Bioinformatics Analysis Of The Volvariella Volvacea Laccase Gene Family And Heterologous Expression Of Laccases In Pichia Pastoris

Laccases(benzenediol:oxygen oxidoreductases,EC 1.10.3.2),belong to the largest subgroup of blue multicopper oxidases(MCO),use the distinctive redox ability of copper ions to catalyze the oxidation of a wide range of aromatic substrates,they play many important roles in fungi.Volvariella volvacea is a commercially important,edible basidiomycete fungus that is cultivated on straw and other carbonaceous wastes in China and other countries in East Asia.In this study,we sequenced the V volvacea PYd21 genome and constructed a digital gene expression(DGE)tag library of different development stages,based on which we identified five new laccase(lac)genes in this fungus.The laccase multigene family in V.volvacea contains eleven genes which are distributed over a 274 kb fragment with tandem in one scaffold(or chromosome),these laccase genes were classifiable into two subfamilies based on the intron phase composition.The genes in this family may play an important role in substrate degradation and fruiting body development.Six laccase genes with signal peptides possess extracellular functions,while five laccase genes without signal peptides play intracellular roles.The genes lac3 and lac4 were found to may be involved in substrate degradation,while lac5 was the only one that was highly expressed in primordia and may be involved in fruiting.Except bioinformatics analysis of V volvacea laccase gene family,two sensu stricto laccase gene(lac3 and lac4)and its open reading frame(ORF)was isolated from the edible fungus V volvace.The lac3 and lac4 gene contain 14 and 18 introns,respectively;and an ORF of 1548 bp and 1689 bp,respectively.In addition,lac3 and lac4 encode a mature protein containing 496 and 539 amino acids(AA)with a signal peptide of 19 and 23 AA,respectively.The ORF(without the signal peptide)of lac3 and lac4 were inserted in P.pastoris GS115 using the plasmid pPIC9k with an alpha-factor signal peptide.The activity of laccase(lac3)secreted in P.pastoris GS115 attained values up to 296.83 U/L.The putative molecular weight of the mature protein was 58 kDa,determined using SDS-PAGE,while the optimal reaction pH and temperature for enzyme activity were 4.5 and 45?,respectively,using ABTS as a substrate.In addition,lac3 was highly sensitive to temperature and was most stable at a pH of 6.5.The effects of four potential inhibitors and nine metal ions on lac3 activity were tested.Enzyme activity was completely inhibited by 40%ethanol and 5 mM Hg2+;however,some metal ions promoted laccase activity while others had no significant effect.This study provides a foundation for the heterologous expression of other V.laccases.