| Lignin is a phenolic heteropolymer derived from the oxidative combinatorial coupling of phenylpropanoids. It is the second most abundant renewable organic polymer on earth, after cellulose. Lignin is intimately interspersed with hemicelluloses forming a matrix that surrounds cellulose microfibrils. It protects the plant from microbial degradation. Biodegradation of lignin is thought to be a rate-limiting step of the carbon cycle in the biosphere. However, lignin is a barrier to the utilization of biomass. Biode gradation of lignin is an important way to achieve the effective utilization of biomass. White rot fungi are the only known organisms that can completely mineralize lignin in nature. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase are the main oxidative enzymes responsible for the initial attack of lignin. However, oxidation with these enzymes in vitro mainly leads to more polymerized lignin because of the phenolic coupling reactions of the radical intermediates. This suggests that white rot fungi must have some unknown enzymes and mechanisms to control the repolymerization of reacted lignin, possibly through the reduction of the phenoxy radials orquinoid intermediates.Pyranose 2-oxidase (P2O) is a FAD-containing enzyme widely distributed among white rot fungi. It catalyzes the oxidation of D-glucose and several other aldopyranoses at the C-2 position yielding the corresponding 2-keto sugars, concomitant with the reduction of O2 to H2O2. The physiological role of P2O in white rot fungi is believed to be providing H2O2 to the lignin-degrading peroxides. However, it is demonstrated that in addition to O2, P2O could also reduce various quinones and radicals, some of which are better electron acceptors than O2. This suggests another possible role of P2O, which is to reduce the radicals and quinoid intermediates produced during ligninolysis, thereby preventing the repolymerization of the lignin fragments. However, direct evidence for such a role of P2O has been lacking. One of the main aims of this study is to verify wether P2O can cooperate with laccase and enhance lignin degradation.Laccase catalyzes the one-electron oxidation of a broad range of substrates. This makes it applicable in many industrial fields such as pulp and paper, wastewater treatment, and organic synthesis. Recently the enzyme has also attracted much attention for its potential use in lignin modification. To find new laccases suitable for industrial applications are research projects of many scientists. Our lab has preserved a number of strains of white rot fungi, among which Trametes trogii YDHSD is an efficient laccase producer. In this thesis, the laccase from T. trogii YDHSD was purified and characterized. Its application in lignin modification and dye decolorization was also investigated. The main results of this thesis are as follows:1. Purification and characterization of P2OApyranose oxidase was purified from the white rot fungus Irpex lacteus dft-1 by using ammonium sulfate fraction, hydrophobic interaction chromatography, anion exchange chromatography, and gel filtration chromatography. The enzyme was pufied 27.9-fold, with a yield of 27.4%. The glucose oxidation product of the enzyme was identified as 2-keto-D-glucose by TLC and GC-MS analysis, which confirmed that the purified enzyme was P2O, not glucose 1-oxidase. Gel filtration chromatography suggested that the molecular weight of the enzyme was 266 kDa, and SDS-PAGE gave a single band at 71 kDa, indicating that the enzyme is composed of four subunits. The enzyme showed typical flavoprotein UV-Vis spectrum. It exhibits maximum activity at pH 6.5 and 55℃ and is rather stable. Several monopyranose including D-glucose, D-galactose, L-sorbose, D-xylose, and D-Glucono-1,5-lactone were readily oxidized by the enzyme. Some disaccharides such as maltose and cellobiose were also oxidized by I. lacteus P2O. Km and Kcat for these sugars suggest that D-glucose is the preferred substrate. Not only was P2O nonspecific with respect to the electron donors used, but in addition to oxygen, it could also transfer electrons to a number of different compounds, mainly substituted benzoquinones and some radicals. Some quinones were even better substrates than oxygen as judged from the catalytic efficiencies. Since radicals and quinoids are key intermediates in the degradation of lignin, it is possible that P2O not only uses oxygen but also various substituted quinones originated from lignin as electron acceptors under natural conditions.2. Cooperation of P2O and laccase in lignin degradationLaccase oxidized various phenolic compounds and lignosulfonate to generate quinoid structures, which could be reduced by P2O as determined by spectrophotometry. The effect of P2O on laccase-catalyzed oxidation of lignin was tested on lignosulfonate, alkali lignin, eucalyptus CEL and bagasse CEL. The lignins were treated with laccase with and witiout the presence of P2O. GPC analysis indicated that the lignins treated by laccase alone polymerized. On the other hand, the addition of P2O effectively inhibited the polymerization, and even caused depolymerization of lignosulfonate. Moreover, when treated with both laccase and P2O, the conjugated carbonyl groups in the lignins increased as demonstrated by the FTIR analysis, which was a result of the Cα-oxidation of the lignin subunits. This indicates that P2O promoted the oxidation of lignin.In conclusion, the present study demonstrates that P2O can reduce the quinoid intermediates, thereby preventing the polymerization and accelerating the degradation of lignin catalyzed by laccase. The prevention of the repolymerization of lignin iragments during ligninolysis may be another role of P2O in addition to supplying H2O2. In view of the predominance of P2O in white rot fungi, the cooperation of P2O with laccase and/or peroxidases to maintain a redox cycle may be a common mechanism used by white rot fungi to regulate lignin polymerization and degradation.3. Purification and characterization of laccaseA blue laccase was purified from the white rot fungus T. trogii YDHSD by using ammonium sulfate fraction, anion exchange chromatography, and gel filtration chromatography. It was a monomeric protein of 64 kDa as determined by SDS-PAGE. The enzyme acted optimally at a pH of 2.2 to 4.5 and a temperature of 70℃ and showed high thermal stability, with a half-life of 1.6 h at 60℃. A broad range of substrates, including the non-phenolic azo dye methyl red, was oxidized by the laccase, and the laccase exhibited high affinities towards ABTS and syringaldazine. Moreover, the laccase was fairly metal-tolerant. These properties make the laccase a good candidate for industrial applications.4. Modification of kraft lignin with laccaseThe laccase was able to catalyze the oxidative polymerization of a high-molecular-weight KL. The polymerization was pH-dependent. The favored pH range was 6 to 6.5, which was higher than that for the oxidation of low-molecular-weight substrates. A maximum of 6.4-fold increase in Mw was achieved at pH 6.5. Notable structural changes were detected in the polymerized KL. The carbonyl and aliphatic hydroxyl groups increased, while the methoxyl groups decreased. The phenolic hydroxyl groups also decreased, but to a lesser extent. Moreover, condensed structures were formed during the oxidation by laccase. These results indicate the potential use of the laccase in lignin modification and may provide useful references for revealing the mechanism of lignin oxidation by laccase.5. Decolorization of synthetic dyes with laccaseT. trogii laccase can directly oxidized many synthetic dyes without any mediators, including the anthraquinone dyes alizarin red S and RBBR, the triphenylmethane dyes bromophenol blue and brilliant green, the azo dyes methyl red, methyl orange, and congo red. The pH, temperature, initial concentration of the dye, and laccase dosage can influence the decolorization rate. The presence of mediators can broaden the range of the dyes oxidized by the laccase. The mediators TEMPO, HBT and ASG exhibited different effects on the decolorization. |
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