| Laccase (EC 1.10.3.2, benzenediol:oxygen oxidoreductase) are copper containing enzymes with potential applications including biobleaching, degradation of environmental contaminants and sewage disposal. Laccase are not only applied for degradation of azo dyes (eg. methyl hesperidin), decoloring of textiles, catalytic oxidation of arylamines such as aniline and phenols, but also for catalytic synthesis of some specific compounds.Therefore, laccase have come to be one of hot pot fields among biology, chemistry and environmental science. In this paper, laccase of Ganoderma lucidum K-17 was selected as object to study its' fermentation and applications.A growing number of researchers attach importance to G. Lucidum K-17 for it can release ligninolytic enzymes when cultured on the medium contained green poison-free agricultural by-products. In this research, three-level three-factor Box-Behnken design was combined with response surface analysis (RSA) to optimize the medium contents. The results showed that the main factors influecing laccase yield were corn powder, wheat bran and peanut shell with other quantitative nutritional ingredients. The optimized medium components included corn powder (17.6g/L), bran (24.7g/L), peanut shells (22.4g/L), KH2PO4 1.5g/L and glucose 20 g/L and enzyme activity of laccase achieved maximum 5242.61U/L under such culture condition. Electrophoresis was employed for isoenzyme analysis of crude laccase of G. Lucidum and the result showed that the laccase was monomeric enzyme without any isoenzyme.Laccase of G. lucidum K-17 was employed to catalyze tea polyphenols to synthesize theaflavin. In organic system of ethyl acetate with 30mg/ml concentration of substrate, the reaction was stired up by adding 1/25(v/v) laccase and continued with water bath oscillating reaction at 40℃and terminated by ultrasonication treatment. The reaction products were extracted with 4-methyl-2-pentanone and confirmed to be theaflavin-3-mongallate and small amounts of their isomer theaflavin-3'-mongallate via HPLC and LC-MS and the concentration of product was 1.37mg/ml.Univariate analysis and response surface designs were applied for extracting theanine from green tea with laccase, pectase and cellulase. Based on single-factor test, Response Surface Analysis was employed to establish the model of enzyme-assisted extraction of theanine, and the validity of the model was verified. The interaction of laccase, pectase and cellulase was investigated and the optimal compound ratio was defined. The results showed that the optimal temperature was 50℃and the optimal extraction time was 2 hours. Response surface analysis also showed that laccase, pectase and cellulase significantly influenced the extracting efficiency of theanine. The extraction yield of theanine reached 11.60% by adding 0.6ml pectase,0.6ml cellulase and 0.48ml laccase into extraction system. Biological enzyme-assisted method provided an easy operating and environment-friendly process for xtracting theanine from green tea without expensive equipment.Quadratic regression model of extraction of lentinan was established through Response Surface Analysis and Box-Benhnken design. RSA-optimized condition for extraction of lentinan was as follows:fixed liquid ratio at 1:15 and reaction temperature at 40℃, the concentrations of pectinase, cellulase and laccase were 7.11%,6.82% and 3.69%, respectively. The yield of lentinan was 37.07% which is substantially increased compared with single enzyme experimental groups. |
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