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Studies On Coriolus Versicolor And Its Laccases As Well As Their Degradation To Environmental Pollutants

Posted on:2003-08-21Degree:DoctorType:Dissertation
Country:ChinaCandidate:K WuFull Text:PDF
GTID:1101360062486000Subject:Environmental Engineering
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Herein a systematic research of Coriolus versicolor and C. versicolor laccases has been made in the article, including election of high laccase-secreted strain, fermental condition for laccase production by C. versicolor, isolation, purification , characterization and enzymatic kinetic of laccases from C. versicolor, dynamics of m-cresol biodegradation as well. A plenty of active results have also been made as fellows:The biodegradation of lignin and cellulose is an extremely slow natural process. The production of laccase and cellulose was assayed in the 60 Deuteromycotina strain and Basidiomycetes strains collected. A highly productive laccase strain-Cono/us versicolor and a highly productive cellulase and hemicellulase strain - Aspergillus niger Ml 5 was screened, of which production of laccase by Coriolus versicolor in liquid fermentation reached 153U/ml, while the highest productivity of cellulase and hemicellulase by Ml 5 in solid fermentation were 5 007U/ml and 2 639U/ml, respectively.The optimal fermentation conditions for laccase production by Coriolus versicolor were studied and as the followings: The composition of fermentation medium was soluble starch 2g/L, NHjCl 24mmol/L, mixed liquid of trace metals 7ml/L, pH 3.0, citric acid-Na2HPO4 buffer O.Olmol/L, KH2PO4 1.4 X 10'2mol/L, MgSO4 ?7H2O 2.03X 10-3mol/L, CaCl2 ?H2O 6.8 X 10-4 mol/L, VB1 2.97 X 10 -6 mol/L, Tween 80 4.0g/L, guaiacol 0.01mmol/L and CuSO4 ?5H2O 0.005mmol/L, and the optimal conditions were the initial pH of medium 3.0, 25ml medium in 250ml-flask, and incubated at 25 癈 and 150r/minfor 9d.Two laccases were purified to electrophoretic homogeneity from Coriolus versicolor culture by the combination of superflltrate and DEAE-Sephadex A-250 ion exchange chromatography. The properties of the isoenzymes were determined by zymogram patterns from polyacrylamide gel and subsequently named Lac A and Lac B electrophoresis(PAGE). The purities were increased 9.3-fold and 3.41-fold with an overall yield of 18.2% for Lac A and 0.2% for Lac B, respectively. The molecular weights of Lac A and Lac B are 66.0 kDa and 30.7kDa as determined by SDS gel electrophoresis(SDS-PAGE) and their isoelectric points(pl) are 4.79 and 5.18 as assayed by polyacrylamide gel isoelectric focusing(PAGIF),82respectively. Both of these enzymes have same pH optimum (pH 4.0) and different temperature optimum (40 癈 for Lac A and 50 癈 for Lac B) for their optical activity. The temperature for half loss activity(t1/2) in 2 hours is 60 癈 for Lac A and 90 癈 for Lac B. The activities are constant at pH value in the range of 2.5~5.5. The Km and Vmax of Lac A for oxidizing guaiacol is 0.2865mmol/L and 292.40 mmoUL/min while those of Lac B is 0.8130 mmol/L and 127.39mmol/L/min, respectively. Their activities were inhibited by A13+, Fe3+, Cu2+, Ag+, Hg2+ and strongest by Ag+ and Fe3+.The degradation reaction process of hydroquinone by laccase and filtrate from C. versicolor was analyzed using the principle of M-M equation, leading to this enzyme catalytic reaction kinetics constant Km 16.55047mg/L and Vmax 1.52346 Img/L/min, However, there appeared the inhibition as the substrate concentration reached up to certain degree. The rs,max--1.021mg/min, Ks--12.86mg/L/min, KSI - 137.17mg/L, Cs.opt~40mg/L, and the reaction rate equation of substrate inhibition of rss=1.021/(l+12.86/Cs + Cs/137.17) was revealed after dealing with properties of the kinetics in different substrate density. The analysis and comparison of the parameters from theoretical equation and the practice assayed results suggested both Km and Vmax given by Lineweaver - Burke plots was quite different from the actual case, indicating it is possible to get kinetics parameter just from Lineweaver-Burke plots would lead to a great deviation.The degradation reaction process of hydroquinone by filtrate from C. versicolor was analyzed using the principle of M--M equation, leading to the kinetics equation of catalytic reaction Vf=1.768347Cs/(3.626834+ Cs). However, there appeared the inhibition as the substr...
Keywords/Search Tags:Coriolus versicolor, Laccase, Purification, Characterization, Kinetics, Dynamics, Model, Degradation, Pollutant, Biodegradation, Aniline, Benzynine, p-Nitrobenzinine, 2,4-Ddinitrochlorobenzene, Pentachlorophenol, Aflavtoxin B1, Hydroquinone, m-Cresol
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