Study On Laccase Character Secreted By White Rot Fungus Pleurotus Sp.

Laccase is an important enzyme of lignin degradation by white rot fungi. It has high redox potential and takes O2 as electron receptor without the aid of H2O2, so it has more practical value than other ligninases. Pleurotus sp. is a very important species of white rot fungi with great activity of producing laccase. Firstly, the influence on the production of laccase by Pleurotus sp. in different fermentation conditions and culture systems has been studied. In the liquid culture system, the optimum fermentation conditions of best growth and excretion laccase are non-buffering system of pH 5.0, rotating speed 150rpm, 100mL culture medium per 250mL flask. The optimum inducing intermediate is solid degradations of corn stalk. The maximal laccase activity can reach 3,748U/mL, which is 10 times than the control. Pleurotus sp. produces more laccase and exhibits more visible multi-peak phenomena in wood chip solid condition than in other solid conditions and any liquid system, but grows worst slowly in it. Adding lignocellulose or intermediates with similar structure into the system of degradating dyes of Pleurotus sp. can achieve cometabolization in both liquid and solid systems. Secondly, the dynamics of growth, laccase production of Pleurotus sp. and lignocellulose degradation, purification and properties of laccase isoforms have been studied in the solid system. Several laccase isoenzymes are the main reason of the multi-peak phenomenon of laccase production by Pleurotus sp.. There are main three kinds of isoenzymes: low molecular weight Lac1, medium-high molecular weight Lac2 and Lac3. The laccase activity is main determined by Lac1. The deeper and longer of band of Lac1 is, the higher the activity is. The other isoenzymes just play a supplementary role. The molecular weight of Lac1 is 33.8kDa, the optimum pH of reaction and stability are 5.0 and 6.0 respectively. The optimum temperature is 25℃. Cl-suppresses the activity of Lac1 strongly, Cu2+ and Co2+ activate obviously the activity of Lac1. Michaelis constant Km of Lac1 for ABTS in wood chip, corn stalk and straw stalk is 0.037727, 0.035377 and 0.032165 mmol/L respectively, a little larger than that of crude laccase corresponding, which means crude laccase has higher appetency with ABTS and laccase isoenzymes have cooperative effects. Its decoloration rate approximately 51.0% of malachite green is better than that of other three dyes. The dynamics of growth, laccase production and lignocellulose degradation of Pleurotus sp. are consistent with one another. Generally, the higher lignified degree is, the more favorable the laccase production is, the more obvious the break of chemical bonds in FTIR patterns is, the higher degrading quantity and selectivity is. At the same time, the more cellulose and hemicellulose is, the more favorable the growth of Pleurotus sp. is. In this thesis, the author for the first time, explained and studied the phenomena of multi-peak and variation of michaelis constant Km with culture time during the process of laccase excretion by white rot fungus, and explored the relations between the dynamics of growth, enzyme production and lignocellulose degradation, being of great important academic and practical significance.