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

从鲜牛奶中分离到1株产β-galactosidase的细菌，经16S rDNA序列比对鉴定为类芽孢菌Paenibacillus sp. 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2028bp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25.06U/mL，高于原始菌株的4.55U/mL，并进一步用亲和层析将该酶进行了纯化。

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

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 DH5α 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-galactosidase activity in E.coli was 25.06U/mL, which was more than 4.55U/mL in the wild strain of Paenibacillus sp. K1. This enzyme was purified using affinity chromatography.