Cloning of laccase gene form Coprinopsis cinerea and study on its expression in Pichia pastoris

The laccase gene 1 was cloned by RT-PCR from Coprinopsis cinerea okayama 7#130,and through analyzing its amino-acid sequence using software SignalP 3.0 Server,a new primer without signal peptide sequence was designed and laccase 1 was obtained through its extension.Construction of Pichia pastoris was conducted for expressing plasmid pPIC9K-lac1,and then transformed into Pichia pastoris GS115 by electric shock and induction expression was effected using methanol.The properties of recombinant enzyme were studied.The full length was 1 593 bp of laccase 1,which coded 530 amino-acids,among them,18 amino acids of signal peptide sequence.SDS-PAGE showed that obvious protein band was 65 kDa.The optimal fermentative conditions of it: 45 ℃,and pH=4.3.The enzyme activity of secretion expression achieved 1.108 U/mL.