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有机磷水解酶的大肠杆菌细胞表面展示 被引量：3

Cell-Surface Display of Organophosphorus Hydrolase in Escherichia coli
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摘要 PCR扩增来自黄杆菌ATCC27551的有机磷水解酶基因opd,直接与XcmⅠ酶切去除Ampr基因片段的pZXL-T连接,将opd克隆于锚定单元Lpp-OmpA编码序列下游,转化E.coliBL21(DE3).经抗性筛选、PCR鉴定和测序证实,成功构建了具有全细胞催化效应的大肠杆菌细胞表面展示工程菌.SDS-PAGE结果表明,工程菌能表达产生51 kD的融合蛋白.细胞表面展示的有机磷水解酶具有较高全细胞酶活性,用蛋白酶K消化处理重组菌表面蛋白可使其全细胞酶活降低90%. Opd genes of Flavobacterium sp. ATCC27551 strain coding organophosphate hydrolase is amplified by PCR, then ligated with vector pZXL-T which is digested with Xcm I to remove Ampr, so opd gene is cloned into anchoring motif Lpp-OmpA downstream and the recombinant plasmid is transformed into E. coli BL21（DE3）.After drug-resistant selection and identification by PCR and DNA sequencing, the cell-surface display engineering strain,which have a whole cell catalytic activity of organophosporus hydrolase, is successfully constructed. SDS-PAGE analyses eonfirm that the fusion protein can be expressed in engineering strain with a molecular of about 51 kD, and it is found that the engineering strain exhibits high whole cell activity of OPH. Additionally, OPH activity declines by 92 % Of total whole cell activity by treating the surface proteins with proteinase K.
作者 黎小军 林陈水 胡军民
机构地区 新余高等专科学校 浙江工业大学药学院 爱思进生物技术有限公司
出处 《江西师范大学学报（自然科学版）》 CAS 北大核心 2010年第1期93-97,共5页 Journal of Jiangxi Normal University(Natural Science Edition)
基金 江西省教育厅青年科学基金(GJJ09625)资助项目
关键词 有机鳞水解酶 细胞表面展示 T载体 大肠杆菌 organophosphorus hydrolase cell-surface display T-vector E. coli
分类号 Q939.9 [生物学—微生物学]
  • 相关文献

参考文献18

  • 1Zhang J L, Lan W H, Qiao C L, et ah Bioremediation of organophosphorus pesticides by surface-expressed carboxylesterase from mosquito in Escherichia coli [J]. Biotechnol Prog, 2004 , 20( 5 ) :1567-1571.
  • 2White B J, Harmon H J. Optical solid-state detection of organophosphates using organophosphorus hydrolase [ J]. Biosensors and Bioel, 2005,20(10) : 1977-1983.
  • 3Singh B K, Walker A. Microbial degradation of organophosphorus compounds [ J ]. FEMS Micmbiol Rev, 2006,30(3) :428-471.
  • 4Bondarenko S, Gan J Y, Hayer D L, et al. Persistence of selected organophosphate and carbamate insecticides in waters from a coastal watemhed [ J]. Envlmn Toxlcol Chem, 2004,23 (11) : 2649-2654.
  • 5Konstantinou I K, Hela D G,Albanis T A. The status of pesticide pollution in surface waters (rivers and lakes)of Greece part I review on occurrence and levels [ J]. Environ Pollut, 2006,141 (3) : 555-570.
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  • 7Mulbry W W, Karns J S. Parathion hydrolase specified by the Flavobacterium opd gene: relation ship between the gene and protein [ J]. J Bacteriol, 1989,171(12) : 6740-6746.
  • 8Dumas D P, Caldwell S R,Wild J R,et al.Purification and properties of the phosphotriesterase from Pseudomonas diminuta [J] .J Biol Chem, 1989,264(33) : 19659-19665.
  • 9Benhar I. Biotechnological applications of phage and cell display [J]. Biotechnol Adv,2001,19( 1 ):1-33.
  • 10Richins R D, Kaneva I, Mulchandani A, etal. Biodegradation of organophosphorus pesticides by surface-expressed organophosphorus hydrolase [J]. Nature Biotechnology, 1997,15:984-987.

同被引文献32

  • 1Bondarenko S,Gan J Y,Hayer D L,et a1.Persistence of selected organophosphate and carbamate insecticides in waters from a coastal watemhed[J].Envlmn Toxlco1 Chem,2004,23(11):2649-2654.
  • 2Singh B K,Walker A.Microbial degradation of organophosphorus compounds[J].FEMS Micmbio1 Rev,2006,30(3):428-471.
  • 3Konstantinou I K,Hela D G,Albanis T A.The status of pesticide pollution in surface waters(rivers and lakes) of Greece.Part I.Review on occurrence and levels[J].Environ Pollut,2006,141(3):555-570.
  • 4Mulbry W W,Karns J S.Parathion hydrolase specified by the Flavobacterium opd gene:relation ship between the gene and protein[J].J Bacteriol,1989,171(12):6740-6746.
  • 5Dumas D P,Caldwell S R,Wild J R,et al.Purification and properties of the phosphotriesterase from Pseudomonas diminuta[J].J Biol Chem,1989,264(33):19659-19665.
  • 6Gopal S,Rastogi V,Ashman W.Mutagenesis of organophosphorus hydrolase to enhance hydrolysis of the nerve agent VX[J].Biochem Bio Res Com,2000,279(2):516-519.
  • 7Shimazu M,Mulchandani A,Chen W.Cell surface display of organophosphorus hydrolase using ice nucleation protein[J].Biotechnol Prog,2001,17(1):76-80.
  • 8Zhang J L, Lan W H, Qiao C L,et al. Bioremediation of organophosphorus pesticides by surface-expressed carboxylesterase from mosquito in Escherichia coli [J]. Biotechnol Prog, 2004,20( 5 ) : 1567-1571.
  • 9White B J, Harmon H J. Optical solid-state detection of organophosphates using organophosphorus hydrolase[J]. Biosensors and Bioel,2005,20(10):1977-1983.
  • 10Singh B K, Walker A. Microbial degradation of organophosphorus compounds [J]. FEMS Micmbiol Rev, 2006,30(3) :428-471.

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江西师范大学学报（自然科学版）
2010年 第1期

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