Fermentation optimization, cloning and sequence analysis of the laccase gene from Shiraia sp SUPER-H168

Incubation with different concentrations of different carbon, nitrogen and metal ion sources was found to significantly influence Shiraia sp. SUPER-H168 laccase production. Based on the magnitude of the range, the factors were ranked as follows: starch > Cu2+ > yeast powder for laccase production. The optimal combination of factors was 20 g/L starch, 4 g/L yeast powder and 0.6 mM Cu2+. Other fermentation media and conditions were also studied. The maximal laccase yield of Shiraia sp. SUPER-H168 varied from 5,058 to 1.105 x 10(5) U/L. Shiraia sp. SUPER-H168 laccase DNA and its corresponding full-length cDNA were cloned and characterized; furthermore, the Shiraia laccase gene sequence is reported for the first time. The coding region of Shiraia sp. SUPER-H168 laccase consisted of a 1,848 bp open reading frame encoding a protein of 615 aa with a 21 aa signal peptide sequence. Three introns of 50, 49 and 51 bp in length were located at upstream of the N terminus. The molecular mass (66.7 kDa) and isoelectric point (7.63) were predicted. Additionally, the protein contained six potential N-glycosylation sites (Asn-X-Ser/Thr) and four conserved copper-binding domains. The neighbor joining phylogenetic tree demonstrated that Shiraia sp. SUPER-H168 laccase is an ascomycetes laccase closely related to Phoma sp. UHH 5-1-03 laccase.