基于VBNC状态复苏的荧光假单胞杆菌CHZYR63对毒死蜱的酶降解效果
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摘要
利用复苏可培养化VBNC(Viable but non-culturable bacteria)资源菌筛选了对毒死蜱有降解性能的菌种及其酶.应用最大或然数计数(MPN)法,添加复苏促进因子Rpf蛋白分子,获取可培养化VBNC菌种,确定菌种的系统进化属性,并用常规培养法筛选了高效毒死蜱降解菌及其酶,探索了适宜的酶降解优化条件.从分离到的可培养化VBNC菌株中筛选出一株降解毒死蜱效果达70%的菌株CHZYR63,经16S r RNA基因序列分析,初步鉴定为荧光假单胞菌(Pseudomonas fluorescens).继而,从该菌株提取出粗酶,其降解毒死蜱能力为50%.降解条件优化研究表明:降解酶促反应的最适温度为35℃,最适p H值为7;在最适条件下该降解酶在30 min内对毒死蜱(100 mg/L)的降解率可达60.26%.
Using MPN system,some strains of the VBNC( viable but non-culturable) bacteria were isolated by Rpf( resuscitation promoting factor),identifying phylogenetic analysis. The Screening of chlorpyrifos-degrading bacteria and corresponding enzymes used routine methods,explored the appropriate optimal conditions of the degradation enzyme. One strain—CHZYR63 was selected from the bacteria which could degrade Chlorpyrifos. Its degradation efficiency could reach 70%. The strain was identified as Pseudomonas fluorescens based on 16 S r RNA sequence analysis. Degradation enzyme,whose degradation rate was 50%,was also extracted. A series of studies were conducted to determine its separation conditions and the characteristics of chlorpyrifos degradation. Studies showed that the optimal temperature and p H of enzymatic reaction was 35 ℃and 7,respectively. Under optimal conditions,its degradation rate of chlorpyrifos( 100 mg / L) was as high as60. 26% within 30 minutes.
Using MPN system,some strains of the VBNC( viable but non-culturable) bacteria were isolated by Rpf( resuscitation promoting factor),identifying phylogenetic analysis. The Screening of chlorpyrifos-degrading bacteria and corresponding enzymes used routine methods,explored the appropriate optimal conditions of the degradation enzyme. One strain—CHZYR63 was selected from the bacteria which could degrade Chlorpyrifos. Its degradation efficiency could reach 70%. The strain was identified as Pseudomonas fluorescens based on 16 S r RNA sequence analysis. Degradation enzyme,whose degradation rate was 50%,was also extracted. A series of studies were conducted to determine its separation conditions and the characteristics of chlorpyrifos degradation. Studies showed that the optimal temperature and p H of enzymatic reaction was 35 ℃and 7,respectively. Under optimal conditions,its degradation rate of chlorpyrifos( 100 mg / L) was as high as60. 26% within 30 minutes.
引文
[1]王利,余贤美,贺春萍.毒死蜱降解细菌WJI-063的鉴定及酶促降解特性[J].热带作物学报,2009,30(3):357-361.
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[14]Obojska A,Ternan N G,Lejczak B,et al.Organophosphonate utilization by the thermophile Geobacillus caldoxylosi lyticus T20[J].Applied and Environmental Microbiology,2002,68(4):2081-2084.
[15]刘新,尤民生,魏英智,等.降解毒死蜱曲霉Y的分离和降解效能测定[J].应用与环境生物学报,2003,9(1):78-80.
[16]王金花,朱鲁生,王军,等.3株真菌对毒死蜱的降解特性[J].应用与环境生物学报,2005,11(2):211-214.
[17]Yang Chao,Liu Na,Guo Xinmin,et al.Cloning of mpd gene from a chlorpyrifos-degrading bacterium and use of this strain in bioremediation of contaminated soil[J].FEMS Microbiology Letters,2006,265(1):118-125.
[18]Xu Gangming,Li Yingying,Zheng Wei,et al.Mineralization of chlorpyrifos by co-culture of Serratia and Trichosporon spp[J].Biotechnology Letters,2007,29(10):1469-1473.
[19]Ding L X.Studies on the isolation of viable but non-culturable bacteria and the phylogenetic analysis of the genus Aquaspirillum[D].Tokyo:The University of Tokyo,2004.
[20]丁林贤,苏晓梅,横田明.活的但非可培养(VBNC)状态菌的研究进展[J].微生物学报,2011,51(7):858-862.
[21]丁林贤,张萍华,洪华嫦,等.藤黄微球菌Rpf活性蛋白的制取及其对红球菌VBNC菌体的复苏作用[J].微生物学报,2012,52(1):77-82.
[22]王金花.毒死蜱降解微生物的筛选及其降解特性研究[D].泰安:山东农业大学,2004:22-23.
[23]谢慧,朱鲁生,王军,等.真菌WZ-I对有机磷杀虫剂毒死蜱的酶促降解[J].环境科学,2005(6):164-168.
[24]Mansour S A.Determination of residues of chlorpyifos and its oxygen analog in dates[J].Pesticide Sic,1985,10:677-688.
[25]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye biding[J].Anal Biochem,1976,72:248-254.
[26]王金花,陆贻通.农药毒死蜱的生态风险及其微生物修复技术研究进展[J].环境污染与防治,2006,28(2):125-127.
[27]Islam S M A,Math R K,Cho K M,et al.Organophosphorus hydrolase(Opd B)of lactobacillus brevis WCP902 from kimchi is able to degrade organophosphorus pesticides[J].Agricultural and Food Chemistry,2010,58(9):5380-5386.
[28]Feng Y,Racke K D,Bollag J M.Isolation and characterization of a chlorinated-pyridinol-degrading bacterium[J].Applied and Environmental Microbiology,1997,63(10):4096-4098.
[2]石利利,林玉锁,徐亦钢,等.毒死蜱农药环境行为研究[J].土壤与环境,2000,9(1):73-74.
[3]Beam S.Pesticides linked with birth weight[J].Environment,2004,46(5):8.
[4]Racke K D.Environmental fate of chlorpyrifos[J].Rev Environ Contam Toxicol,1993,131:1-150.
[5]Singh B K,Walker A,Wright D J.Degradation of pesticides in combination and their effect on soil microbial activity[J].British Crop Protection Council Monograph,2001,78:145-150.
[6]Liu B,Mc Connell L L,Torents A.Hydrolysis of chlorpyrifos in natural waters of the Chesapeake Bay[J].Chemosphere,2001,44(6):1315-1323.
[7]Phillips T A,Summerfelt R C,Wu J.Toxicity of chlorpyrifos adsorbed on humic colloids to larval walleye(Stizostedion vitreum)[J].Archives of Environmental Contamination and Toxicology,2003,45(2):258-263.
[8]Kumar Singh B,Walker A,Wright D J.Persistence of chlorpyrifos,fenamiphos,chlorothalorill and pendimethalin in soil and their effects on soil microbial characteristics[J].Bull Environ Contam Toxicol,2002,69(2):181-188.
[9]Lalah J O,Ondieki D,Wandiga S O.Dissipation,distribution,and uptake of14C-chlorpyrifos in a model tropical seawater/sediment/fish ecosystem[J].Bull Environ Contam Toxicol,2003,70(5):883-890.
[10]虞云龙,樊德方,陈鹤鑫.农药微生物降解的研究现状与发展策略[J].环境科学进展,1996,4(3):8-36.
[11]李艳,张有林,王宏.农药残留降解方法研究[J].食品研究与开发,2004,25(6):32.
[12]Singh B K,Walker A,Morgan J A W,et al.Effects of soil p H on the biodegradation of chlorpyrifos and isolation of a chlorpyrifos degrading bacterium[J].Applied and Environmental Microbiology,2003,69(9):5198-5206.
[13]Bending G D,Friloux M,Walker A.Degradation of contrasting pesticides by white rot fungi and its relationship with ligninolytic potential[J].FEMS Microbiology Letters,2002,212(1):59-63.
[14]Obojska A,Ternan N G,Lejczak B,et al.Organophosphonate utilization by the thermophile Geobacillus caldoxylosi lyticus T20[J].Applied and Environmental Microbiology,2002,68(4):2081-2084.
[15]刘新,尤民生,魏英智,等.降解毒死蜱曲霉Y的分离和降解效能测定[J].应用与环境生物学报,2003,9(1):78-80.
[16]王金花,朱鲁生,王军,等.3株真菌对毒死蜱的降解特性[J].应用与环境生物学报,2005,11(2):211-214.
[17]Yang Chao,Liu Na,Guo Xinmin,et al.Cloning of mpd gene from a chlorpyrifos-degrading bacterium and use of this strain in bioremediation of contaminated soil[J].FEMS Microbiology Letters,2006,265(1):118-125.
[18]Xu Gangming,Li Yingying,Zheng Wei,et al.Mineralization of chlorpyrifos by co-culture of Serratia and Trichosporon spp[J].Biotechnology Letters,2007,29(10):1469-1473.
[19]Ding L X.Studies on the isolation of viable but non-culturable bacteria and the phylogenetic analysis of the genus Aquaspirillum[D].Tokyo:The University of Tokyo,2004.
[20]丁林贤,苏晓梅,横田明.活的但非可培养(VBNC)状态菌的研究进展[J].微生物学报,2011,51(7):858-862.
[21]丁林贤,张萍华,洪华嫦,等.藤黄微球菌Rpf活性蛋白的制取及其对红球菌VBNC菌体的复苏作用[J].微生物学报,2012,52(1):77-82.
[22]王金花.毒死蜱降解微生物的筛选及其降解特性研究[D].泰安:山东农业大学,2004:22-23.
[23]谢慧,朱鲁生,王军,等.真菌WZ-I对有机磷杀虫剂毒死蜱的酶促降解[J].环境科学,2005(6):164-168.
[24]Mansour S A.Determination of residues of chlorpyifos and its oxygen analog in dates[J].Pesticide Sic,1985,10:677-688.
[25]Bradford M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye biding[J].Anal Biochem,1976,72:248-254.
[26]王金花,陆贻通.农药毒死蜱的生态风险及其微生物修复技术研究进展[J].环境污染与防治,2006,28(2):125-127.
[27]Islam S M A,Math R K,Cho K M,et al.Organophosphorus hydrolase(Opd B)of lactobacillus brevis WCP902 from kimchi is able to degrade organophosphorus pesticides[J].Agricultural and Food Chemistry,2010,58(9):5380-5386.
[28]Feng Y,Racke K D,Bollag J M.Isolation and characterization of a chlorinated-pyridinol-degrading bacterium[J].Applied and Environmental Microbiology,1997,63(10):4096-4098.