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

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

陆文伟, 孔文涛, 孙芝兰, 孔健*, 季明杰

山东大学微生物技术国家重点实验室，山东济南 250100

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2006-10-24 发布日期:2006-10-24
通讯作者: 孔健

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

LU Wen-wei, KONG Wen-tao, SUN Zhi-lan, KONG Jian*, JI Ming-jie

State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Shandong, China

Received:1900-01-01 Revised:1900-01-01 Online:2006-10-24 Published:2006-10-24
Contact: KONG Jian

摘要/Abstract

摘要：

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。

关键词:

类芽孢杆菌, DNA文库

, β-半乳糖苷酶

Abstract:

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.

Key words: DNA library, β-galactosidase, Paenibacillus

中图分类号:

Q781

陆文伟,孔文涛,孙芝兰,孔健*,季明杰 . Paenibacillus sp. K1 β-半乳糖苷酶基因的克隆及在大肠杆菌中的表达[J]. J4, 2008, 43(7): 69-73 .

LU Wen-wei,KONG Wen-tao,SUN Zhi-lan,KONG Jian*,JI Ming-jie

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

[J]. J4, 2008, 43(7): 69-73 .

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