Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis

A novel phytase gene (phyL) was cloned from Bacillus licheniformis by multiple steps of degenerate and inverse PCR. The coding region of the phyL gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. This gene, together with a phytase gene (168phyA) identified in the B. subtilis strain 168 genome by a homology search, was cloned and over-expressed in B. subtilis using a φ105MU331 prophage vector system. Up to 35 units of phytase/ml were secreted into the culture media; and mature enzymes of around 44-47 kDa were purified for characterization. Both phytases exhibited broad temperature and pH optima and showed high thermostability. Of the two, the phytase encoded by phyL exhibited higher thermostability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95°C for 10 min. With their neutral pH optima and good temperature stabilities, these Bacillus phytases are good candidates for animal feed applications and transgenic studies.Department of Applied Biology and Chemical Technolog