Purification, sequencing and evaluation of a divergent phytase from Penicillium oxalicum KCTC6440

Abstract

A fungal strain producing high levels of phytase was purified to homogeneity from Penicillium oxalicum KCTC6440 (PhyA). The molecular mass of the purified PhyA was 65 kDa and optimal activity occurred at 55°C. The enzyme was stable in a pH range of 4.5–6.5, with an optimum performance at pH 5.5. The K_m value for the substrate sodium phytate was 0.48 mM with a V_max of 672 U/mg. The enzyme was inhibited by Ca²⁺, Cu²⁺, and Zn²⁺, and slightly enhanced by EDTA. The PhyA efficiently released phosphate from feedstuffs such as soybean, rich bran and corn meal. The PhyA gene was cloned in two steps of degenerate PCR and inverse PCR and found to comprise 1501 bp and encode 461 amino acid residues. The enzyme was found to have only 13 amino acids differing to the known PhyA from other Penicillium sp., but has distinct enzyme characteristics. Computational analysis showed that PhyA possessed more positively charged residues in the active sites compared to other PhyA molecules, which may explain the broader pH spectrum.