Cloning and heterologous expression of laccaes isozyme B gene from Trametes sp. 420

A novel laccase gene(lacB) was cloned from the genomic DNA isolated from Trametes sp.420,using the degenerate primers based on the conserved copper-binding regions in fungal laccases.Long distance-inverse PCR(LD-IPCR) was used to amplify the flanking sequences,generating the entire lacB DNA sequence including the 2255-bp structure gene and 5′-and 3′-noncoding sequences.The lacB gene is interrupted by 12 introns.The lacB cDNA is 1560 bp long,encoding a 519 amino acid polypeptide composed of a 24 amino acid signal sequence and a 495 amino acid mature protein.In addition,lacB displays a relatively high homology with laccase genes from other different fungi but a low homology of less than 25% with those from plants,bacteria and insects.Furthermore,the lacB cDNA without its signal sequence was cloned into the expression vector pPIC9K through the pPIC9 plasmid and transformed into the Pichia pastoris strain GS115.The laccase-producing transformants were screened out from the BMM plate containing substrate ABTS(2,2'-azino-bis-(3ethylbenztbiozoline 6-sulfonic acid).