Characterization?Application Of Laccase From Ganoderma And The Expression Of Laccase Gene In Pichia Patoris

Laccase?EC 1.10.3.2?is a copper-containing polyphenol oxidase,which has great potential for application because of its wide range of substrates and no harm to the environment.However,its large-scale application has been constrained by the economy and efficiency of the enzyme.The screening of laccase strains with excellent properties has important value and significance.Laccase-producing strain SYBC L48 was identified by molecular identification.The composition of laccase-producing medium,the purification of laccase and its enzymatic properties were investigated.The application of laccase in decolorization of synthetic dyes was also studied.The laccase gene was cloned and heterologously expressed.The main results are as follows:?1?A laccase-producing fungus preserved in laboratory was identified as Ganoderma sp.and was named Ganoderma sp.SYBC L48.The optimized fermentation medium for enzyme production was:sucrose 12 g·L^-1?soybean meal 6 g·L^-1?CuSO₄·5H₂O 0.5 mmol·L^-1?KCl0.5 g·L^-1?KH₂PO₄ 1 g·L^-1?MgSO₄·7H₂O 0.5 g·L^-1?MnSO₄·H₂O 0.01 g·L^-1.Under the optimized medium,the highest laccase activity of Ganoderma sp.SYBC L48 was 2598 U·L^-1,which was 35.1%higher than that before optimization.?2?The purified Ganoderma sp.SYBC L48 laccase was obtained by ammonium sulfate fractionation and DEAE-FF ion exchange chromatography.Its purity and yield were 13.2 folds and 21.5%of the crude extract,respectively.The molecular mass of the purified laccase was about 60.0 kDa on SDS-PAGE.The optimum temperature of laccase was 60°C,the optimum pH was 2.5.The enzyme activity exhibited excellent stability at pH 5.0⁹.0 and 20⁶0°C respectively.Co²⁺,Cr³⁺,Fe³⁺inhibited the activity of laccase.The Km of ABTS?2,2'-azinebis?3-ethylbenzothiazole-6-sulfonic acid??was 0.044 mmol·L^-1.?3?The purified laccase enzyme has obvious decolorization effect on remazol brilliant blue R,acid red 1 and malachite green,the decolorization rates were 76.6%,90.3%and 58.1%after120 min,respectively.The toxicity of dyes and their laccase degradation products was analyzed using wheat seed and Chlorella vulgaris.The results showed that the toxicity of dyes after the enzyme treatment was decreased.?4?The Ganoderma sp.SYBC L48 laccase full-length structural gene lac1 and the corresponding cDNA sequence were obtained by cloning.Bioinformatics analysis indicated that the lac1 laccase gene encodes 525 amino acids,of which the first 22 amino acids were predicted to be signal peptide sequences.The predicted molecular weight of lac1 laccase protein was 57.2 kDa?excluding glycosylation?and its isoelectric point was 5.23.The heterologous expression of lac1 laccase gene in Pichia pastoris was successfully achieved.