Efficient Expression Of Laccase Genes In Pichia Pastoris

A novel laccase gene (lacE) 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 of the gene. The DNA sequence of lacE obtained was 2717 base pairs (bp) in length, including the entire structure gene (2130 bp) and the 5'- and 3'-noncoding regions. Specific primers, used to amplify the cDNA sequences of lacB from Trametes sp. AH28-2 and lacA, lacB, lacC, lacD and lacE from Trametes sp. 420, were designed according to their corresponding DNA sequences. These cDNA sequences obtained from reverse transcription products ranged from 1560 bp to 1578 bp. The six genes display relatively high identity of over 50% with other laccase genes from different fungal strains. Based on the amino acid sequences deduced from the cDNA sequences, the conserved amino acid residues in copper-binding regions, including ten His and one Cys residues, and several putative N-glycosilyzation sites were found in the peptide chains of these laccase genes.Both Trametes sp. AH28-2 lacB and Trametes sp. 420 lacC were inserted into the expression vector pPIC9K and heterologously expressed in the Pichia pastoris strain GS115 using the leader sequence of α-factor as a secretion signal, respectively. Laccase-producing transformants were screened out by MD plates and BMM plates containing substrate ABTS. Several target clones were shakingly cultured at 20 ℃ in BMM medium containing 0.3 mmol/L CuSO₄ and 0.8 % alanine. The laccase activity of transformant GSb1 with lacB reached 3.2×10⁴U/L after 13-day culture and the laccase activity of transformant GSc1 with lacC reached 1.62×10⁴ U/L after 9-day cell growth.The apparent K_m value (667 μmol/L) of the recombinant laccase rLacB, derived from lacB, was 4 folds to that of the corresponding native laccase (nLacB). The optimal temperature of rLacB optimal for oxidizing substrate ABTS is 45 ℃, lower than that of nLacB (55 ℃). nLacB is most stable at pH 5.4 whereas rLacC is most stable at pH 7.4. Furthermore, rLacB has a broader pH range (pH 3:0-9.0) for the stability in comparison with nLacB.The purified laccases of rLacB and nLacB and the culture supernatant of recombinant...