。酶学特性表明:SDS-PAGE电泳在37kD左右有表达蛋白带,该工程菌最适酶活29.4U/mL,是出发菌的15倍,最适温度80℃,最适pH9。该工程菌可作为耐热性材料构建耐热性好、酶活高的杂合基因工程菌。
A gene encoding a β-glucanase from Clostridium. sp has been cloned and expressed in Escherichia coli. The whole length of the gene is 1 040 bp, containing an open reading frame （ORF） of 1 003 bp, which encodes 334 amino acids. The enzyme showsed that a molecular weight of 37.8 kDa and a pI of 7.67. The nucleotide sequence of the gene shares high homology to that of Clostridium thermocellum accessed in GeneBank, and the similarity is 99%. The gene has been registered in GeneBank （Accession number is AY225218） . The gene from recombinant cloning plasmid was subcloned into Bam H Ⅰ and Xho Ⅰ site of expression plasmid pET-clo in E. coli. The recombinant expression plasmid was transformed into E. coli strain BL 21. The result of SDS-PAGE showed that about 37 kDa protein of the β-1, 3-1, 4-glucanase was expressed in E. coli strain BL 21. The enzyme activity of E. coli expressed strain was 29.4 U/ mL, which was about 15-fold higher than that of original strain. The pH and temperature at which the highest activity was found were 9 and 80 ℃ respectively, It＇s good material for gene engineer to construct highly active and thermophilic enzymatic gene.
出处
《中国食品学报》
EI
CAS
CSCD
2006年第1期320-324,共5页
Journal of Chinese Institute Of Food Science and Technology
基金
国家自然科学基金项目(No.30000118)
