Cui L, Li Y, Liu B
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing.
Yi Chuan Xue Bao. 1993;20(6):561-70.
Our research showed that propionyl acylase gene is on 4.16kb insert DNA fragment originated from S. mycarofaciens in recombinant plasmid pIJM9. For localization 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.
我们的研究表明，丙酰化酶基因位于重组质粒pIJM9中源自弗氏链霉菌的4.16kb插入DNA片段上。为了将该基因定位在4.16kb插入DNA片段上，已对经BamHI消化的质粒pIJM9进行重新连接进行亚克隆。在转化子中，5号转化子中携带的重组质粒pIJM95的分子量为5.0kb。该质粒中插入DNA片段的分子量为0.53kb。在对5号转化子的螺旋霉素进行生物转化实验中，用薄层层析（TLC）、生物自显影、高效液相色谱（HPLC）和质谱（FAB）对生物转化产物进行了分析。结果表明，生物转化产物为丙酰螺旋霉素。5号转化子能够将螺旋霉素转化为丙酰螺旋霉素。丙酰化酶基因定位在重组质粒pIJM95的0.53kb插入DNA片段上。DNA序列分析结果表明，0.53kb插入DNA片段的G + C含量为68.2%。该片段上有70多种限制性内切酶的切割位点。从第54至393个核苷酸处，有一个开放阅读框，其编码一个由122个氨基酸组成的多肽。起始密码子为ATG，终止密码子为TGA。
