【摘要】 我们已往的工作已经确定在重组质粒pIJM9中,生米卡链霉菌来源的4.16kb插入DNA片段带有丙酰化酶基因,为了进一步确定该基因在4.16kb插入片段中的位置,我们以BamHI对pIJM9进行酶切,在此基础上进行缺失重组亚克隆,在所获得的转化子中,No.5转化子含重组质粒pIJM95,分子量为5.0kb,插入DNA片段为0.53kb。以No.5转化子对螺旋霉素进行生物转化实验,其转化产物经薄层层析,生物显迹,高压液相色谱及质谱(FAB)分析表明,与标准丙酰螺旋霉素一致,这说明No.5转化子具有将螺旋霉素生物转化为丙酰螺旋霉素的能力,丙酰化酶基因位于重组质粒pIJM95 0.53kb插入DNA片段上。 DNA序列测定结果表明,该片段G+C含量为68.2%,70多种限制性内切酶具有酶切位点。从No.54至No.393核苷酸有一开放阅读框架,编码一个122个氨基酸的多肽,起始密码子为ATG,终止密码子为TGA。更多还原

【Abstract】 Our research showed that propionyl acyla.se gene is on 4.16kb insert DNA fragment originated from S. mycarofacicns in recombinant plasmid pIJM9. For locoli-zation of this gene on 4.16kb insert DNA fragment, sub-cloning has been carried out with religation of BamHI digested plasmid pIJM9. Among the transformants, molecular weight of recombinant plasmid pIJM95 harbouring in No. 5 transformant is 5.0kb. Molecular weight of insert DNA fragment is 0.53kb in this plasmid. In bioconversion experiment for spiramycin of No. 5 transformant, the bioconversion product was analysed with TLC, bioautography, HPLC and mass spectrum (FAB). Results showed that the bioconvert product is propionylspiramycin. No. 5 transformant is able to transform spiramycin into propionylspiramycin. Propionyl acylase gene was locolized on 0.53kb insert DNA fragment of recombinant plasmid pIJM95.Analysis result of DNA sequence showed that content of G + C is 68.2% for 0.53kb insert DNA fragment. More than 70 kinds of restriction endonuclease have cut sit on this fragment. From No.54-No.393 nucleotide, there is an open reading frame, which codes a polypeptide consisted of 122 amino acids. Start codon is ATG, stop codon is TGA.更多还原