Preliminary Research On Properties And Function Of Laccase From White Rot Fungi 1j-1

In order to improve lignin degradation efficiency, we isolated and characterized a fungus that has good properties. This fungus can secrete the most laccases of all, and grows fast. The laccase is somewhat different from other normal laccases such as good thermostability and unexpectedly low optimal pH, which make it a good candidate for theoretical and applied research. The fungus was characterized as Trametes sp. lj-1 according to its morphology and the reference book. By studying biomass and enzyme production curve of this fungus, we well know its growth pattern. We analyzed the laccase production influence factors of lj-1 with the method of orthogonal experimental design, factorial design, steepest ascent, and response surface methodology. We found the C/N ratio was a key factor. According to the model, the maximum production of laccase was obtained at the concentration of 10.45 gl-1 glucose and 1.28 gl-1 ammonium dibasic phosphate. The maximum laccase activity was about 33.48 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.The purification of laccase was carried out on the basis of the traditional method. After a crude enzyme preparation and purification, we obtained electrophoretically pure laccase. The laccase had a molecular weight of 60 kDa according to the results of SDS-PAGE. UV scanning revealed that there was an absorption peak at 600nm, indicating that the laccase was one of common laccases. pH 3.0 was optimal for the oxidation of ABTS and pH 2.6 for 2,6-DMP. Although the laccase was very unstable at its optimal pH, pH 4.5was suited for both of substance. Moreover temperature 40℃was optimal for 2,6-DMP oxidation and it was very stable at its optimal temperature. When the laccase was incubated at 40℃for 24h there was negligible loss of activity.With microwave radiation we processed cellulytic enzyme lignin (CEL) through the purified laccase, and made a research on the change in the content of phenolic hydroxyl by UV-Vis spectrophotometry and Infrared spectroscopy. We hope to clarify the mechanism of laccase. The results above revealed that microwave radiation did not lead enzyme inactivation at its optimal temperature. Compared with the control experiment, we found that microwave radiation could cause the increase of phenolic hydroxyl of CEL in a short of time. Images produced by UV spectrum and FT-IR also supported the conclusion. Because of its omnidirectional heating, microwave radiation can accelerate the lignin degradation, and some researchers made a hypothesis that non-phenolic substrates may turn into phenolic substrates in lignin degradation, and then were degraded by oxidase. Our experiments can also provide evidences for the hypothesis.