有机磷农药降解菌的分离鉴定、生物学特性和有机磷水解酶基因（ophc2）的克隆及表达
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摘要
从长期受有机磷农药污染的土壤中分离到辛硫磷降解菌XSP-1和甲基对硫磷降解菌SMSP-1。经生理生化鉴定和16S rRNA基因同源性比较,将它们初步鉴定为戴尔福特菌属(Delftia sp.)XSP-1(GenBank Accession No.EF061135),及寡养单胞菌属(Stenotrophomonas sp.)SMSP-1(GenBank Accession No.EU312979)。
菌株XSP-1在pH5.0-9.0范围内生长良好;最适生长温度为30℃;好氧性生长;在5-35g╱L NaCl范围生长较好;在以果糖为碳源,以有机氮为氮源的培养基中生长较好。菌株XSP-1能在7小时内完全降解100mg╱L的辛硫磷;最适降解条件为pH为7.0,35℃。该菌株对甲基对硫磷、倍硫磷和杀螟硫磷也有较好的降解能力,对毒死蜱降解能力较差。
菌株SMSP-1在pH5.0-10.0范围内生长良好;最适生长温度为30℃和35℃;好氧性生长;在5-45g╱L NaCl范围生长较好;在以葡萄糖为碳源,以有机氮为氮源的培养基中生长较好。SMSP-1在48h内能将20mg/L的甲基对硫磷完全降解为对硝基酚,但是对对硝基酚没有进一步的降解能力;降解甲基对硫磷的最适条件为pH7.0、30℃。菌株SMSP-1还能降解杀螟硫磷、乙基对硫磷、倍硫磷和辛硫磷,对毒死蜱没有降解能力。通过构建基因组文库,克隆到菌株SMSP-1的有机磷水解酶基因,运用在线分析软件,推断出有机磷水解酶的结构基因、信号肽及启动子序列。通过在线比对同源性,该基因与Pseudomonas pseudoalcaligenes C2-1中的ophc2基因(GenBank accessionNo.AJ605330)同源性为98%,这是国际上第二次克隆到有机磷水解酶基因ophc2,也是第一次在寡养单胞菌属中克隆到这个基因。根据基因序列分析结果设计特异性引物,通过PCR扩增到带有信号肽和不带有信号肽的ophc2结构基因,分别将其构建到表达载体pET29a中,转化到E.coli BL21(DE3)中进行了表达。根据蛋白电泳结果推断,在有机磷水解酶OPHC2中可能存在着一个能被菌株BL21(DE3)胞内的某个蛋白酶识别并切割的位点,并且该位点位于OPHC2的C-端。
Phoxim-degrdading strain XSP-1 and methyl parathion-degrading strain SMSP-1 were isolated from organophosphate pesticides-contaminated soil. These bacterium were preliminarily identified as Delftia sp. and Stenotrophomonas sp. based on the physiological and biochemical analysis and 16S rRNA gene series source analysis. They were designated as XSP-1 (GenBank Accession No. EF061135) and SMSP-1 (GenBank Accession No. EU312979) respectively.
Biological properties were analyzed respectively. The optimal pH value for the growth of strain XSP-1 was 5.0-9.0, the optimal temperature was 30℃. The optimal osmotic pressure for strain XSP-1 was 5-35g NaCl/L. The optimal medium for the growth of strain XSP-1 was fructose as carbon source and organic nitrogen as nitrogen source. Strain XSP-1 could completely degrade phoxim within 7h, the optimal phoxim-degrading condition was pH7.0, 30℃. Strain XSP-1 could also degrade fenitrothion, methyl parathion and fenthion rapidly, but had little degrading ability towards chlorpyrifos.
Strain SMSP-1 could grow well through pH5.0-10.0, the optimal temperature were 30℃and 35℃, the optimal osmotic pressure was 5-45g NaCl/L. The optimal medium for growth was glucose as carbon source and organic nitrogen as nitrogen source. SMSP-1 could completely hydrolyze methyl parathion to p-nitrophenol (PNP) within 48h, but it had no further degrading ability towards PNP. The optimal MP-degrading condition was pH7.0, 30℃. Strain SMSP-1 could also degrade ethyl parathion, fenthion, fenitrothion and phoxim, but could not hydrolyse chlorpyrifos. Through the construction of genomic DNA library of strain SMSP-1, we obtained ophc2 gene encoding the organic phosphorus hydrolyzing enzyme. According to the analysis of online-software and GenBank database, this sequence had 98% similarity to ophc2 gene (GenBank Accession No. AJ605330) and contained a 24-aa signal peptide, and it was the second report of cloning the ophc2 gene and the first report of this gene from the genus of Stenotrophomonas up to now. The ophc2 gene with and without signal peptide were ligated into expression vector pET29a, then transformed into E.coli BL21 (DE3) and overexpessed. The map of SDS-PAGE showed that there might exist a protease-cleavage site other than the signal peptide site, which could be recognized by some kind of proteases in BL21 (DE3) and cleaved, and this cleavage site was located at the C-terminal of OPHC2.
菌株XSP-1在pH5.0-9.0范围内生长良好;最适生长温度为30℃;好氧性生长;在5-35g╱L NaCl范围生长较好;在以果糖为碳源,以有机氮为氮源的培养基中生长较好。菌株XSP-1能在7小时内完全降解100mg╱L的辛硫磷;最适降解条件为pH为7.0,35℃。该菌株对甲基对硫磷、倍硫磷和杀螟硫磷也有较好的降解能力,对毒死蜱降解能力较差。
菌株SMSP-1在pH5.0-10.0范围内生长良好;最适生长温度为30℃和35℃;好氧性生长;在5-45g╱L NaCl范围生长较好;在以葡萄糖为碳源,以有机氮为氮源的培养基中生长较好。SMSP-1在48h内能将20mg/L的甲基对硫磷完全降解为对硝基酚,但是对对硝基酚没有进一步的降解能力;降解甲基对硫磷的最适条件为pH7.0、30℃。菌株SMSP-1还能降解杀螟硫磷、乙基对硫磷、倍硫磷和辛硫磷,对毒死蜱没有降解能力。通过构建基因组文库,克隆到菌株SMSP-1的有机磷水解酶基因,运用在线分析软件,推断出有机磷水解酶的结构基因、信号肽及启动子序列。通过在线比对同源性,该基因与Pseudomonas pseudoalcaligenes C2-1中的ophc2基因(GenBank accessionNo.AJ605330)同源性为98%,这是国际上第二次克隆到有机磷水解酶基因ophc2,也是第一次在寡养单胞菌属中克隆到这个基因。根据基因序列分析结果设计特异性引物,通过PCR扩增到带有信号肽和不带有信号肽的ophc2结构基因,分别将其构建到表达载体pET29a中,转化到E.coli BL21(DE3)中进行了表达。根据蛋白电泳结果推断,在有机磷水解酶OPHC2中可能存在着一个能被菌株BL21(DE3)胞内的某个蛋白酶识别并切割的位点,并且该位点位于OPHC2的C-端。
Phoxim-degrdading strain XSP-1 and methyl parathion-degrading strain SMSP-1 were isolated from organophosphate pesticides-contaminated soil. These bacterium were preliminarily identified as Delftia sp. and Stenotrophomonas sp. based on the physiological and biochemical analysis and 16S rRNA gene series source analysis. They were designated as XSP-1 (GenBank Accession No. EF061135) and SMSP-1 (GenBank Accession No. EU312979) respectively.
Biological properties were analyzed respectively. The optimal pH value for the growth of strain XSP-1 was 5.0-9.0, the optimal temperature was 30℃. The optimal osmotic pressure for strain XSP-1 was 5-35g NaCl/L. The optimal medium for the growth of strain XSP-1 was fructose as carbon source and organic nitrogen as nitrogen source. Strain XSP-1 could completely degrade phoxim within 7h, the optimal phoxim-degrading condition was pH7.0, 30℃. Strain XSP-1 could also degrade fenitrothion, methyl parathion and fenthion rapidly, but had little degrading ability towards chlorpyrifos.
Strain SMSP-1 could grow well through pH5.0-10.0, the optimal temperature were 30℃and 35℃, the optimal osmotic pressure was 5-45g NaCl/L. The optimal medium for growth was glucose as carbon source and organic nitrogen as nitrogen source. SMSP-1 could completely hydrolyze methyl parathion to p-nitrophenol (PNP) within 48h, but it had no further degrading ability towards PNP. The optimal MP-degrading condition was pH7.0, 30℃. Strain SMSP-1 could also degrade ethyl parathion, fenthion, fenitrothion and phoxim, but could not hydrolyse chlorpyrifos. Through the construction of genomic DNA library of strain SMSP-1, we obtained ophc2 gene encoding the organic phosphorus hydrolyzing enzyme. According to the analysis of online-software and GenBank database, this sequence had 98% similarity to ophc2 gene (GenBank Accession No. AJ605330) and contained a 24-aa signal peptide, and it was the second report of cloning the ophc2 gene and the first report of this gene from the genus of Stenotrophomonas up to now. The ophc2 gene with and without signal peptide were ligated into expression vector pET29a, then transformed into E.coli BL21 (DE3) and overexpessed. The map of SDS-PAGE showed that there might exist a protease-cleavage site other than the signal peptide site, which could be recognized by some kind of proteases in BL21 (DE3) and cleaved, and this cleavage site was located at the C-terminal of OPHC2.
引文
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[1]Xie X P,Yah Y C,Liu P P.Isolation,degradation and characterization of methylparathion degradative strain L4[J].Acta Scientiae Circumstantiae,2006,26(10):1637-1642.
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[1]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.176-182.
[2]张瑞福,朱卫,崔中利,李顺鹏.辛硫磷降解菌X-1的分离鉴定及降解性状的初步研究[J].环境科学学报,2003,3(23):411-413
[3]Walter W,Mulbry,Jeffrey S.Karns purification and characterization of three parathion hydrodase from gram-negatiive bacterial strains[J].Appl Environ Microbiol,1989,55:289-293
[4]戴树桂,庄源益,陈勇生,等.两种假单胞菌中二氯酚降解酶活性及其定域研究[J].环境科学学报,1996,16(2):173-178
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[1]Sethunathan N,Yoshida T.A Flavobacterium sp.that degrades diazinon and parathion[J].Can.J.Microbiol.,1973,19:873-875
[2]Sedar CM,Gibson DT,Munnecke DM,et al.Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta[J].Appl.Environ.Microbiol.,1982,44:246-249
[3]Home I,Sutherland TD,Harcourt RL,et al.Identification of an opd(organophosphate degradation)gene in an Agrobacterium isolate[j].Appl.Environ.Microbiol.,2002,68:3371-3376
[4]Cui ZL,Li SP,Fu GP.Isolation of methyl parathion-degrading strain M6 and cloning of the methyl parathion hydrolase gene[j].Appl.Environ.Microbiol.,2001,67:4922-4925
[5]Masahito H,Motoko H,Shinichi T.Involvement of two plasmids in fenitrothion degradation by Burkholderia sp.Strain NF100[j].Appl.Environ.Microbiol.,2000,66:1737-1740
[6]张忠辉,洪青,李顺鹏,等.杀螟硫磷降解菌FDS-1的分离鉴定及其降解特性研究[J].中国环境科学,2005,25(1):52-56
[7]Miller SA,Dykes DD & Polesky HF.A simple salting out procedure for extracting DNA from human nucleated cells[J].Nucleic.Acids.Res.,1988,16:1215