CAJ | 학술논문

目的：克隆酮古龙酸菌D-2-羟酸脱氢酶（2-hydroxyacid dehydrogenase ， HADH）基因hadh，并在大肠杆菌中进行表达、纯化和酶学性质检测。方法 PCR扩增酮古龙酸菌D-2-羟酸脱氢酶基因hadh，构建表达载体pTIG-hadh，转化大肠杆菌BL21（DE3），对重组菌进行诱导表达，表达产物经Ni＋亲和层析柱进行纯化后进行酶学性质检测。结果 SDS-PAGE分析结果显示，重组菌中HADH表达量可达菌体总蛋白的50％以上，相对分子质量约35×103。酶学性质检测结果表明，纯化的HADH以2-酮基古龙酸（2-keto-gulonic acid，2-KGA）为底物，最适pH 8．0，最适温度45℃，几种常见金属离子和螯合剂对HADH的活性影响微弱或无影响，以2-KGA为底物时的HADH最大反应速度27 U/mg，Km值为2．6 mmol/L，体内酶活检测结果表明，HADH能够代谢2-KGA。结论重组HADH在大肠杆菌中获得了高效表达，并研究了其酶学性质，为后续山梨糖代谢通路研究和进一步提高糖酸转化率奠定了基础。
목적：극륭동고룡산균D-2-간산탈경매（2-hydroxyacid dehydrogenase ， HADH）기인hadh，병재대장간균중진행표체、순화화매학성질검측。방법 PCR확증동고룡산균D-2-간산탈경매기인hadh，구건표체재체pTIG-hadh，전화대장간균BL21（DE3），대중조균진행유도표체，표체산물경Ni＋친화층석주진행순화후진행매학성질검측。결과 SDS-PAGE분석결과현시，중조균중HADH표체량가체균체총단백적50％이상，상대분자질량약35×103。매학성질검측결과표명，순화적HADH이2-동기고룡산（2-keto-gulonic acid，2-KGA）위저물，최괄pH 8．0，최괄온도45℃，궤충상견금속리자화오합제대HADH적활성영향미약혹무영향，이2-KGA위저물시적HADH최대반응속도27 U/mg，Km치위2．6 mmol/L，체내매활검측결과표명，HADH능구대사2-KGA。결론중조HADH재대장간균중획득료고효표체，병연구료기매학성질，위후속산리당대사통로연구화진일보제고당산전화솔전정료기출。
Objective To clone 2-hydroxyacid dehydrogenase (HADH) gene from Ketogulonigenium vulgare(KGV) Y25 and investigate its expression , purification, and enzymatic characterics .Methods The hadh gene was amplified from ketogulonigenium vulgare Y 25 and cloned into the expression plasmid pITG .The recombinant plasmid was transformed into Escherichia coli BL21(DE3).HADH was then successfully expressed with induction .To explore its enzymatic characteris-tics,HADH was purified by Ni +exchange chromatography .Results HADH constituted more than 50% of the total cell proteins analyzed by SDS-PAGE,with a relative molecular mass of about 35 ×103.With 2-keto-gulonic acid(2-KGA) as substrate, the optimal pH of HADH was at 8.0 ,while the optimal temperature of the purified HADH was at 45℃.Mean-while, such metal ions and chelating agents as Cu 2+, Ca2+, Mg2+, EDTA, and DEPC exerted little effect on enzymatic activities.The maximum initial activity of the enzyme towards 2-KGA reached 27 U/mg, and the Km was calculated as 2.6 mmol/L.The results of in vivo enzyme activity assay showed that HADH could metabolize 2-KGA.Conclusion The HADH gene form Y25 is successfully expressed in E.coli BL21 ( DE3 ) and the enzymatic characteristics of HADH are explored, which will facilitate subsequent studies on sorbose metabolic pathways and sugar acid conversion .