缺失氨肽酶N的变铅青链霉菌的构建和鉴定

用PCR方法扩增变铅青链霉菌(Streptomyces lividans)TK24氨肽酶N基因,体外插入卡那霉素抗性基因进行失活,然后利用不含链霉菌复制起点的重组质粒pPEPN-KAN进行同源重组,获得了氨肽酶N缺失的菌株PEPN-.突变株的胞外氨肽酶N活性与原株基本相同,而胞内氨肽酶N的活性明显低于原株,为原株的42.5±5.7%.

CONSTRUCTION AND CHARACTERIZATION OF AMINOPEPTIDASE N DEFICIENT STREPTOMYCES LIVIDANS TK24

N terminal degradation has been noted for some homologous and heterologous proteins expressed in Streptomyces lividans , and genes of some aminopeptidases have been cloned Pep N is the one which has broad substrate specificities and the major leucine aminopeptidase activity in S lividans We cloned the pepN gene by PCR and inactivated it by homologous recombination to replace the wild type chromosomal pepN with a mutant gene in which kanamycin resistant gene was inserted The recombinational exchange of mutant and wild type allele was effected using a non replicating plasmid pPEPN KAN (derivative of pUC19) The intracellular and extracellular PepN activities of wild type and negative strains were detected The ability of the negative strains (designated PEPN -) to hydrolyze Leu pNA was significantly reduced compared to the wild type