[Cloning of a laccase gene from Flammulina velutipes and study on its expression in Pichia pastoris].

Laccase(EC1.10.3.2) can be used for enzymatic detoxification of lignocellulosic hydrolysates. By using molecular techniques such as RACE (rapid amplification of cDNA ends) and Genome-Walking, a laccase gene and its corresponding full-length cDNA were cloned from Flammulina velutipes and designated as glccFv and IccFv. The sequences were submitted to GenBank, and the accession numbers obtained were AY485826 and AY450406, respectively. Analysis of amino acids sequence suggested that one laccase from Polyporus ciliatus possessed the highest homology with the protein encoded by lccFv showing for 72%. The ORF (open reading frame) of lccFv was transformed into Pichia pastoris strain GS115 through the P. pastoris expression vector pHBM906, which contains both the promoter and transcription terminator of the AOX1 gene. The recombinant laccase LCCFv was detected from the engineering strain GS115 (pHBM557) which was fermented with BMMY liquid medium and induced by 1.0% (V/V) methanol at 20 degrees C with the highest expression level (0.1070 U/mL). The optimal reaction temperature of LCCFv that secreted from P. pastoris GS115(pHBM557) was 45 degrees C, the optimal reaction pH value was pH3.9 and the thermostability and pH stability were very well under the optimal conditions.