嗜热革节孢多糖单加氧酶氧化方式
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摘要
多糖单加氧酶(polysaccharidemonooxygenase,PMO)是一种铜离子依赖的氧化酶,属于辅助活性酶类第九家族(auxiliary activity 9,AA9),在存在电子供体维生素C(vitamin C,Vc)的情况下,可以氧化裂解纤维素的多糖链,显著提高纤维素的酶解效率。本文克隆了嗜热革节孢Scytalidium thermophilumAA9家族的一个编码基因pmo7651,并在毕赤酵母GS115进行诱导表达,通过His标签获得了重组蛋白PMO7651-His。以磷酸膨胀纤维素(PASC)为底物进行酶促反应,薄层层析法(TLC)结果显示PMO7651酶解产物主要为纤维二糖至纤维五糖;飞行时间质谱法(MALDI-TOF-MS)和溴氧化法确定PMO7651具有C1、C4、C6位的氧化活性;底物结合平面的3个芳香族氨基酸位点突变对酶的活性具有不同程度的影响;在PMO7651帮助下,纤维素酶的降解效率均具有不同程度的提高。
Polysaccharide monooxygenase(PMO) is a copper ion-dependent oxidase and belongs to the auxiliary activity family 9(AA9). PMO can oxidize and cleave the polysaccharide chains of cellulose with vitamin C, and significantly increase hydrolysis efficiency. In this study, a gene of auxiliary activity family 9(AA9) from Scytalidium thermophilum named pmo7651 was cloned and the enzyme PMO7651 was successfully expressed in Pichia pastoris. The recombinant protein PMO7651 was obtained by nickel affinity chromatography. PMO7651 with substrate phosphoric acid-swollen cellulose(PASC) was incubated in pH 5.0,50°C for 48 hours. Thin layer chromatography(TLC) analysis showed that the hydrolysis products were mainly cello-oligosaccharides with a degree of polymerization(DP) from DP2 to DP5. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and bromine oxidation analyses showed that PMO7651 possessed the function of C1, C4 and C6 oxidation to cellulose. The mutation of the three aromatic amino acid sites exhibited different hydrolysis activities and final products.In addition, the enzymatic hydrolysis efficiency was improved at different degrees with the enzyme of PMO7651.
Polysaccharide monooxygenase(PMO) is a copper ion-dependent oxidase and belongs to the auxiliary activity family 9(AA9). PMO can oxidize and cleave the polysaccharide chains of cellulose with vitamin C, and significantly increase hydrolysis efficiency. In this study, a gene of auxiliary activity family 9(AA9) from Scytalidium thermophilum named pmo7651 was cloned and the enzyme PMO7651 was successfully expressed in Pichia pastoris. The recombinant protein PMO7651 was obtained by nickel affinity chromatography. PMO7651 with substrate phosphoric acid-swollen cellulose(PASC) was incubated in pH 5.0,50°C for 48 hours. Thin layer chromatography(TLC) analysis showed that the hydrolysis products were mainly cello-oligosaccharides with a degree of polymerization(DP) from DP2 to DP5. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and bromine oxidation analyses showed that PMO7651 possessed the function of C1, C4 and C6 oxidation to cellulose. The mutation of the three aromatic amino acid sites exhibited different hydrolysis activities and final products.In addition, the enzymatic hydrolysis efficiency was improved at different degrees with the enzyme of PMO7651.
引文
Aachmann FL,S?rlie M,Skj?k-Br?k G,Eijsink VG,Vaaje-Kolstad G,2012.NMR structure of a lytic polysaccharide monooxygenase provides insight into copper bingding,protein dynamics,and substrate Interactions.Proceedings of the Nationnal Academy of Sciences of the United States of America,109(46):18779-18784
Beeson WT,Phillips CM,Gate JH,Marletta MA,2012.Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases.Journal of the American Chemical Society,134(2):890-892
Bennati-Granier C,Garajova S,Champion C,Grisel S,Haon M,Zhou S,Fanuel M,Ropartz D,Rogniaux H,Gimbert I,Record E,Berrin JG,2015.Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.Biotechnology for Biofuels,8:90
Borisova AS,Isksen T,Dimarogona M,Kognole AA,Mathiesen G,Varnal A,R?hr?K,Payne CM,S?rlie M,Sandgren M,Eijsink VG,2015.Structural and functional characterization of a lytic polysaccharide monooxygenase with broad substrate specificity.The Journal of Biological Chemistry,290(38):22955-22969
Cantarel BL,Coutinho PM,Rancurel C,Bernard T,Lombard V,Henrissat B,2009.The carbohydrate-active enzymes database(CAZy):an expert resource for glycogenomics.Nucleic Acids Research,37(Database issue):D233-D238
Chen C,Chen J,Geng Z,Wang M,Liu N,Li D,2018.Regioselectivity of oxidation by a polysaccharide monooxygenase from Chaetomium thermophilum.Biotechnology for Biofuels,11:155
Dimarogona M,Topakas E,Christakopoulos P,2013.Recalcitrant polysaccharide degradation by novel oxidative biocatalysts.Applied Microbiology and Biotechnology,97(19):8455-8465
Forsberg Z,Vaaje-Kolstad G,Westereng B,Bun?s AC,Stenstr?m Y,MacKenzie A,S?rlie M,Horn SJ,Eijsink VG,2011.Cleavage of cellulose by a CBM33 protein.Protein Science,20(9):1479-1483
Harris PV,Welner D,McFarland KC,Re E,Navarro Poulsen JC,Brown K,Salbo R,Ding H,Vlasenko E,Merino S,Xu F,Cherry J,Larsen S,Lo Leqqio L,2010.Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61:structure and function of a large,enigmatic family.Biochemistry,49(15):3305-3316
Horn SJ,Vaaje-Kolstad G,Westereng B,Eijsink VG,2012.Novel enzymes for the degradation of cellulose.Biotechnology for Biofuels,5:45
Koseki T,Mese Y,Fushinobu S,Masaki K,Fujii T,Ito K,Shiono Y,Murayama T,Iefuj H,2008.Biochemical characterization of a glycoside hydrolase family 61endoglucanase from Aspergillus kawachii.Applied Microbiology and Biotechnology,77(6):1279-1285
Langston JA,Shanghai T,Abbate E,Xu F,Vlasenko E,Sweeney MD,2011.Oxidoreductive cellulose depolymerizaion by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61.Applied and Environmengtal Microbiology,77(19):7007-7015
Levasseur A,Drula E,Lombard V,Coutinho PM,Henrissat B,2013.Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes.Biotechnology for Biofuels,6(1):41
Li X,Beeson WT,Phillips CM,Marletta MA,Cate JH,2012.Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases.Structure,20(6):1051-1061
Merino ST,Cherry J,2007.Progress and challenges in enzyme development for biomass utilization.Advances in Biochemical Engineering,108:95-120
Phillips CM,Beeson WT,Cate JH,Marletta MA,2011.Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa.ACS Chemical Biology,6(12):1399-1406
Quinlan RJ,Sweeney MD,Lo Leggio L,Otten H,Poulsenb JC,Johansenc KS,kroghc KB,J?rgensen CI,Tovborg M,Anthonsen A,Tryfona T,Walter CP,Dupree P,Xua F,Davies GJ,Walton PH,2011.Insight into the oxidative degration of cellulose by a copper metalloenzyme that exploits biomass components.Proceedings of the National Academy of Sciences of the United States of America,108(37):15079-15084
Simmons TJ,Frandsen KEH,Ciano L,Tryfona T,Lenfant N,Poulsen JC,Wilson LFL,Tandrup T,Tovborg M,Schnorr K,Johansen KS,Henrissat B,Walton PH,Lo Leggio L,Dupree P,2017.Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates.Nature Communications,8(1):1064
Sweeney MD,Xu F,2012.Biomass converting enzymes as industrial biocatalysts for fuels and chemicals:recent developments.Catalysts,2(2):244-263
Vaaje-Kolstad G,Westereng B,Horn SJ,Liu Z,Zhai H,S?rlie M,Eijsink VG,2010.An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides.Science,330(6001):219-222
Vaaje-Kolstad G,Horn SJ,van Aalten DM,Synstad B,Eijsink VG,2005.The non-catalytic chitin-binding protein CBP21from Serratia marcescens is essential for chitin degradation.Journal of Biological Chemistry,280(31):28492-28497
Vu VV,Beeson WT,Phillips CM,Cate JH,Marletta MA,2014.Determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases.Journal of the American Chemical Society,136(2):562-565
Beeson WT,Phillips CM,Gate JH,Marletta MA,2012.Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases.Journal of the American Chemical Society,134(2):890-892
Bennati-Granier C,Garajova S,Champion C,Grisel S,Haon M,Zhou S,Fanuel M,Ropartz D,Rogniaux H,Gimbert I,Record E,Berrin JG,2015.Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.Biotechnology for Biofuels,8:90
Borisova AS,Isksen T,Dimarogona M,Kognole AA,Mathiesen G,Varnal A,R?hr?K,Payne CM,S?rlie M,Sandgren M,Eijsink VG,2015.Structural and functional characterization of a lytic polysaccharide monooxygenase with broad substrate specificity.The Journal of Biological Chemistry,290(38):22955-22969
Cantarel BL,Coutinho PM,Rancurel C,Bernard T,Lombard V,Henrissat B,2009.The carbohydrate-active enzymes database(CAZy):an expert resource for glycogenomics.Nucleic Acids Research,37(Database issue):D233-D238
Chen C,Chen J,Geng Z,Wang M,Liu N,Li D,2018.Regioselectivity of oxidation by a polysaccharide monooxygenase from Chaetomium thermophilum.Biotechnology for Biofuels,11:155
Dimarogona M,Topakas E,Christakopoulos P,2013.Recalcitrant polysaccharide degradation by novel oxidative biocatalysts.Applied Microbiology and Biotechnology,97(19):8455-8465
Forsberg Z,Vaaje-Kolstad G,Westereng B,Bun?s AC,Stenstr?m Y,MacKenzie A,S?rlie M,Horn SJ,Eijsink VG,2011.Cleavage of cellulose by a CBM33 protein.Protein Science,20(9):1479-1483
Harris PV,Welner D,McFarland KC,Re E,Navarro Poulsen JC,Brown K,Salbo R,Ding H,Vlasenko E,Merino S,Xu F,Cherry J,Larsen S,Lo Leqqio L,2010.Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61:structure and function of a large,enigmatic family.Biochemistry,49(15):3305-3316
Horn SJ,Vaaje-Kolstad G,Westereng B,Eijsink VG,2012.Novel enzymes for the degradation of cellulose.Biotechnology for Biofuels,5:45
Koseki T,Mese Y,Fushinobu S,Masaki K,Fujii T,Ito K,Shiono Y,Murayama T,Iefuj H,2008.Biochemical characterization of a glycoside hydrolase family 61endoglucanase from Aspergillus kawachii.Applied Microbiology and Biotechnology,77(6):1279-1285
Langston JA,Shanghai T,Abbate E,Xu F,Vlasenko E,Sweeney MD,2011.Oxidoreductive cellulose depolymerizaion by the enzymes cellobiose dehydrogenase and glycoside hydrolase 61.Applied and Environmengtal Microbiology,77(19):7007-7015
Levasseur A,Drula E,Lombard V,Coutinho PM,Henrissat B,2013.Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes.Biotechnology for Biofuels,6(1):41
Li X,Beeson WT,Phillips CM,Marletta MA,Cate JH,2012.Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases.Structure,20(6):1051-1061
Merino ST,Cherry J,2007.Progress and challenges in enzyme development for biomass utilization.Advances in Biochemical Engineering,108:95-120
Phillips CM,Beeson WT,Cate JH,Marletta MA,2011.Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa.ACS Chemical Biology,6(12):1399-1406
Quinlan RJ,Sweeney MD,Lo Leggio L,Otten H,Poulsenb JC,Johansenc KS,kroghc KB,J?rgensen CI,Tovborg M,Anthonsen A,Tryfona T,Walter CP,Dupree P,Xua F,Davies GJ,Walton PH,2011.Insight into the oxidative degration of cellulose by a copper metalloenzyme that exploits biomass components.Proceedings of the National Academy of Sciences of the United States of America,108(37):15079-15084
Simmons TJ,Frandsen KEH,Ciano L,Tryfona T,Lenfant N,Poulsen JC,Wilson LFL,Tandrup T,Tovborg M,Schnorr K,Johansen KS,Henrissat B,Walton PH,Lo Leggio L,Dupree P,2017.Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates.Nature Communications,8(1):1064
Sweeney MD,Xu F,2012.Biomass converting enzymes as industrial biocatalysts for fuels and chemicals:recent developments.Catalysts,2(2):244-263
Vaaje-Kolstad G,Westereng B,Horn SJ,Liu Z,Zhai H,S?rlie M,Eijsink VG,2010.An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides.Science,330(6001):219-222
Vaaje-Kolstad G,Horn SJ,van Aalten DM,Synstad B,Eijsink VG,2005.The non-catalytic chitin-binding protein CBP21from Serratia marcescens is essential for chitin degradation.Journal of Biological Chemistry,280(31):28492-28497
Vu VV,Beeson WT,Phillips CM,Cate JH,Marletta MA,2014.Determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases.Journal of the American Chemical Society,136(2):562-565