不同酿酒酵母菌株来源异麦芽糖酶IMA1的克隆、表达及表征（英文）
|
摘要
【目的】异麦芽糖酶IMA1在充分利用含有α-1,6-O-糖苷键的低聚糖中起着关键作用。【方法】在本研究中,对来自4株酿酒酵母菌株(包括3株嗜酸性菌株)来源的异麦芽糖酶IMA1进行克隆、表达、纯化和表征。【结果】研究发现,4种异麦芽糖酶IMA1表现出类似的pH和温度依赖性,但表现出不同的动力学参数和热稳定性。IMA1-A对α-MG(α-甲基葡糖苷)表现出最高的结合亲和力、转换数、催化效率和热稳定性。结构和序列分析表明,2个远离活性位点和底物结合位点的氨基酸的差异对异麦芽糖酶IMA1的动力学参数和热稳定性有重要影响。【结论】本研究结果对进一步研究异麦芽糖酶IMA1的结构-功能关系奠定了基础。
[Objective] Isomaltases IMA1 play key roles in full utilization of oligosaccharides containing α-1,6-O-glucosidic bonds. [Methods] We cloned, overexpressed, purified and characterized four isomaltases IMA1 from four strains of S. cerevisiae including three acidophilic ones. [Results] They showed similar pH and temperature dependence, but different kinetic parameters and thermostability. IMA1-A exhibited the highest binding affinity for α-MG(α-methylglucoside), turnover number, catalytic efficiency, and thermostability. Structure and sequence analysis revealed that even variation in two remote amino acids from the active residues and the substrate binding site could also lead to significantly different kinetic behavior and thermostability of isomaltases IMA1. [Conclusion] Our results will be useful for further investigation into the structure-function relationship of isomaltases IMA1.
[Objective] Isomaltases IMA1 play key roles in full utilization of oligosaccharides containing α-1,6-O-glucosidic bonds. [Methods] We cloned, overexpressed, purified and characterized four isomaltases IMA1 from four strains of S. cerevisiae including three acidophilic ones. [Results] They showed similar pH and temperature dependence, but different kinetic parameters and thermostability. IMA1-A exhibited the highest binding affinity for α-MG(α-methylglucoside), turnover number, catalytic efficiency, and thermostability. Structure and sequence analysis revealed that even variation in two remote amino acids from the active residues and the substrate binding site could also lead to significantly different kinetic behavior and thermostability of isomaltases IMA1. [Conclusion] Our results will be useful for further investigation into the structure-function relationship of isomaltases IMA1.
引文
[1]Cantarel BL,Coutinho PM,Rancurel C,Bernard T,Lombard V,Henrissat B.The carbohydrate-active enzymes database(CAZy):an expert resource for Glycogenomics.Nucleic Acids Research,2009,37(S1):D233-D238.
[2]Henrissat B.A classification of glycosyl hydrolases based on amino acid sequence similarities.Biochemical Journal,1991,280(2):309-316.
[3]Henrissat B,Bairoch A.Updating the sequence-based classification of glycosyl hydrolases.Biochemical Journal,1996,316(2):695-696.
[4]Khan NA,Eaton NR.Purification and characterization of maltase andα-methyl glucosidase from yeast.Biochimica et Biophysica Acta,1967,146(1):173-180.
[5]Yamamoto K,Miyake H,Kusunoki M,Osaki S.Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltose.The FEBSJournal,2010,277(20):4205-4214.
[6]Ten Berge AMA.Genes for the fermentation of maltose and?-methylglucoside in Saccharomyces carlsbergensis.Molecular and General Genetics,1972,115(1):80-88.
[7]Khan NA,Haynes RH.Genetic redundancy in yeast:non-identical products in a polymeric gene system.Molecular and General Genetics,1972,118(3):279-285.
[8]Teste MA,Fran?ois JM,Parrou JL.Characterization of a new multigene family encoding isomaltases in the yeast Saccharomyces cerevisiae,the IMA family.Journal of Biological Chemistry,2010,285(35):26815-26824.
[9]Naumov GI,Naumoff DG.Molecular genetic differentiation of yeastα-glucosidases:Maltase and isomaltase.Microbiology,2012,81(3):276-280.
[10]Deng X,Petitjean M,Teste MA,Kooli W,Tranier S,Fran?ois JM,Parrou JL.Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae.FEBS Open Bio,2014,4:200-212.
[11]Yamamoto K,Miyake H,Kusunoki M,Osaki S.Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae.Journal of Bioscience and Bioengineering,2011,112(6):545-550.
[12]Yamamoto K,Nakayama A,Yamamoto Y,Tabata S.Val216decides the substrate specificity ofα-glucosidase in Saccharomyces cerevisiae.European Journal of Biochemistry,2004,271(16):3414-3420.
[13]Dwiarti L,Otsuka M,Miura S,Yaguchi M,Okabe M.Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1.Bioresource Technology,2007,98(17):3329-3337.
[14]Sharma A,Kawarabayasi Y,Satyanarayana T.Acidophilic bacteria and archaea:acid stable biocatalysts and their potential applications.Extremophiles,2012,16(1):1-19.
[15]Sambrook J,Fritsch EF,Maniatis T.Molecular cloning:a laboratory manual.2nd ed.Cold Spring Harbor:Cold Spring Harbor Laboratory Press,1989.
[16]Komeda H,Ishikawa N,Asano Y.Enhancement of the thermostability and catalytic activity of D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution.Journal of Molecular catalysis B:Enzymatic,2003,21(4/6):283-290.
[17]Zhao HM,Arnold FH.Directed evolution converts subtilisin E into a functional equivalent of thermitase.Protein Engineering,1999,12(1):47-53.
[18]Serour E,Antranikian G.Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae.Antonie van Leeuwenhoek,2002,81(1/4):73-83.
[19]Schepers B,Thiemann V,Antranikian G.Characterization of a novel glucoamylase from the thermoacidophilic Archaeon Picrophilus torridus heterologously expressed in E.coli.Engineering in Life Sciences,2006,6(3):312-317.
[20]Oshima T,Arakawa H,Baba M.Biochemical studies on an acidophilic,thermophilic bacterium,Bacillus acidocaldarius:isolation of bacteria,intracellular pH,and stabilities of biopolymers.The Journal of Biochemistry,1977,81(4):1107-1113.
[2]Henrissat B.A classification of glycosyl hydrolases based on amino acid sequence similarities.Biochemical Journal,1991,280(2):309-316.
[3]Henrissat B,Bairoch A.Updating the sequence-based classification of glycosyl hydrolases.Biochemical Journal,1996,316(2):695-696.
[4]Khan NA,Eaton NR.Purification and characterization of maltase andα-methyl glucosidase from yeast.Biochimica et Biophysica Acta,1967,146(1):173-180.
[5]Yamamoto K,Miyake H,Kusunoki M,Osaki S.Crystal structures of isomaltase from Saccharomyces cerevisiae and in complex with its competitive inhibitor maltose.The FEBSJournal,2010,277(20):4205-4214.
[6]Ten Berge AMA.Genes for the fermentation of maltose and?-methylglucoside in Saccharomyces carlsbergensis.Molecular and General Genetics,1972,115(1):80-88.
[7]Khan NA,Haynes RH.Genetic redundancy in yeast:non-identical products in a polymeric gene system.Molecular and General Genetics,1972,118(3):279-285.
[8]Teste MA,Fran?ois JM,Parrou JL.Characterization of a new multigene family encoding isomaltases in the yeast Saccharomyces cerevisiae,the IMA family.Journal of Biological Chemistry,2010,285(35):26815-26824.
[9]Naumov GI,Naumoff DG.Molecular genetic differentiation of yeastα-glucosidases:Maltase and isomaltase.Microbiology,2012,81(3):276-280.
[10]Deng X,Petitjean M,Teste MA,Kooli W,Tranier S,Fran?ois JM,Parrou JL.Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae.FEBS Open Bio,2014,4:200-212.
[11]Yamamoto K,Miyake H,Kusunoki M,Osaki S.Steric hindrance by 2 amino acid residues determines the substrate specificity of isomaltase from Saccharomyces cerevisiae.Journal of Bioscience and Bioengineering,2011,112(6):545-550.
[12]Yamamoto K,Nakayama A,Yamamoto Y,Tabata S.Val216decides the substrate specificity ofα-glucosidase in Saccharomyces cerevisiae.European Journal of Biochemistry,2004,271(16):3414-3420.
[13]Dwiarti L,Otsuka M,Miura S,Yaguchi M,Okabe M.Itaconic acid production using sago starch hydrolysate by Aspergillus terreus TN484-M1.Bioresource Technology,2007,98(17):3329-3337.
[14]Sharma A,Kawarabayasi Y,Satyanarayana T.Acidophilic bacteria and archaea:acid stable biocatalysts and their potential applications.Extremophiles,2012,16(1):1-19.
[15]Sambrook J,Fritsch EF,Maniatis T.Molecular cloning:a laboratory manual.2nd ed.Cold Spring Harbor:Cold Spring Harbor Laboratory Press,1989.
[16]Komeda H,Ishikawa N,Asano Y.Enhancement of the thermostability and catalytic activity of D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution.Journal of Molecular catalysis B:Enzymatic,2003,21(4/6):283-290.
[17]Zhao HM,Arnold FH.Directed evolution converts subtilisin E into a functional equivalent of thermitase.Protein Engineering,1999,12(1):47-53.
[18]Serour E,Antranikian G.Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae.Antonie van Leeuwenhoek,2002,81(1/4):73-83.
[19]Schepers B,Thiemann V,Antranikian G.Characterization of a novel glucoamylase from the thermoacidophilic Archaeon Picrophilus torridus heterologously expressed in E.coli.Engineering in Life Sciences,2006,6(3):312-317.
[20]Oshima T,Arakawa H,Baba M.Biochemical studies on an acidophilic,thermophilic bacterium,Bacillus acidocaldarius:isolation of bacteria,intracellular pH,and stabilities of biopolymers.The Journal of Biochemistry,1977,81(4):1107-1113.