Paenibacillus sp.K1 β-半乳糖苷酶基因的克隆及在大肠杆菌中的表达

Cloning and expression of the beta-galactosidase gene of Paenibacillus sp.K1 in E.coli

从鲜牛奶中分离到1株产-βgalactosidase的细菌,经16S rDNA序列比对鉴定为类芽孢菌Paenibacillussp.K1。提取该菌株的染色体DNA,以pUC18(lac-)为载体,构建其DNA文库;在含有X-gal的LB平板上筛选该文库,得到6个蓝色菌落;对阳性克隆中插入的DNA片段序列测定,鉴定出1个编码全长为2 028 bp并携带有组成型启动子的β-半乳糖苷酶基因。将该基因导入大肠杆菌BL21(DE3)中,实现了β-半乳糖苷酶高效表达,其酶活为25.06 U/mL,高于原始菌株的4.55 U/mL,并进一步用亲和层析将该酶进行了纯化。

A bacteria strain with β-galactosidase activity was isolated from fresh milk on LB plates containing X-gel. It was identified as Paenibacillus sp. K1 by 16S rDNA analysis. The genomic DNA library of Paenibacillus sp. K1 was constructed in Escherichia coli DH5a with the vector pUC18 （lac-）. A gene of β-galactosidase was obtained by sequencing a positive clone with potential β-galactosidase activity from the DNA library. The full length of the gene is 2028 bp. A constructive promoter was found upstream of the ORF （open reading frame）. The overproduction of the β-galactosidase was carried out in E. coli BL21 （DE3）, and the β-galaetosidase activity in E. coli was 25.06 U/mL, which was more than 4.55 U/mL in the wild strain of Paenibacillus sp. K1. This enzyme was purified using affinity chromatography.