Molecular cloning, characterization, and expression of the phytase gene from marine yeast Kodamaea ohmeri BG3

摘要

The extracellular phytase structural gene was isolated from the cDNA of the marine yeast, Kodamaea ohmeri BG3, using the switching mechanism at 5'-end of RNA transcript (SMART)trade mark rapid-amplification of cDNA ends (RACE) cDNA amplification kit. The gene had an open reading frame of 1389bp and the coding region of the gene had no intron. It encoded 462 amino acid residues of a protein with a putative signal peptide of 15 amino acids. The protein sequence deduced from the extracellular phytase structural gene contained the consensus motifs (RHGXRX P and HD), which are conserved among histidine acid phosphatases, and six conserved putative N-glycosylation sites. According to the phylogenetic tree of the phytase, the phytase from K. ohmeri BG3 was closely related to Candida albicans (XP_713452) and Pichia stipitis (XP_001385108) phytase proteins and more distantly related to other phytases. The mature peptide encoding cDNA was subcloned into the pET-24a (+) expression vector. The recombinant plasmid [pET-24a (+)PHY1] was expressed in Escherichia coli BL21 (DE3). The expressed fusion protein was analysed by SDS-PAGE and Western blotting, and a specific band with a molecular mass of about 51kDa was found. An enzyme activity assay verified the recombinant protein as a phytase. A maximum activity of 16.5Umg(-1) was obtained from the cellular extract of E. coli BL21 (DE3) harbouring pET-24a (+)PHY1. The optimal pH and temperature of the crude recombinant lipase were 5 and 65 degrees C, respectively, and the crude recombinant phytase had hydrolytic activity towards phytate.