Expression Of A Laccase Gene From Lentinula Edodes In Pichia Pastoris And Degradation Of Triarylmethane Dyes By Laccase In The Presence Of Natural Mediators

Laccases (EC 1.10.3.2) belong to the family of the multicopper oxidases and catalyse the four-electron reduction of O2 to H2O with the concomitant oxidation typically of a phenolic substrate. They belong to the group of the ligninolytic enzyme systems. Compared with other ligninolytic enzymes, it has two advantages:one is that it need not the assistant of H2O2 to oxidize the substrate, the other is it won't cause contamination due to the reduction of O2 to H2O. Laccases have more potential industrial value and been widely used in dyes decolourisation, paper bleaching, biological detecting and so on. As the white-rot fungi, Lentinula edodes can secrete more laccases into the medium and degrade the dyes. However, the levels of laccases synthesis from Lentinula edodes grown on the artificial or natural medium are usually low. Moreover, it is very difficult to ferment it on a large-scale industrialization, due to the difficult conditions of culture and the complex products of fermentation. Thus laccases genes cloning and high production in hetero-hosts can offer an important alternative way to work out the shortage of enzymes.Based on the sequence of the LeLccl gene (GenBank accession No. AB035409) from L. edodes (shiitake mushroom), using L. edodes cDNA as template, we designed two outside primers. The forward primer (5'-GGATCCATGTTTTACTTCTCATCTTTCCTG-3') and the reverse primer (5'GAGCTCTTAATTTCCACCAAGTTGTGCAGG-3') were used to amplify the LeLccl gene and to introduce BamH I at 5'end and Sac I at 3'end. The BamH I and Sac I restriction sites in Leccl gene and native signal peptide (18 amino acids) were deleted using site-directed mutagenesis. After BamH I and Sac I digestion, the gene secretion expression vector pYM8837 in Pichia pastoris was constructed after inserting the LeLccl gene into a modified pPIC9K in which the Sac I site in the AOX promoter and Xho I site in the G418 cassette were removed by site-directed mutagenesis. Meanwhile, 6XHis-tag was addded after KEX2 protease cleavage site of the MFa signal sequence. The pYM8837 vector was linearized with Bgl II and transformed into P. pastor is GS115 by electroporation method. According to SDS-PAGE, the purified laccase was estimated to have a molecular mass of around 62 KD and the rate of glycosylation might be 15.4%. The pH profile for recombinant laccase (rLccl) activity against ABTS showed one peak of maximum activity at pH 2.4 and the enzyme was stable at 30℃. Compared with the native laccase, they have similar enzymatic properties in many aspects. They both were inhibited by Ba2+, Ca2+, Zn2+, and A13+. The rLccl was strongly inhibited by the copper chelating agents, sodium azide and DTT, but not by EDTA, which is typical of other previously characterized laccases. L-Alanine, used to keep pH stable during the expression of a laccase gene in P. pastoris, was found to have positive effect on laccase, especially 0.1 mM L-Alanine. In addition, methanol and Cu2+play important role in the level of laccases expression and were studied in our work. It was shown that the maximal level of laccases expression was obtained in the BMM media with 0.2 mM copper and daily addition of 1.5%methanol.The declourisation rates of malachite green, crystal violet and brilliant were very low by the purified rLccl. The conditions of decolourisation dyes by Laccase-ABTS system were optimized. The optimal conditions for malachite green were at pH 2.4-3.0,40℃with 10μM ABTS; crystal violet at pH 2.4-3.6,45℃with 10μM ABTS; brilliant green at pH 3.0-4.0,50℃with 4μM ABTS.Acetosyringone and syringaldehyde were selected as laccase mediators after screening 8 different natural compounds with a recalcitrant dye (malachite green) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times by the use of the rLccl and were compared with ABTS. In the presence of the two mediators, a nearly complete decolourisation (99%-100%) was attained after 18h for malachite green. With the two mediators, the two other dyes (crystal violet and brilliant green) attained about 65%decolourisation. Mediator concentration and pH have an effect on decolourisation of the dyes. The two natural mediators reached their most efficient decolourisation levels at pH 4.0 for malachite green and crystal violet, and at pH 3.6 for brilliant green. In addition, HBT has no mediating effect on decolourisation of the recalcitrant dyes.