海洋弧菌(Vibrio sp. QD-5)褐藻胶裂解酶基因的克隆和生物信息学分析
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摘要
以海洋弧菌(Vibrio sp.QD-5)的基因组为模板,使用设计的褐藻胶裂解酶引物进行PCR扩增。将目的基因克隆至pet-22b(+)载体后进行测序。测序结果显示,基因aly-Ⅱ的大小为2 170bp,预测编码含有723个氨基酸的蛋白质。对该蛋白质进一步进行了生物信息学分析。利用ProtParam和ProtScale对蛋白质的理化性质进行分析,结果表明,蛋白质Aly-Ⅱ的理论分子质量为85.6kDa,理论等电点为5.1,并且是一种亲水性蛋白。利用软件MEG 6.0对蛋白质Aly-Ⅱ进行系统发育分析,结果显示,蛋白质Aly-Ⅱ很可能是PL-17家族的褐藻胶裂解酶。利用同源建模法构建蛋白质Aly-Ⅱ的三维结构,Aly-Ⅱ含有2个结构域,分别为(α/α)6toroid结构域和β-sheet结构域。
Using designed specific primers,the target aly-Ⅱ gene was amplified by PCR from the genomic DNA of Vibrio sp.QD-5,cloned into pet-22 b(+)vector and then sequenced.The result showed that the cloned gene was 2 170 bp,encoding 723 amino acid residues.The bioinformatics analysis of Aly-Ⅱ protein was further conducted using ProtParam and ProtScale to analyze the physical and chemical properties of the protein.The theoretical molecular weight and pI of Aly-Ⅱ were 85.6 kDa and 5.1,respectively.The protein had hydrophilic property.The phylogenetic trees were constructed by MEG 6.0 and phylogenetic analysis showed that,Aly-Ⅱ was most likely an alginate lyase of PL-17 family.The three-dimensional structure of Aly-Ⅱ was constructed using homology modeling method.The structure of Aly-Ⅱ displayed that it contained two domains,i.e.,(α/α)6 toriod domain andβ-sheet domain.
Using designed specific primers,the target aly-Ⅱ gene was amplified by PCR from the genomic DNA of Vibrio sp.QD-5,cloned into pet-22 b(+)vector and then sequenced.The result showed that the cloned gene was 2 170 bp,encoding 723 amino acid residues.The bioinformatics analysis of Aly-Ⅱ protein was further conducted using ProtParam and ProtScale to analyze the physical and chemical properties of the protein.The theoretical molecular weight and pI of Aly-Ⅱ were 85.6 kDa and 5.1,respectively.The protein had hydrophilic property.The phylogenetic trees were constructed by MEG 6.0 and phylogenetic analysis showed that,Aly-Ⅱ was most likely an alginate lyase of PL-17 family.The three-dimensional structure of Aly-Ⅱ was constructed using homology modeling method.The structure of Aly-Ⅱ displayed that it contained two domains,i.e.,(α/α)6 toriod domain andβ-sheet domain.
引文
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[33]REHM B H,VALLA S.Bacterial alginates:biosynthesis and applications[J].Applied Microbiology and Biotechnology,1997,48(3):281-288.
[34]LAIRSON L L,HENRISSAT B,DAVIES G J,et al.Glycosyltransferases:structures,functions,and mechanisms[J].Annual Review of Biochemistry,2008(77):521-555.
[35]LOMBARD V,BERNARD T,RANCUREL C,et al.A hierarchical classification of polysaccharide lyases for glycogenomics[J].Biochemical Journal,2010,432(3):437-444.
[36]PARK D,JAGTAP S,NAIR S K.Structure of a PL17family alginate lyase demonstrates functional similarities among exotype depolymerases[J].Journal of Biological Chemistry,2014,289(12):8645-8655.
[2]GIMMESTAD M,ERTESVAG H,HEGGESET T M,et al.Characterization of three newAzotobacter vinelandii alginate lyases,one of which is involved in cyst germination[J].Journal of Bacteriology,2009,191(15):4845-4853.
[3]FREMOND B,MALANDAIN C,GUYOMARD C,et al.Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver[J].Cell Transplantation,1993,2(6):453-460.
[4]STEINLECHNER-MARAN R,EBERL T,KUNC M,et al.Oxygen dependence of respiration in coupled and uncoupled endothelial cells[J].American Journal of Physiology,1996,271:C2053-C2061.
[5]ATALA A,KIM W,PAIGE K T,et al.Endoscopic treatment of vesicoureteral reflux with a chondrocyte-alginate suspension[J].Journal of Urology,1994,152:641-644.
[6]WONG T Y,PRESTON L A,Schiller N L.Alginate lyase:review of major sources and enzyme characteristics,structure-function analysis,biological roles,and applications[J].Annual Review of Microbiology,2000,54(1):289-340.
[7]GARRON M L,CYGLER M.Structural and mechanistic classification of uronic acid-containing polysaccharide lyases[J].Glycobiology,2010,20(12):1547-1573.
[8]SUN Y,CHEN H,JIANG G L,Rerearch progress of alginate lyase[J].China Brewing,2017,36(3):14-18.孙艳宾,陈洪基,姜国良.褐藻酸裂解酶的研究进展[J].中国酿造,2017,36(3):14-18.
[9]LI L,JIANG X,GUAN H,et al.Preparation,purification and characterization of alginate oligosaccharides degraded by alginate lyase from Pseudomonas sp.HZJ 216[J].Carbohydrate Research,2011,346(6):794-800.
[10]JONATHAN B,JAMES R T.Structural studies of Alginic acid,using a bacterial poly-α-L-guluronate lyase[J].Carbohydrate Research,1978,66(1):187-194.
[11]CHITNIS C E,OHMAN D E.Cloning of Pseudomonas aeruginosa algG,which controls alginate structure[J].Journal of Bacteriology,1990,172(6):2894-900.
[12]DAVIDSON I W,SUTHERLAND I W,LAWSON C J.Purification and properties of an alginate lyase from a marine bacterium[J].Biochemical Journal,1976,159(3):707.
[13]FAVOROV V V,VOZHOVA E I,DENISENKO V A,et al.A study of the reaction catalyzed by alginate lyase VI from the sea mollusc,Littorina sp.[J].Biochimica et Biophysica Acta-Enzymology,1979,569(2):259-266.
[14]FUJIBAYASHI S,HABE H,NISIZAWA K.Heterogeneity of alginate in special reference to the enzymatic degradation[J].Journal of Biochemistry,1970,67(1):37-45.
[15]HEYRAUD A,COLIN-MOREL P,GIROND S,et al.HPLC analysis of saturated or unsaturated oligoguluronates and oligomannuronates-application to the determination of the action pattern of haliotis tuberculata alginate lyase[J].Carbohydrate Research,1996,291(38):115-126.
[16]MIN K H,SASAKI S F,KASHIWABARA Y,et al.Fine structure of SMG alginate fragment in the light of its degradation by alginate lyases of Pseudomonas sp.[J].Journal of Biochemistry,2008,81(3):555.
[17]NAKADA H I,SWEENY P C.Alginic acid degradation by eliminases from abalone hepatopancreas[J].Journal of Biological Chemistry,1967,242(5):845.
[18]OSTGAARD K.Enzymatic microassay for the determination and characterization of alginates[J].Carbohydrate Polymers,1992,19(1):51-59.
[19]STGAARD K.Determination of alginate composition by a simple enzymatic assay[J].Hydrobiologia,1993,260-261(1):513-520.
[20]ROMEO T,PRESTON III J F.Depolymerization of alginate by an extracellular alginate lyase from a marine bacterium:substrate specificity and accumulation of reaction products[J].Biochemistry,1986,25(26):8391-8396.
[21]INOUE A,MASHINO C,KODAMA T,et al.Protoplast preparation from Laminaria japonica with recombinant alginate lyase and cellulase[J].Marine Biotechnology,2011,13(2):256-263.
[22]HU X,JIANG X,GUAN H.Isolation of protoplasts from undaria pinnatifida,by alginate lyase digestion[J].Journal of Ocean University of Qingdao,2003,2(1):58-61.
[23]ISLAN G A,BOSIO V E,CASTRO G R.Alginate lyase and ciprofloxacin co-immobilization on biopolymeric microspheres for cystic fibrosis treatment[J].Macromolecular Bioscience,2013,13(9):1238-1248.
[24]ISLAN G A,MARTINEZ Y N,ILLANES A,et al.Development of novel alginate lyase cross-linked aggregates for the oral treatmentof cystic fibrosis[J].RSC Advances,2014,4(23):11758-11765.
[25]GAGNEUX P,VARKI A.Evolutionary considerations in relating oligosaccharide diversity to biological function[J].Glycobiology,1999,9(8):747-755.
[26]SCHAEFER L,SCHAEFER R M.Proteoglycans:from structural compounds to signaling molecules[J].Cell and Tissue Research,2010,339(1):237-246.
[27]MYTHREYE K,BLOBE G C.Proteoglycan signaling co-receptors:roles in cell adhesion,migration and invasion[J].Cellular Signalling,2009,21(11):1548-1558.
[28]CHEN D,WU X Z,WEN Z Y.Sulfated polysaccharides and immune response:promoter or inhibitor?[J].Panminerva Medica,2008,50(2):177-183.
[29]MARTH J D,GREWAL P K.Mammalian glycosylation in immunity[J].Nature Reviews Immunology,2008,8(11):874-887.
[30]BONECA I.The role of peptidoglycan in pathogenesis[J].Current Opinion in Microbiology,2005,8(1):46-53.
[31]VLODAVSKY I,ILAN N,NADIR Y,et al.Heparanase,heparin and the coagulation system in cancer progression[J].Thrombosis Research,2007,120(S2):S112-S120.
[32]IOZZO R V,ZOELLER J J,NYSTRM A.Basement membrane proteoglycans:Modulators Par Excellence of cancer growth and angiogenesis[J].Molecules and Cells,2009,27(5):503-513.
[33]REHM B H,VALLA S.Bacterial alginates:biosynthesis and applications[J].Applied Microbiology and Biotechnology,1997,48(3):281-288.
[34]LAIRSON L L,HENRISSAT B,DAVIES G J,et al.Glycosyltransferases:structures,functions,and mechanisms[J].Annual Review of Biochemistry,2008(77):521-555.
[35]LOMBARD V,BERNARD T,RANCUREL C,et al.A hierarchical classification of polysaccharide lyases for glycogenomics[J].Biochemical Journal,2010,432(3):437-444.
[36]PARK D,JAGTAP S,NAIR S K.Structure of a PL17family alginate lyase demonstrates functional similarities among exotype depolymerases[J].Journal of Biological Chemistry,2014,289(12):8645-8655.
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