CAJ | 학술논문

应用PCR从杜仲内生Pseudomonas koreensis JDM-2株基因组中扩增出ACC脱氨酶基因acdS,将其克隆到pMD18-T载体并段进行DNA测序,通过BamHⅠ和HindⅢ酶切从重组质粒pMD18ACDS中切下acdS基因序列,将其连接到pQE30质粒的BamHⅠ和HindⅢ位点,构建表达质粒pQE30ACDS,转化大肠杆菌BL21,利用比色法测定ACC脱氨酶活力.测序结果表明,JDM-2菌株acdS基因大小为1 017bp,与已报道不同菌种acdS基因的同源性在86%～99%之间.SDS-PAGE结果显示在大肠杆菌BL21中表达了分子量为38kDa的ACC脱氨酶.酶活性检测结果显示重组菌ACC脱氨酶的酶活力为0.214U/mg.
응용PCR종두중내생Pseudomonas koreensis JDM-2주기인조중확증출ACC탈안매기인acdS,장기극륭도pMD18-T재체병단진행DNA측서,통과BamHⅠ화HindⅢ매절종중조질립pMD18ACDS중절하acdS기인서렬,장기련접도pQE30질립적BamHⅠ화HindⅢ위점,구건표체질립pQE30ACDS,전화대장간균BL21,이용비색법측정ACC탈안매활력.측서결과표명,JDM-2균주acdS기인대소위1 017bp,여이보도불동균충acdS기인적동원성재86%～99%지간.SDS-PAGE결과현시재대장간균BL21중표체료분자량위38kDa적ACC탈안매.매활성검측결과현시중조균ACC탈안매적매활력위0.214U/mg.
The acdS gene encoding an ACC deaminase was amplified by PCR from genomic DNA of Pseudomonas koreensis JDM-2,and the PCR product was ligated into the pMD18-T vector and then sequenced. The acdS gene was excised from plasmid pMD18ACDS by double digestion with BanH Ⅰ and Hind Ⅲ ,and cloned into the prokaryotic expression vector pQE30 to provide plasmid of pQE30ACDS. The ACC deaminase was expressed in Escherichia coli BL21 harboring the plasmid pQE30ACDS by IPTG induction. The sequence analyses showed that the acdS gene was 1 017 bp in length,DNA homolo- gy of the acdS genes between the Pseudomonas koreensis JDM-2 and the previously reported different bacterial strains in GenBank was 86% to 99 %. The SDS-PAGE analyses showed a single protein band with a molecular weight of 39 kDa expressed in Escherichia coli BL21 ,and its enzyme activity was 0. 214 U/mg.