【摘要】 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。更多还原

【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 25.06 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.更多还原