Molecular Cloning And Heterogenous Expression Of Laccase Gene From Auricularia Auricula

The degradation of lignin in nature is mainly by laccase which secreted from white-rot fungus. Laccase is a polyphenol oxidase containing copper, it can catalyze oxidation phenolic compounds, and has strong ability of decomposition to many compounds that structure similar with lignin. Laccase has a wide application value in many fields.over the years, although has achieved breakthrough and remarkable achievements, but laccase primarily produced from white-rot fungus, and its capacity is low. Auricularia auricula is a kind of white-rot fungi, is also a kind of edible and medicinal supplements black health food. Therefore screen high production of laccase strains has important significance, but no reports of domestic. We screening a strain with high production of laccase from long dozen of Auricularia Auricula named Dimao-1. All strains grown on PDA plate containing 0.04% guaiacol. We selected according to their diameters of coloration circle and enzyme activity.We cloning its laccase gene and studied its heteroexpression,and providing theoretical basis for the expression of laccase engineering strains and industrialized production.Through Primer 5.0 Analysis on Auricularia Auricula laccase lac1(GenBank No. AY450405), and devise primer, we cloned laccase gene cDNA and gDNA from Dimao-1 by RT-PCR and PCR, its length was 1788 and 2824bp, respectively. The gDNA conclude 18 intron by Blast alignment, the length of which rank from 50bp to 70bp. And most of them subject to GT-AG rule.The sequences are submit to GenBank and obtain accession number GU370887 and GU553092. We BLAST cDNA in NCBI found its homology is 99% with AY450405.Construction of Pichia pastoris expression plasmid pPIC9K-cDNA which containedα-factor instead of signal peptide itself. The plasmid were transformed into the Pichia pastoris GS115, and the genetically engineered microorganism known as pPIC9K-cDNA-GS115, the effective expression was detected by SDS-PAGE and the determined result of enzyme activity with fermentation liquor. The optimum temperature were 50℃, the optimum pH value recombinant laccase of P.pastoris and Auricularia Auricula laccase were 2.8 and 3.2 respectively.The perdiction of laccase senior structure. We predicted the senior structure of laccase with the help of Bioinformatics. The secondary structure are displayed by the soft of CLC protein work bench and pymol. The laccase gene with theα-factor signal sequence was inserted into the expression vector of pPIC9K and transformed into Picha pastoris GS115. Though G418 screening and active validation of MM flat which contain ABTS, screen a transformation of high production of laccase After the 6 days of induction with 0.5 % methanol at 28-30℃,the produced crude enzyme was detected to reach the highest enzyme activity.SDS-PAGE Analysis shows that in the recombinant fermentation liquor just contain one protein, and facilitate to the downstream purification.