Study Of The Atypical Laccase From Trametes Hirsuta Lg-9

The people should seek solutions to the energy, environmental, and food challenges in the 21st century. The biofuel is a good candidate for fossil fuel. Biomass is the most abundant renewable resource in the world. The biomass is mainly composed of cellulose, hemicellulose and lignin, and it is of good use in fuel, silage, and chemical materials. Generation Liquid Biofuels from Biomass is one of the key strategies for sustainable development. Lignin is one of the key barriers to the utilization of biomass. The research on the degradation of lignin is signification to the application of biomass. The contaminations in pulp and dyes waste water are main small molecular lignin, the derivate of the monomer of lignin, and polyphenols. Lignin is mainly composed with three vinyl alcohols. The degradation of lignin does not make any pollution to the nature, while the most of the industry pollutions are aromatic substances.Laccases have a wide range of substrates (typically mono-, di-, and polyphenols, aromatic amines, methoxyphenols and ascorbate). This oxidation is coupled to the four-electron reduction of dioxygen to water molecules. Laccases also generate free radicals, which mediate a variety of subsequent reactions. Laccase is a main enzyme in the degradation of lignin. But it was ignored for a long time because it could not directly oxidize non phenol compounds. The discovery of the mediator attracts more attentions of the researchers. Laccases have great biotechnological potential because of their broad substrate specificity. They can be used in the paper industry, the food industry, in dye or stain bleaching, bioremediation, plant fibre modification, ethanol production, biosensors, biofuel cells, organic synthesis, and drug synthesisIn our study, we isolated and characterized a fungus. The laccase was purified and characterized. The main jobs are as follows:I. Isolation and characterization of laccase production fungusTrametes hirsuta lg-9 (CGMCC No.2422) was isolated from Mengshan Mountain (in Shandong Province, China) and characterized by our laboratory. This fungus can secrete more laccases than the other fungi in our lab, school and those were purchased from China General Microbiological Culture Collection Center (CGMCC). The fungus grows faster. The fungus was characterized as T. hirsuta lg-9 according to its' morphology and ITS. The fungus was kept in CGMCC and the number was CGMCC No.2422.Ⅱ. The laccase production influence factors and the influence of C/NWe analyzed the laccase production influence factors of the fungus T. hirsuta lg-9 with the method of orthogonal experimental design, factorial design, steepest ascent, and response surface methodology (Fig.1). A quadratic model was obtained using the design expert, and the model was proved to be validated. According to the model, the maximum production of laccase was obtained at the concentration of 10.31 g l-1 glucose and 1.32 g l-1 ammonium dibasic phosphate, and the C/N ratioFig.1 the protocol of experiment condition optimization was 343.67 mM (glucose-C):19.99 mM (NH4+-N). The maximum laccase activity was about 16.56 U ml-1. We could reduce the nitrogen source in the laccase fermentation to reduce the cost according to the optimization. The result indicated that the secretion of laccase could be induced by the nitrogen starvation. Ⅲ. Purification and characterization of the laccaseThe purification of the laccase was carried according to the common method (Fig. 2). The characterization of the laccase was well studied. We classified the laccase from T. hirsuta lg-9 as a novel "white" laccase because of its atypical spectrum. White laccases lack absorption at 600 nm, and they also have been called "yellow"Fig.2 purification of laccase laccases (Fig.3). The laccase had a molecular weight of 90 kD. The isoelectric point of the enzyme was about 4.3. The metal content was determined by atomic absorption. The laccase from T. hirsuta lg-9 showed valued of 2.86±0.21 copper atoms and 0.82±0.27 manganese atoms per protein molecular. Zinc and iron was not detected. The N-terminal amino acid sequence of the purified protein was determined up to 13 amino acids as AIGPTADLTQSQA. A pH of 2.4 was optimal for the oxidation of ABTS and a pH of 2.5 was optimal for DMP. A temperature of 85℃was optimal for DMP oxidation. The half-life of this laccase was 70 min at 75℃, and 5 hours at 65℃.Fig.3 UV/visible spectrum of laccase from T. hirsuta lg-9In our study, we found that different concentration of oxalic acid can caused different inference to the laccase activity determination with different substrate, and the laccase from T. hirsuta lg-9 can directly oxidize some non-phenolic compounds. Both of them are worth further study. IV Comparative Study of the influences of organic compounds on laccase activity testsThe influence of oxalic acid and ethylenediaminetetraacetic acid disodium salt-2-hydrate (EDTA Na2) on laccase from T. hirsuta lg-9 and Rhus vernificera in different test systems utilizing 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP) as enzyme substrates were tested. Our study indicated that oxalic acid can influence the laccase activity determination mainly by changing the pH of the reaction system. The influences of both oxalic acid and EDTA on the laccase activity determination with different substrates were different. The results indicated that both oxalic acid and EDTA could influence the laccase activity determination by influencing and the binding of laccases substrates but not by chelating metal of the laccase. The organic compounds also can influence the laccase activity determination by changing the pH of the reaction system.V oxidation of non-phenol dye methyl red and conditions optimizationUV-Vis spectrophotometry was used for process monitoring of the oxidation products of methyl red. High performance liquid chromatography (HPLC) was used for detecting the products. Additionally, the structures of products were elucidated with electrospray injection mass spectroscopy. Probably degradation mechanism of the azo dye methyl red oxidized by the atypical laccase directly had been proposed (Fig.4).As methyl red can not be oxidized to generate a phenolic radical, the oxidation mechanism of the atypical laccase to oxidize non-phenolic methyl red may be also different from the oxidation mechanism of phenolic azo dyes with common laccase. UV-Vis spectrophotometry was used for process monitoring of the oxidation products of methyl red. The results of UV-Vis spectrophotometry indicated the productions of methyl red increased with the oxidation time running. HPLC was used for detecting the products. The results of HPLC indicate the quantities of the products are no less than three. Additionally, the structures of products were elucidated with electrospray injection mass spectroscopy. The structures of products indicate the oxidation mechanisms of the atypical laccase and common laccase are similar but different. The cleavage sites of them are the same, while the products are different. There were no quinones detected. We speculate the atypical laccase can directly attack the dye, which causes the N-demethylation of the dye. Similar reactions have been reported in decolorization by laccase-mediator system and laccase-ultrasound treatment. The formed amino-group can be further oxidized by laccase. Then, the atypical laccase attack the azo linkage, which induces a variety of subsequent reactions. Leontievsky et al. proposed that yellow laccases were formed by modification of blue laccases by mediators, so they can directly oxidize non-phenolic compound and hydroxy polyaromatic dye. The white laccases are formed by the replace of T1 Cu with other transition metals. The T1 site functions as the primary electron acceptor and is important to the redox potential of laccase. The laccase from T. hirsuta lg-9 may belong to white laccase which may be formed by the replace of T1 Cu with Mn. This is probably the main reason of direct oxidation of methyl red. Fig.4 The probably reaction scheme of methyl red directly oxidized by the atypical laccase from T. hirsuta lg-9Factorial design, steepest ascent design and central composite design were successfully applied to optimize the decolorization conditions of methyl red. The equations given below are based on the statistical analysis of the experimental data.The results of analysis of variance (ANOVA) imply the model is significant. We can calculate the value of Y was maximum when the algebraic solutions were X1= 0.66, X2=-0.11 and X4= 0.34. These values correspond to the uncoded value of CEnzyme= 1.8 U l-1, pH= 4.93 and Time= 130.23 min. The maximum predicated decolorization of methyl red was 79.41%. These optimum values were checked with experiments. The actual values of the methyl red decolorization were at an average of 80%. The error was less than 5%, and the model is significant.