不动杆菌JH250-8邻苯二酚双加氧酶基因的克隆及分析
|
摘要
目的研究不动杆菌JH250-8的石油降解机制,克隆并分析其邻苯二酚双加氧酶基因.方法应用数据库的同源序列设计引物,对不动杆菌JH250-8邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)基因进行扩增,并用生物信息学软件及在线生物分析工具等分析编码蛋白质.结果不动杆菌JH250-8中同时含有C12O和C23O两种邻苯二酚双加氧酶;扩增的DNA序列长度分别为1 299和1 118 bp,蛋白质编码区长度为1047和930 bp,分别编码358和309个氨基酸;两个邻苯二酚双加氧酶等电点(p I)分别为5.03和4.79,均为酸性蛋白质;酶分子中都含有较多极性和可电离氨基酸,在水中有较好的溶解性,但由于N端都有一段疏水性肽段,可能会影响其水溶液的稳定性;在蛋白质二级结构上,两个酶分子中都有较丰富的二级结构单元,既有较好的稳定性,又有较好的底物适应性.C12O的三级结构由同型二聚体构成,每个亚基上都结合有Fe3+;C23O三级结构的核心由两组β-折叠构成,另有4个α-螺旋结构分布于外侧.结论不动杆菌JH250-8有两种邻苯二酚双加氧酶,两种酶均为酸性蛋白质,有较好的水溶性及结构稳定性.分析结果对邻苯二酚双加氧酶的后续研究有重要的参考价值及指导意义.
Objective To investigate the oil degradation mechanism,the genes of catechol dioxygenases from strain JH250-8 of Acinetobacter were cloned and analyzed. Method Homologous sequences from data base online were used to design PCR primers. Then the genes of catechol 1,2-dioxygenase( C12O) and catechol 2,3-dioxygenase( C23O) from JH250-8 were amplified and analyzed by bioinformatics software and online bioanalysis tools. Results Both catechol dioxygenases( C12 O and C23O) were detected in the strain JH250-8.The length of DNA sequences were 1 299 bp and 1 118 bp and coding regions were 1 047 bp and 930 bp separately. The length of amino acid sequences were 358 and 309. Both catechol dioxygenases are acidic proteinwith p I value 5. 03 and 4. 79. For the presence of polar amino acids and charged amino acids,the two proteins were more stable in aqueous solution,while the hydrophobic peptides exist at the N-terminus should affect their stabilities in water. Prediction of protein secondary structure results showed that,for the rich of constitutional units both C12 O and C23 O were stable in structure and adaptable for substrates. The results of tertiary structure prediction showed that there is Fe3+binding site in C12 O,and the nature structure C12 O might be a dimer formed by two enzyme molecules. While there was a hydrophobic core formed with two groups of β-folding structures,and four α-helical structures distribute outside of protein. Conclusion There are two kinds of catechol dioxygenases in strain JH250-8. Both of them are belong to acidic protein,and with better water solubility and structural stability. Those analytical results were important to investigate catechol dioxygenases of JH250-8.
Objective To investigate the oil degradation mechanism,the genes of catechol dioxygenases from strain JH250-8 of Acinetobacter were cloned and analyzed. Method Homologous sequences from data base online were used to design PCR primers. Then the genes of catechol 1,2-dioxygenase( C12O) and catechol 2,3-dioxygenase( C23O) from JH250-8 were amplified and analyzed by bioinformatics software and online bioanalysis tools. Results Both catechol dioxygenases( C12 O and C23O) were detected in the strain JH250-8.The length of DNA sequences were 1 299 bp and 1 118 bp and coding regions were 1 047 bp and 930 bp separately. The length of amino acid sequences were 358 and 309. Both catechol dioxygenases are acidic proteinwith p I value 5. 03 and 4. 79. For the presence of polar amino acids and charged amino acids,the two proteins were more stable in aqueous solution,while the hydrophobic peptides exist at the N-terminus should affect their stabilities in water. Prediction of protein secondary structure results showed that,for the rich of constitutional units both C12 O and C23 O were stable in structure and adaptable for substrates. The results of tertiary structure prediction showed that there is Fe3+binding site in C12 O,and the nature structure C12 O might be a dimer formed by two enzyme molecules. While there was a hydrophobic core formed with two groups of β-folding structures,and four α-helical structures distribute outside of protein. Conclusion There are two kinds of catechol dioxygenases in strain JH250-8. Both of them are belong to acidic protein,and with better water solubility and structural stability. Those analytical results were important to investigate catechol dioxygenases of JH250-8.
引文
[1]余萃,廖先清,刘子国,等.石油污染土壤的微生物修复研究进展[J].湖北农业科学,2009,48(5):1260-1263.
[2]王悦明,王继富,李鑫,等.石油污染土壤微生物修复技术研究进展[J].环境工程,2014,32(8):157-161.
[3]Ji Y,Mao G,Wang Y,et al.Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases[J].Front Microbiol,2013,4(5):Artical 58.
[4]Coon M J.Omega oxygenases:nonheme-iron enzymes and P450 cytochromes[J].Biochem Biophys Res Commun,2005,338(1):378-385.
[5]花莉,洛晶晶,彭香玉,等.产表面活性剂降解石油菌株产物性质及降解性能研究[J].生态环境学报,2013,22(12):1945-1950.
[6]Thavasi R,jayalakshmi S,Banat I M.Application of biosurfactant produced from peanut oil cake by Lactobacillus delbrueckii in biodegradation of crude oil[J].Bioresour Technol,2011,102(3):3366-3372.
[7]Wang W,Cai B,Shao Z.Oil degradation and biosurfactant production by the deep sea bacterium Dietzia maris As-13-3[J].Front Microbiol,2014,5:Article711.
[8]杨智,陈吉祥,秦波,等.3株石油降解红球菌(Rhodococcus spp.)特性及相关基因分析[J].应用与环境生物学报,2015,21(5):805-812.
[9]杨庆丽,董艳,姬妍茹,等.5株石油降解菌降解相关基因的研究[J].环境科技,2015,28(4):1-4.
[10]陆健.高效烷烃降解菌XCZ的分离、鉴定及其烷烃单加氧酶基因alk B功能的研究[D].南京:南京农业大学,2007.
[11]王万鹏,邵宗泽.红灯食烷菌(Alcanivorax hongdengensis)黄素结合单加氧酶(Alm A)的基因克隆及其烷烃诱导表达[J].微生物学报,2010,50(8):1051-1057.
[12]梁强.南极海洋石油烃低温降解菌Shewanella sp.NJ49加氧酶及其基因研究[D].青岛:国家海洋局第一海洋研究所,2011.
[13]Van Beilen J B,Panke S,Lucchini S,et al.Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences:evolution and regulation of the alk genes[J].Microbiology,2001,147(6):1621-1630.
[14]Van Beilen J B,Marin M M,Smits T H,et al.Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis[J].Environ Microbiol,2004,6(3):264-273.
[15]Wang R,Ding Y,Cheng X,et al.Cloning and expression analysis of Alkane monooxygenases from oil degrading Acinetobacte[C]//Puerto Vallarta:World Automation Congress(WAC),2012.
[16]Whyte L G,Smits T H,Labbe D,et al.Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531[J].Appl Environ Microbiol,2002,68(12):5933-5942.
[17]胡日查,孙立波.低温1,2,4-TCB降解菌的选育、降解特性及邻苯二酚1,2-双加氧酶基因表达水平[J].环境工程学报,2013,7(2):777-782.
[18]吴宇澄,骆永明,滕应,等.石油污染土壤中芳烃降解菌及邻苯二酚2,3双加氧酶的克隆[J].土壤,2006,38(5):640-644.
[19]Bugg T D H.Dioxygenase enzymes:catalytic mechanisms and chemical models[J].Tetrahedron,2003,59(36):7075-7101.
[20]Wang R,Wang D,Shi Y,et al.Isolation,identification and characterization of four oil degrading strains[C]//Puerto Vallarta:World Automation Congress(WAC),2012.
[21]H ein P,Powlowski J,Barriault D,et al.Biphenylassociated meta-cleavage dioxygenases from Comamonas testosteroni B-356[J].Can J Microbiol,1998,44(1):42-49.
[22]Pearl L H,Neidle S.Origins of stereospecificity in DNA damage by anti-benzo pyrene diol-epoxides.A molecular modelling study[J].FEBS Lett,1986,209(2):269-276.
[23]Chatfield L K,Williams P A.Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes[J].J Bacteriol,1986,168(2):878-885.
[24]K eil H.Molecular cloning and expression of a novel catechol 2,3-dioxygenase gene from the benzoate metacleavage pathway in Azotobacter vinelandii[J].J Gen Microbiol,1990,136(4):607-613.
[25]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,2007,23(4):51-54.
[26]宋蕾,王慧,施汉昌.氯代邻苯二酚1,2-双加氧酶基因的克隆及其定量表达[J].环境科学,2009,30(2):606-609.
[27]马忠华,罗如新,夏怡丰,等.邻单胞菌邻苯二酚1,2-双加氧酶基因(tfd C)的克隆及其在大肠杆菌中表达[J].微生物学报,2000,40(6):579-585.
[28]K ita A,Kita S,Fujisawa I,et al.An archetypical extradiol-cleaving catecholic dioxygenase:the crystal structure of catechol 2,3-dioxygenase(metapyrocatechase)from Pseudomonas putida mt-2[J].Structure,1999,7(1):25-34.
[29]蔡在龙,季朝能,王学敏,等.编码耐热邻苯二酚2,3-双加氧酶的phe B基因在大肠杆菌中的亚克隆与高表达[J].第二军医大学学报,1997,18(2):5-8.
[30]Oh J M,Kang E,Min K R,et al.Structure of catechol 2,3-dioxygenase gene encoded in TOM plasmid of Pseudomonas cepacia G4[J].Biochem Biophys Res Commun,1997,234(3):578-581.
[31]Kang B S,Ha J Y,Lim J C,et al.Structure of catechol 2,3-dioxygenase gene from Alcaligenes eutrophus 335[J].Biochem Biophys Res Commun,1998,245(3):791-796.
[32]Moon J,Kang E,Min K R,et al.Characterization of the gene encoding catechol 2,3-dioxygenase from Achromobacter xylosoxidans KF701[J].Biochem Biophys Res Commun,1997,238(2):430-435.
[33]Duffner F M,Muller R.A novel phenol hydroxylase and catechol 2,3-dioxygenase from the Thermophilic Bacillus thermoleovorans strain A2:nucleotide sequence and analysis of the genes[J].FEMS Microbiol Lett,1998,161(1):37-45.
[34]Benkert P,Biasini M,Schwede T.Toward the estimation of the absolute quality of individual protein structure models[J].Bioinformatics,2011,27(3):343-350.
[35]Arnold K,Bordoli L,Kopp J,et al.The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195-201.
[2]王悦明,王继富,李鑫,等.石油污染土壤微生物修复技术研究进展[J].环境工程,2014,32(8):157-161.
[3]Ji Y,Mao G,Wang Y,et al.Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases[J].Front Microbiol,2013,4(5):Artical 58.
[4]Coon M J.Omega oxygenases:nonheme-iron enzymes and P450 cytochromes[J].Biochem Biophys Res Commun,2005,338(1):378-385.
[5]花莉,洛晶晶,彭香玉,等.产表面活性剂降解石油菌株产物性质及降解性能研究[J].生态环境学报,2013,22(12):1945-1950.
[6]Thavasi R,jayalakshmi S,Banat I M.Application of biosurfactant produced from peanut oil cake by Lactobacillus delbrueckii in biodegradation of crude oil[J].Bioresour Technol,2011,102(3):3366-3372.
[7]Wang W,Cai B,Shao Z.Oil degradation and biosurfactant production by the deep sea bacterium Dietzia maris As-13-3[J].Front Microbiol,2014,5:Article711.
[8]杨智,陈吉祥,秦波,等.3株石油降解红球菌(Rhodococcus spp.)特性及相关基因分析[J].应用与环境生物学报,2015,21(5):805-812.
[9]杨庆丽,董艳,姬妍茹,等.5株石油降解菌降解相关基因的研究[J].环境科技,2015,28(4):1-4.
[10]陆健.高效烷烃降解菌XCZ的分离、鉴定及其烷烃单加氧酶基因alk B功能的研究[D].南京:南京农业大学,2007.
[11]王万鹏,邵宗泽.红灯食烷菌(Alcanivorax hongdengensis)黄素结合单加氧酶(Alm A)的基因克隆及其烷烃诱导表达[J].微生物学报,2010,50(8):1051-1057.
[12]梁强.南极海洋石油烃低温降解菌Shewanella sp.NJ49加氧酶及其基因研究[D].青岛:国家海洋局第一海洋研究所,2011.
[13]Van Beilen J B,Panke S,Lucchini S,et al.Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences:evolution and regulation of the alk genes[J].Microbiology,2001,147(6):1621-1630.
[14]Van Beilen J B,Marin M M,Smits T H,et al.Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis[J].Environ Microbiol,2004,6(3):264-273.
[15]Wang R,Ding Y,Cheng X,et al.Cloning and expression analysis of Alkane monooxygenases from oil degrading Acinetobacte[C]//Puerto Vallarta:World Automation Congress(WAC),2012.
[16]Whyte L G,Smits T H,Labbe D,et al.Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531[J].Appl Environ Microbiol,2002,68(12):5933-5942.
[17]胡日查,孙立波.低温1,2,4-TCB降解菌的选育、降解特性及邻苯二酚1,2-双加氧酶基因表达水平[J].环境工程学报,2013,7(2):777-782.
[18]吴宇澄,骆永明,滕应,等.石油污染土壤中芳烃降解菌及邻苯二酚2,3双加氧酶的克隆[J].土壤,2006,38(5):640-644.
[19]Bugg T D H.Dioxygenase enzymes:catalytic mechanisms and chemical models[J].Tetrahedron,2003,59(36):7075-7101.
[20]Wang R,Wang D,Shi Y,et al.Isolation,identification and characterization of four oil degrading strains[C]//Puerto Vallarta:World Automation Congress(WAC),2012.
[21]H ein P,Powlowski J,Barriault D,et al.Biphenylassociated meta-cleavage dioxygenases from Comamonas testosteroni B-356[J].Can J Microbiol,1998,44(1):42-49.
[22]Pearl L H,Neidle S.Origins of stereospecificity in DNA damage by anti-benzo pyrene diol-epoxides.A molecular modelling study[J].FEBS Lett,1986,209(2):269-276.
[23]Chatfield L K,Williams P A.Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes[J].J Bacteriol,1986,168(2):878-885.
[24]K eil H.Molecular cloning and expression of a novel catechol 2,3-dioxygenase gene from the benzoate metacleavage pathway in Azotobacter vinelandii[J].J Gen Microbiol,1990,136(4):607-613.
[25]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,2007,23(4):51-54.
[26]宋蕾,王慧,施汉昌.氯代邻苯二酚1,2-双加氧酶基因的克隆及其定量表达[J].环境科学,2009,30(2):606-609.
[27]马忠华,罗如新,夏怡丰,等.邻单胞菌邻苯二酚1,2-双加氧酶基因(tfd C)的克隆及其在大肠杆菌中表达[J].微生物学报,2000,40(6):579-585.
[28]K ita A,Kita S,Fujisawa I,et al.An archetypical extradiol-cleaving catecholic dioxygenase:the crystal structure of catechol 2,3-dioxygenase(metapyrocatechase)from Pseudomonas putida mt-2[J].Structure,1999,7(1):25-34.
[29]蔡在龙,季朝能,王学敏,等.编码耐热邻苯二酚2,3-双加氧酶的phe B基因在大肠杆菌中的亚克隆与高表达[J].第二军医大学学报,1997,18(2):5-8.
[30]Oh J M,Kang E,Min K R,et al.Structure of catechol 2,3-dioxygenase gene encoded in TOM plasmid of Pseudomonas cepacia G4[J].Biochem Biophys Res Commun,1997,234(3):578-581.
[31]Kang B S,Ha J Y,Lim J C,et al.Structure of catechol 2,3-dioxygenase gene from Alcaligenes eutrophus 335[J].Biochem Biophys Res Commun,1998,245(3):791-796.
[32]Moon J,Kang E,Min K R,et al.Characterization of the gene encoding catechol 2,3-dioxygenase from Achromobacter xylosoxidans KF701[J].Biochem Biophys Res Commun,1997,238(2):430-435.
[33]Duffner F M,Muller R.A novel phenol hydroxylase and catechol 2,3-dioxygenase from the Thermophilic Bacillus thermoleovorans strain A2:nucleotide sequence and analysis of the genes[J].FEMS Microbiol Lett,1998,161(1):37-45.
[34]Benkert P,Biasini M,Schwede T.Toward the estimation of the absolute quality of individual protein structure models[J].Bioinformatics,2011,27(3):343-350.
[35]Arnold K,Bordoli L,Kopp J,et al.The SWISS-MODEL workspace:a web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195-201.