中国抗生素杂志
中國抗生素雜誌
중국항생소잡지
CHINESE JOURNAL OF ANTIBIOTICS
2008年
3期
140-144
,共5页
焦豫良%王梁华%董晓毅%宗英%高云%任娜%郭爱芸%张兴群%焦炳华
焦豫良%王樑華%董曉毅%宗英%高雲%任娜%郭愛蕓%張興群%焦炳華
초예량%왕량화%동효의%종영%고운%임나%곽애예%장흥군%초병화
聚酮合酶%酰基转移酶%乙酰基转移酶%结合常数%组合生物合成
聚酮閤酶%酰基轉移酶%乙酰基轉移酶%結閤常數%組閤生物閤成
취동합매%선기전이매%을선기전이매%결합상수%조합생물합성
目的 研究新型聚酮合酶(PKS)基因簇EF568935(Gen Bank登录号)中酰基转移酶(AT)EF080951(GenBank登录号)底物特异性,为PKS的功能研究及组合生物合成研究提供新组件.方法 从酰基转移酶AT-EF080951结构域两端的连接肽区设计引物,通过PCR克隆其结构域基因at-EF080951;连接入原核表达载体pMAL-c2X,与麦芽糖结合蛋白(MBP)融合表达;直链淀粉树脂柱亲和层析纯化MBP-AT-EF08095l;融合蛋白用Xa因子切除MBP,得到AT-EF080951单体;以MBP、小牛血清白蛋白(BSA)为对照蛋白,用紫外分光光度法测定MBP-AT-EF080951与AT-EF080951对底物的结合能力,计算结合常数(Ka,μmol/L),分析其结合底物的特异性.结果 (1)MBP-AT-EF080951与底物的结合常数为:甲基丙二酰辅酶A,0.0049±0.001;丙二酰辅酶A,0.0049±0.003;乙酰辅酶A,0.107±0.002;辅酶A,0.005±0.003.(2)AT-EF080951与底物的结合常数为:甲基丙二酰辅酶A,0.005±0.002;丙二酰辅酶A,0.0041±0.002;乙酰辅酶A,0.120±0.001;辅酶A,0.0042±0.003.结论 MBP-AT-EF080951和AT-EF080951都特异性结合乙酰辅酶A,AT-EF080951可能是乙酰转移酶.
目的 研究新型聚酮閤酶(PKS)基因簇EF568935(Gen Bank登錄號)中酰基轉移酶(AT)EF080951(GenBank登錄號)底物特異性,為PKS的功能研究及組閤生物閤成研究提供新組件.方法 從酰基轉移酶AT-EF080951結構域兩耑的連接肽區設計引物,通過PCR剋隆其結構域基因at-EF080951;連接入原覈錶達載體pMAL-c2X,與麥芽糖結閤蛋白(MBP)融閤錶達;直鏈澱粉樹脂柱親和層析純化MBP-AT-EF08095l;融閤蛋白用Xa因子切除MBP,得到AT-EF080951單體;以MBP、小牛血清白蛋白(BSA)為對照蛋白,用紫外分光光度法測定MBP-AT-EF080951與AT-EF080951對底物的結閤能力,計算結閤常數(Ka,μmol/L),分析其結閤底物的特異性.結果 (1)MBP-AT-EF080951與底物的結閤常數為:甲基丙二酰輔酶A,0.0049±0.001;丙二酰輔酶A,0.0049±0.003;乙酰輔酶A,0.107±0.002;輔酶A,0.005±0.003.(2)AT-EF080951與底物的結閤常數為:甲基丙二酰輔酶A,0.005±0.002;丙二酰輔酶A,0.0041±0.002;乙酰輔酶A,0.120±0.001;輔酶A,0.0042±0.003.結論 MBP-AT-EF080951和AT-EF080951都特異性結閤乙酰輔酶A,AT-EF080951可能是乙酰轉移酶.
목적 연구신형취동합매(PKS)기인족EF568935(Gen Bank등록호)중선기전이매(AT)EF080951(GenBank등록호)저물특이성,위PKS적공능연구급조합생물합성연구제공신조건.방법 종선기전이매AT-EF080951결구역량단적련접태구설계인물,통과PCR극륭기결구역기인at-EF080951;련접입원핵표체재체pMAL-c2X,여맥아당결합단백(MBP)융합표체;직련정분수지주친화층석순화MBP-AT-EF08095l;융합단백용Xa인자절제MBP,득도AT-EF080951단체;이MBP、소우혈청백단백(BSA)위대조단백,용자외분광광도법측정MBP-AT-EF080951여AT-EF080951대저물적결합능력,계산결합상수(Ka,μmol/L),분석기결합저물적특이성.결과 (1)MBP-AT-EF080951여저물적결합상수위:갑기병이선보매A,0.0049±0.001;병이선보매A,0.0049±0.003;을선보매A,0.107±0.002;보매A,0.005±0.003.(2)AT-EF080951여저물적결합상수위:갑기병이선보매A,0.005±0.002;병이선보매A,0.0041±0.002;을선보매A,0.120±0.001;보매A,0.0042±0.003.결론 MBP-AT-EF080951화AT-EF080951도특이성결합을선보매A,AT-EF080951가능시을선전이매.
