碳源和氮源对木蹄层孔菌产漆酶的影响及酶学性质研究
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摘要
对不同碳源和氮源及碳氮比对木蹄层孔菌产漆酶的影响进行了研究,以提高漆酶产量,同时分析了该漆酶的酶学性质。漆酶合成的最适碳源是麦麸,最适氮源是蛋白胨,C∶N为10.4,优化后漆酶产量增加了约7.88倍;反应最适pH为3.0,最适反应温度为50℃,K m为0.20 mmol/L,V max为2.58 mmol/L·min;DTT 0.1mmol/L和NaN310mmol/L几乎能抑制漆酶全部活性,终浓度为10mmol/L的Ba2+,Ca2+,Co2+和Fe2+也几乎能抑制该酶的全部活性。该研究将为木蹄层孔菌漆酶的生产及在环境工程领域的应用提供基础信息。
The production of laccase by Fomes fomentarius was studied.Cultures conditions involving variations in carbon and nitrogen sources and different C∶ N ratios were examined at constant temperature and pH,with the aim of increasing yield of laccase.And its enzymatic properties were determined.The best results were obtained when using wheat bran and peptone as carbon and nitrogen sources respectively with a C∶ N ratio of 10.4.Compared to the initial medium,the highest laccase yield observed is approximately increased by 7.88 times under the optimized conditions.The optimum pH and temperature for its activity is 3.0 and 50℃ with the corresponding K m and V max of 0.20 mmol / L and 2.58 mmol / L·min respectively.DTT(0.1mmol / L) and NaN3(10mmol/L) nearly inhibit all activity of the laccase,as well as the metal ions especially Ba2 +,Ca2 +,Co2 +and Fe2 +(10mmol/L).In summary,our results will provide basic information to the production of laccase by Fomes fomentarius and the utilization of environmental engineering in the future.
The production of laccase by Fomes fomentarius was studied.Cultures conditions involving variations in carbon and nitrogen sources and different C∶ N ratios were examined at constant temperature and pH,with the aim of increasing yield of laccase.And its enzymatic properties were determined.The best results were obtained when using wheat bran and peptone as carbon and nitrogen sources respectively with a C∶ N ratio of 10.4.Compared to the initial medium,the highest laccase yield observed is approximately increased by 7.88 times under the optimized conditions.The optimum pH and temperature for its activity is 3.0 and 50℃ with the corresponding K m and V max of 0.20 mmol / L and 2.58 mmol / L·min respectively.DTT(0.1mmol / L) and NaN3(10mmol/L) nearly inhibit all activity of the laccase,as well as the metal ions especially Ba2 +,Ca2 +,Co2 +and Fe2 +(10mmol/L).In summary,our results will provide basic information to the production of laccase by Fomes fomentarius and the utilization of environmental engineering in the future.
引文
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[14]Lu L,Zhao M,Zhang B B,et al.Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme.Applied Microbiology and Biotechnology,2007,74(6):1232-1239.
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[2]Claus H.Laccases:structure,reactions,distribution.Micron,2004,35(1):93-96.
[3]刘欣,赵敏,王秋玉.5种木材腐朽菌的生物学特性及对白桦木材腐朽能力的分析.东北林业大学学报,2008,36(3):41-44.Liu X,Zhao M,Wang Q Y.Biological Characters of five species of wood rot fungi and decay capacity to Betula platyphylla.Journal of Northeast Forestry University,2008,36(3):41-44.
[4]Murugesan K,Yang I H,Kim Y M,et al.Enhanced transformation of malachite green by laccase of Ganoderma lucidum in the presence of natural phenolic compounds.Applied Microbiology and Biotechnology,2009,82(2):341-350.
[5]Singh S,Pakshirajan K,Daverey A.Enhanced decolourization of Direct Red-80 dye by the white rot fungus Phanerochaete chrysosporium employing sequential design of experiments.Biodegradation,2010,21(4):501-511.
[6]黄茜,黄凤洪,江木兰,等.木质素降解菌的筛选及混合菌发酵降解秸秆的研究.中国生物工程杂志,2008,8(2):66-70.Huang Q,Huang F H,Jiang M L,et al.The selection of lignindegrading fungus and the straw fermentation by mixed strains.China Biotechnology,2008,8(2):66-70.
[7]Tien M,Kirk T K.Lignin-degrading enzyme from the hymenomycete Phanerochaete chrysosporium Burds.Science(Washington),1983,221(4611):661-662.
[8]林俊芳,刘志明,陈晓阳,等.真菌漆酶的酶活测定方法评价.生物加工过程,2009,7(4):1-8.Lin J F,Liu ZH M,Chen X Y,et al.Evaluation of assay methods for determining fungal laccase activity.Chinese Journal of Bioprocess Engineering,2009,7(4):1-8.
[9]Wolfenden B S,Willson R L.Radical-cations as reference chromogens in kinetic studies of ono-electron transfer reactions.J Chem Soc Perkin Trans,1982,2:805-812.
[10]Srinivasan C,Dsouza T M,Boominathan K,et al.Demonstration of Laccase in the White rot Basidiomycete Phanerochaete chrysosporium BKM-F1767.Applied and Environmental Microbiology,1995,61(12):4274-4277.
[11]Schückel J,Matura A,Van Pee K H.One-copper laccase-related enzyme from Marasmius sp.:Purification,characterization and bleaching of textile dyes.Enzyme and Microbial Technology,2011,48(3):278-284.
[12]Wu Y R,Luo Z H,Kwok-Kei Chow R,et al.Purification and characterization of an extracellular laccase from the anthracenedegrading fungus Fusarium solani MAS2.Bioresource Technology,2010,101(24):9772-9777.
[13]Okamoto K,Yanagi S O,Sakai T.Purification and characterization of extracellular laccase from Pleurotus ostreatus.Mycoscience,2000,41(1):7-13.
[14]Lu L,Zhao M,Zhang B B,et al.Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme.Applied Microbiology and Biotechnology,2007,74(6):1232-1239.
[15]Park K M,Park S S.Purification and characterization of laccase from basidiomycete Fomitella fraxinea.Journal of Microbiology and Biotechnology,2008,18(4):670.
[16]Baldrian P.Purification and characterization of laccase from the white-rot fungus Daedalea quercina and decolorization of synthetic dyes by the enzyme.Applied Microbiology and Biotechnology,2004,63(5):560-563.
[17]Wells A,Teria M,Eve T.Green oxidations with laccase-mediator systems.Biochemical Society Transactions,2006,34(2):304.
[18]Solomon E I,Sundaram U M,Machonkin T E.Multicopper oxidases and oxygenases.Chemical Reviews,1996,96(7):2563-2606.