A novel laccase from white rot fungus Trametes orientalis:Purification,biochemical characterization,and application
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- 英文论文题名：A novel laccase from white rot fungus Trametes orientalis:Purification,biochemical characterization,and application
- 论文作者：Fei Zheng ; Bao-Kai Cui ; Jing Si
- 英文论文作者：Fei Zheng ; Bao-Kai Cui ; Jing Si ; Institute of Microbiology ; Beijing Forestry University
- 年：2016
- 作者机构：Institute of Microbiology,Beijing Forestry University;
- 英文论文关键词：Trametes orientalis ; laccase ; purification ; biochemical characterization ; application
- 会议召开时间：2016-08-19
- 会议录名称：中国菌物学会2016年学术年会论文摘要集
- 英文会议录名称：Abstracts of 2016 Mycological Society of China Annual Meeting
- 语种：英文
- 分类号：Q936
- 学会代码：ZGVL
- 会议名称：中国菌物学会2016年学术年会
- 会议地点：中国福建福州
- 主办单位：中国菌物学会
- 学会名称：中国菌物学会
- 页数：3
- 文件大小：778k
- 原文格式：D
- 会议级别：全国
摘要
In this study,an extracellular laccase(Tolacc) from the culture filtrate of white rot fungus Trametes orientalis had been purified by a three-step method,i.e.ammonium sulfate fractional precipitation,DEAE-cellulose DE52 anionic exchange,and Sepharose CL-6B column chromatography.The sodium dodecyl sulphate poly-acrylamide gel electrophoresis(SDS-PAGE) exhibited a single protein band indicating that the enzyme preparation is a monomeric protein,and its molecular mass 44.0 k Da.When using 2,2'-azino-bis-(3-ethylbonzthiazoline-6-sulfonic acid)(ABTS) as the substrate,the Michaelis-Menten constant(Km),catalytic rate constant(kcat),and catalytic efficiency(kcat/Km) values of the laccase were 333.3 μM,21.81 s-1,and 7.0 s-l μM-1 respectively.Tolacc is highly stable and resistant under acidic conditions,with p H values ranging from 1.0 to 6.0.Remarkably,the temperature optima were 80 °C.Enzyme activity was enhanced in the presence of metal ions Cu2+,Mn2+,Mg2+,and Zn2+ at 25.0 m M.Various concentrations of inhibitors L-cysteine,dithiothreitol(DTT),and Na N3 had strong inhibitory effects on Tolacc activity,whereas no reduction occurred with metal ion chelator ethylene diamine tetraacetic acid(EDTA) under the same experimental conditions.These unusual properties demonstrated that the new fungal laccase Tolacc has potentials for the specific industrial or environmental applications.
In this study,an extracellular laccase(Tolacc) from the culture filtrate of white rot fungus Trametes orientalis had been purified by a three-step method,i.e.ammonium sulfate fractional precipitation,DEAE-cellulose DE52 anionic exchange,and Sepharose CL-6B column chromatography.The sodium dodecyl sulphate poly-acrylamide gel electrophoresis(SDS-PAGE) exhibited a single protein band indicating that the enzyme preparation is a monomeric protein,and its molecular mass 44.0 k Da.When using 2,2'-azino-bis-(3-ethylbonzthiazoline-6-sulfonic acid)(ABTS) as the substrate,the Michaelis-Menten constant(Km),catalytic rate constant(kcat),and catalytic efficiency(kcat/Km) values of the laccase were 333.3 μM,21.81 s-1,and 7.0 s-l μM-1 respectively.Tolacc is highly stable and resistant under acidic conditions,with p H values ranging from 1.0 to 6.0.Remarkably,the temperature optima were 80 °C.Enzyme activity was enhanced in the presence of metal ions Cu2+,Mn2+,Mg2+,and Zn2+ at 25.0 m M.Various concentrations of inhibitors L-cysteine,dithiothreitol(DTT),and Na N3 had strong inhibitory effects on Tolacc activity,whereas no reduction occurred with metal ion chelator ethylene diamine tetraacetic acid(EDTA) under the same experimental conditions.These unusual properties demonstrated that the new fungal laccase Tolacc has potentials for the specific industrial or environmental applications.
In this study,an extracellular laccase(Tolacc) from the culture filtrate of white rot fungus Trametes orientalis had been purified by a three-step method,i.e.ammonium sulfate fractional precipitation,DEAE-cellulose DE52 anionic exchange,and Sepharose CL-6B column chromatography.The sodium dodecyl sulphate poly-acrylamide gel electrophoresis(SDS-PAGE) exhibited a single protein band indicating that the enzyme preparation is a monomeric protein,and its molecular mass 44.0 k Da.When using 2,2'-azino-bis-(3-ethylbonzthiazoline-6-sulfonic acid)(ABTS) as the substrate,the Michaelis-Menten constant(Km),catalytic rate constant(kcat),and catalytic efficiency(kcat/Km) values of the laccase were 333.3 μM,21.81 s-1,and 7.0 s-l μM-1 respectively.Tolacc is highly stable and resistant under acidic conditions,with p H values ranging from 1.0 to 6.0.Remarkably,the temperature optima were 80 °C.Enzyme activity was enhanced in the presence of metal ions Cu2+,Mn2+,Mg2+,and Zn2+ at 25.0 m M.Various concentrations of inhibitors L-cysteine,dithiothreitol(DTT),and Na N3 had strong inhibitory effects on Tolacc activity,whereas no reduction occurred with metal ion chelator ethylene diamine tetraacetic acid(EDTA) under the same experimental conditions.These unusual properties demonstrated that the new fungal laccase Tolacc has potentials for the specific industrial or environmental applications.
引文
Chaurasia PK,Yadav A,Yadav RSS,Yadava S,2013.Purification and characterization of laccase secreted by Phellinus linteus MTCC-1175 and its role in the selective oxidation of aromatic methyl group.Applied Biochemistry and Microbiology,49:592-599.
Chaurasia PK,Yadav RSS,Yadava S,2014.Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes.Process Biochemistry,49:1647-1655.
El-Batal AI,El Kenawy NM,Yassin AS,Amin MA,2015.Laccase production by Pleurotus ostreatus and its application in synthesis of gold nanoparticles.Plant Biotechnology Reports,5:31-39.
Guan ZB,Zhang N,Song CM,Zhou W,Zhou LX,Zhao H,Xu CW,Cai YJ,Liao XR,2014.Molecular cloning,characterization,and dye-decolorizing ability of a temperature-and p H-stable laccase from Bacillus subtilis X1.Applied Biochemistry and Biotechnology,172:1147-1157.
Hu X,Wang CY,Wang L,Zhang RR,Chen H,2014.Influence of temperature,p H and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis.World Journal of Microbiology Biotechnology,30:1285-1290.
Johannes C,Majcherczyk A,2000.Laccase activity tests and laccase inhibitors.Journal of Biotechnology,78:193-199.
Kalaiarasan E,Arulkumar M,Palvannan T,2014.Aqueous thermostabilization of Pleurotus florida NCIM1243 laccase and its potential application in Acid Black 10BX dye decolorization:process optimization.Journal of the Taiwan Institute of Chemical Engineers,45:2403-2410.
Kalyani D,Dhiman SS,Kim H,Jeya M,Kim IW,Lee JK,2012.Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass.Process Biochemistry,47:671-678.
Mogharabi M,Faramarzi MA,2014.Laccase and laccase-mediated systems in the synthesis of organic compounds.Advanced Synthesis&Catalysis,356:897-927.
Park N,Park SS,2014.Purification and characterization of a novel laccase from Fomitopsis pinicola mycelia.International Journal of Biological Macromolecules,70:583-589.
Si J,Peng F,Cui BK,2013.Purification,biochemical characterization and dye decolorization capacity of an alkali-resistant and metal-tolerant laccase from Trametes pubescens.Bioresource Technology,128:49-57.
Sun J,Peng RH,Xiong AS,Tian YS,Zhao W,Xu H,Liu DT,Chen JM,Yao QH,2012.Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris.Molecular Biology Reports,39:3807-3814.
Thurston CF,1994.The structure and function of fungal laccases.Microbiology,140:19-26.
Wang B,Wang LJ,Lin YQ,Han Q,Han J,Gao JJ,Tian YS,Zhao W,Peng RH,Yao QH,2014.Purification and characterization of a laccase from Coprinopsis cinerea in Pichia pastoris.World Journal of Microbiology and Biotechnology,30:1199-1206.
Wang CL,Zhao M,Lu L,Wei XD,Li TL,2011.Characterization of spore laccase from Bacillus subtilis WD23 and its use in dye decolorization.African Journal of Biotechnology,10:2186-2192.
Wang TN,Lu L,Wang JY,Xu TF,Li J,Zhao M,2015.Enhanced expression of an industry applicable Cot Alaccase from Bacillus subtilis in Pichia pastoris by non-repressing carbon sources together with p Hadjustment:recombinant enzyme characterization and dye decolorization.Process Biochemistry,50:97-103.
Wang ZX,Cai YJ,Liao XR,Tao GJ,Li YY,Zhang F,Zhang DB,2010.Purification and characterization of two thermostable laccases with high cold adapted characteristics from Pycnoporus sp.SYBC-L1.Process Biochemistry,45:1720-1729.
Xu F,2005.Applications of oxidoreductases:recent progress.Industrial Biotechnology,1:38-50.
You LF,Liu ZM,Lin JF,Guo LQ,Huang XL,Yang HX,2014.Molecular cloning of a laccase gene from Ganoderma lucidum and heterologous expression in Pichia pastoris.Journal of Basic Microbiology,54:S134-141.
Zhang C,Zhang S,Diao HW,Zhao HZ,Zhu XY,Lu FX,Lu ZX,2013.Purification and characterization of a temperature-and p H-stable laccase from the spores of Bacillus vallismortis fmb-103 and its application in the degradation of malachite green.Journal of Agricultural Food Chemistry,61:5468-5473.
Zhuo R,He F,Zhang XY,Yang Y,2015.Characterization of a yeast recombinant laccase r LAC-EN3-1 and its application in decolorizing synthetic dye with the coexistence of metal ions and organic solvents.Biochemical Engineering Journal,93:63-72.
Chaurasia PK,Yadav RSS,Yadava S,2014.Purification and characterization of yellow laccase from Trametes hirsuta MTCC-1171 and its application in synthesis of aromatic aldehydes.Process Biochemistry,49:1647-1655.
El-Batal AI,El Kenawy NM,Yassin AS,Amin MA,2015.Laccase production by Pleurotus ostreatus and its application in synthesis of gold nanoparticles.Plant Biotechnology Reports,5:31-39.
Guan ZB,Zhang N,Song CM,Zhou W,Zhou LX,Zhao H,Xu CW,Cai YJ,Liao XR,2014.Molecular cloning,characterization,and dye-decolorizing ability of a temperature-and p H-stable laccase from Bacillus subtilis X1.Applied Biochemistry and Biotechnology,172:1147-1157.
Hu X,Wang CY,Wang L,Zhang RR,Chen H,2014.Influence of temperature,p H and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis.World Journal of Microbiology Biotechnology,30:1285-1290.
Johannes C,Majcherczyk A,2000.Laccase activity tests and laccase inhibitors.Journal of Biotechnology,78:193-199.
Kalaiarasan E,Arulkumar M,Palvannan T,2014.Aqueous thermostabilization of Pleurotus florida NCIM1243 laccase and its potential application in Acid Black 10BX dye decolorization:process optimization.Journal of the Taiwan Institute of Chemical Engineers,45:2403-2410.
Kalyani D,Dhiman SS,Kim H,Jeya M,Kim IW,Lee JK,2012.Characterization of a novel laccase from the isolated Coltricia perennis and its application to detoxification of biomass.Process Biochemistry,47:671-678.
Mogharabi M,Faramarzi MA,2014.Laccase and laccase-mediated systems in the synthesis of organic compounds.Advanced Synthesis&Catalysis,356:897-927.
Park N,Park SS,2014.Purification and characterization of a novel laccase from Fomitopsis pinicola mycelia.International Journal of Biological Macromolecules,70:583-589.
Si J,Peng F,Cui BK,2013.Purification,biochemical characterization and dye decolorization capacity of an alkali-resistant and metal-tolerant laccase from Trametes pubescens.Bioresource Technology,128:49-57.
Sun J,Peng RH,Xiong AS,Tian YS,Zhao W,Xu H,Liu DT,Chen JM,Yao QH,2012.Secretory expression and characterization of a soluble laccase from the Ganoderma lucidum strain 7071-9 in Pichia pastoris.Molecular Biology Reports,39:3807-3814.
Thurston CF,1994.The structure and function of fungal laccases.Microbiology,140:19-26.
Wang B,Wang LJ,Lin YQ,Han Q,Han J,Gao JJ,Tian YS,Zhao W,Peng RH,Yao QH,2014.Purification and characterization of a laccase from Coprinopsis cinerea in Pichia pastoris.World Journal of Microbiology and Biotechnology,30:1199-1206.
Wang CL,Zhao M,Lu L,Wei XD,Li TL,2011.Characterization of spore laccase from Bacillus subtilis WD23 and its use in dye decolorization.African Journal of Biotechnology,10:2186-2192.
Wang TN,Lu L,Wang JY,Xu TF,Li J,Zhao M,2015.Enhanced expression of an industry applicable Cot Alaccase from Bacillus subtilis in Pichia pastoris by non-repressing carbon sources together with p Hadjustment:recombinant enzyme characterization and dye decolorization.Process Biochemistry,50:97-103.
Wang ZX,Cai YJ,Liao XR,Tao GJ,Li YY,Zhang F,Zhang DB,2010.Purification and characterization of two thermostable laccases with high cold adapted characteristics from Pycnoporus sp.SYBC-L1.Process Biochemistry,45:1720-1729.
Xu F,2005.Applications of oxidoreductases:recent progress.Industrial Biotechnology,1:38-50.
You LF,Liu ZM,Lin JF,Guo LQ,Huang XL,Yang HX,2014.Molecular cloning of a laccase gene from Ganoderma lucidum and heterologous expression in Pichia pastoris.Journal of Basic Microbiology,54:S134-141.
Zhang C,Zhang S,Diao HW,Zhao HZ,Zhu XY,Lu FX,Lu ZX,2013.Purification and characterization of a temperature-and p H-stable laccase from the spores of Bacillus vallismortis fmb-103 and its application in the degradation of malachite green.Journal of Agricultural Food Chemistry,61:5468-5473.
Zhuo R,He F,Zhang XY,Yang Y,2015.Characterization of a yeast recombinant laccase r LAC-EN3-1 and its application in decolorizing synthetic dye with the coexistence of metal ions and organic solvents.Biochemical Engineering Journal,93:63-72.