响应面法优化芘降解菌的降解条件及降解基因分析
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摘要
采用响应面法进行实验设计,选择p H值、温度、天数为影响因素,对芘浓度为50 mg/L无机盐培养基进行wp3-1菌株降解条件优化,得到了芘降解率与3种因素间的非线性回归方程,确定了降解芘的最优工艺条件:在温度为34.15?C,p H值为6.98,降解时间为4.28天时,预测芘的降解率为86.42%,实验验证值为85.70%,结果显示,建立的模型具有较高的精度.采用PCR技术克隆邻苯二酚-2,3-双加氧酶基因,生物信息学分析结果表明:C23O基因所编码的氨基酸序列具有双加氧酶基因保守结构域,编码的蛋白质也是一种稳定的酸性亲水蛋白质,分子量为35.01k D,等电点为5.45,二级结构由17%α-螺旋、36%β-折叠和45%的无规则卷曲组成,具有跨膜结构,不具有信号肽.三级结构预测结果与二级结构一致,三级结构有Fe2+结合位点.
Response Surface Methodology(RSM)was employed to optimize some factors such as the culture temperature, p H value, and culture time, to enhance the biodegradation process. The degradation conditions of wp3-1 strain were optimized by using pyrene concentration 50 mg/L inorganic medium, and the nonlinear regression equation between pyrene degradation rate and the 3 factors was obtained. The optimum process parameters for pyrene-degrading rates were determined as the following: the incubation temperature of 34.15?C, the p H value of 6.98 and the culture time of 4.28 days. Under these optimum conditions, the predicted pyrene-degrading rate was 86.42%, whereas the observed value was 85.70%. Experimental results showed that the proposed model had higher accuracy. Catechol-2,3-dioxygenase gene(C23O)was cloned by PCR. Bioinformatic analytical results of the amino acid sequence indicated that there was dioxygenase conserved domain in this sequence, and the molecular weight of the protein component was 35.01 k D. The p I was 5.45, and it was stable protein. The prediction of the secondary structure showed that there were 17%α-helix, 36% β-fold and 45% random coils. The subcellular localization signal peptide and transmembrane prediction and the signal analysis showed that C23O is transmembrane proteins, but do not contain a signal peptide. Teritary structure of C23O was similar with its secondary structure. From this model, we can know that there are Fe2+binding sites of C23O.
Response Surface Methodology(RSM)was employed to optimize some factors such as the culture temperature, p H value, and culture time, to enhance the biodegradation process. The degradation conditions of wp3-1 strain were optimized by using pyrene concentration 50 mg/L inorganic medium, and the nonlinear regression equation between pyrene degradation rate and the 3 factors was obtained. The optimum process parameters for pyrene-degrading rates were determined as the following: the incubation temperature of 34.15?C, the p H value of 6.98 and the culture time of 4.28 days. Under these optimum conditions, the predicted pyrene-degrading rate was 86.42%, whereas the observed value was 85.70%. Experimental results showed that the proposed model had higher accuracy. Catechol-2,3-dioxygenase gene(C23O)was cloned by PCR. Bioinformatic analytical results of the amino acid sequence indicated that there was dioxygenase conserved domain in this sequence, and the molecular weight of the protein component was 35.01 k D. The p I was 5.45, and it was stable protein. The prediction of the secondary structure showed that there were 17%α-helix, 36% β-fold and 45% random coils. The subcellular localization signal peptide and transmembrane prediction and the signal analysis showed that C23O is transmembrane proteins, but do not contain a signal peptide. Teritary structure of C23O was similar with its secondary structure. From this model, we can know that there are Fe2+binding sites of C23O.
引文
[1]许丽,高振,罗霂,等.一株高效降解芘的细菌分离、鉴定及其降解效果[J].微生物学报,2011,51(03):313-319.
[2]陈志丹,晁群芳,杨滨银,等.一株芘降解菌B2的降解条件优化及降解基因[J].环境工程学报,2012,6(10):3795-3800.
[3]孙艳,钱世钧.芳香族化合物生物降解的研究进展[J].生物工程进展,2001,21(01):42-46.
[4]Liang Y,Gardner D R,Miller C D,et al.Study of biochemical pathways and enzymes involved in pyrene degradation by Mycobacterium sp.strain KMS[J].Appl Envion Microbiol,2006,72(12):7821-7828.
[5]郝婧,高磊,吴春旭,等.石油降解菌Pseudomonas stutzeri TH-31的分离与降解条件优化[J].环境工程学报,2015,9(04):1771-1777.
[6]Chakraborty J,Ghosal D,Dutta A,et al.An insight into the origin and functional evolution of bacterial aromatic ringhydroxylating oxygenases[J].Journal of Biomolecular Structure&Dynamics,2012,30(30):419-436.
[7]周鑫淼,陈洁君,耿立召,等.邻苯二酚-2,3-双加氧酶的结构和功能研究进展[J].生物技术通报,2007,2007(04):51-54.
[8]Toyama T,Sei K,Yu Ning,et al.Enrichment of bacteria possessing catechol dioxygenase genes in the rhizosphere of Spirodela polyrrhiza:Amechanism of accelerated biodegradation of phenol[J].Water Research,2009,43(15):3765-3776.
[9]Ferraroni M,Kolomytseva M,Scozzafava A,et al.X-ray structures of 4-chlorocatechol-1,2-dioxygenase adducts with substituted catechols:New perspectives in the molecular basis of intradiol ring cleaving dioxygenases specificity[J].Journal of Structural Biology,2013,181(3):274-282.
[10]Okuta A,Ohnishi K,Yagame S,et al.Intersubunit interaction and catalytic activity of catechol2,3-dioxygenases[J].Biochemical Journal,2003,371(2):557-564.
[11]史延华,任磊,贾阳,等.施氏假单胞菌YC-YH1的萘降解特性及产物分析[J].微生物学通报,2015,42(10):1866-1876.
[12]牛志睿,山宝琴,刘羽,等.响应面法优化石油降解菌性能的研究[J].环境工程学报,2016,10(03):1527-1532.
[13]张涵,韩雨彤,张守娟,等.响应曲面优化固定化微生物修复石油污染土壤[J].石油学报(石油加工),2015,31(04):1028-1034.
[14]张秀霞,吴佳东,滕芝,等.响应面法优化固定化微生物降解石油污染物[J].石油学报(石油加工),2012,28(05):876-882.
[15]周德平,闵航,夏颖,等.少动鞘氨醇单胞菌ZX4儿茶酚-2,3-双加氧酶基因的克隆与序列分析[J].农业生物技术学报,2004,12(2):192-196.
[16]赵化冰,杨健,任何山,等.萘和苯酚降解菌中儿茶酚-2,3-双加氧酶基因的类似性分析及杂种酶构建[J].南开大学学报(自然科学版),2008,41(04):36-41.
[17]Vedler E,Heinaru E,Jutkina J,et al Limnobacter spp.as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes[J].Systematic and Applied Microbiology,2013,36(8):525-532.
[18]Fiona M,Duffner R M.A novel phenol hydroxylase and catechol2,3-dioxygenase from the thermophilic Bacillus thermoleovorans strain A2:nucleotide sequence and analysis of the genes[J].Fems Microbiology Letters,1998,161(1):37-45.
[19]卫昆.蜡状芽胞杆菌对芘的生物降解及其降解基因的克隆与表达[D].广州:暨南大学,2015:1-58.
[20]聂麦茜,张志杰,雷萍.优势短杆菌对多环芳烃的降解性能[J].环境科学,2001,22(06):83-85.
[21]杨轩,张威,李师翁,等.多环芳烃降解菌的分离鉴定及其生理特性研究[J].环境科学学报,2012,32(05):1033-1040.
[22]王红意,翟红,王玉萍,等.30个中国甘薯主栽品种的RAPD指纹图谱构建及遗传变异分析[J].分子植物育种,2009,7(05):879-884.
[23]Schwede T,Kopp J,Guex N,et al.SWISS-MODEL:an automated protein homology-modeling server[J].Nucleic Acids Research,2003,31(13):3381-3385.
[24]Maddison W,Maddison D,Maddison W P,et al.SWISS-MODEL[J].Evolution,2008,62(5):1103-1118.
[2]陈志丹,晁群芳,杨滨银,等.一株芘降解菌B2的降解条件优化及降解基因[J].环境工程学报,2012,6(10):3795-3800.
[3]孙艳,钱世钧.芳香族化合物生物降解的研究进展[J].生物工程进展,2001,21(01):42-46.
[4]Liang Y,Gardner D R,Miller C D,et al.Study of biochemical pathways and enzymes involved in pyrene degradation by Mycobacterium sp.strain KMS[J].Appl Envion Microbiol,2006,72(12):7821-7828.
[5]郝婧,高磊,吴春旭,等.石油降解菌Pseudomonas stutzeri TH-31的分离与降解条件优化[J].环境工程学报,2015,9(04):1771-1777.
[6]Chakraborty J,Ghosal D,Dutta A,et al.An insight into the origin and functional evolution of bacterial aromatic ringhydroxylating oxygenases[J].Journal of Biomolecular Structure&Dynamics,2012,30(30):419-436.
[7]周鑫淼,陈洁君,耿立召,等.邻苯二酚-2,3-双加氧酶的结构和功能研究进展[J].生物技术通报,2007,2007(04):51-54.
[8]Toyama T,Sei K,Yu Ning,et al.Enrichment of bacteria possessing catechol dioxygenase genes in the rhizosphere of Spirodela polyrrhiza:Amechanism of accelerated biodegradation of phenol[J].Water Research,2009,43(15):3765-3776.
[9]Ferraroni M,Kolomytseva M,Scozzafava A,et al.X-ray structures of 4-chlorocatechol-1,2-dioxygenase adducts with substituted catechols:New perspectives in the molecular basis of intradiol ring cleaving dioxygenases specificity[J].Journal of Structural Biology,2013,181(3):274-282.
[10]Okuta A,Ohnishi K,Yagame S,et al.Intersubunit interaction and catalytic activity of catechol2,3-dioxygenases[J].Biochemical Journal,2003,371(2):557-564.
[11]史延华,任磊,贾阳,等.施氏假单胞菌YC-YH1的萘降解特性及产物分析[J].微生物学通报,2015,42(10):1866-1876.
[12]牛志睿,山宝琴,刘羽,等.响应面法优化石油降解菌性能的研究[J].环境工程学报,2016,10(03):1527-1532.
[13]张涵,韩雨彤,张守娟,等.响应曲面优化固定化微生物修复石油污染土壤[J].石油学报(石油加工),2015,31(04):1028-1034.
[14]张秀霞,吴佳东,滕芝,等.响应面法优化固定化微生物降解石油污染物[J].石油学报(石油加工),2012,28(05):876-882.
[15]周德平,闵航,夏颖,等.少动鞘氨醇单胞菌ZX4儿茶酚-2,3-双加氧酶基因的克隆与序列分析[J].农业生物技术学报,2004,12(2):192-196.
[16]赵化冰,杨健,任何山,等.萘和苯酚降解菌中儿茶酚-2,3-双加氧酶基因的类似性分析及杂种酶构建[J].南开大学学报(自然科学版),2008,41(04):36-41.
[17]Vedler E,Heinaru E,Jutkina J,et al Limnobacter spp.as newly detected phenol-degraders among Baltic Sea surface water bacteria characterised by comparative analysis of catabolic genes[J].Systematic and Applied Microbiology,2013,36(8):525-532.
[18]Fiona M,Duffner R M.A novel phenol hydroxylase and catechol2,3-dioxygenase from the thermophilic Bacillus thermoleovorans strain A2:nucleotide sequence and analysis of the genes[J].Fems Microbiology Letters,1998,161(1):37-45.
[19]卫昆.蜡状芽胞杆菌对芘的生物降解及其降解基因的克隆与表达[D].广州:暨南大学,2015:1-58.
[20]聂麦茜,张志杰,雷萍.优势短杆菌对多环芳烃的降解性能[J].环境科学,2001,22(06):83-85.
[21]杨轩,张威,李师翁,等.多环芳烃降解菌的分离鉴定及其生理特性研究[J].环境科学学报,2012,32(05):1033-1040.
[22]王红意,翟红,王玉萍,等.30个中国甘薯主栽品种的RAPD指纹图谱构建及遗传变异分析[J].分子植物育种,2009,7(05):879-884.
[23]Schwede T,Kopp J,Guex N,et al.SWISS-MODEL:an automated protein homology-modeling server[J].Nucleic Acids Research,2003,31(13):3381-3385.
[24]Maddison W,Maddison D,Maddison W P,et al.SWISS-MODEL[J].Evolution,2008,62(5):1103-1118.