Novel Low-Temperature-Active Phytase from Erwinia carotovora var. carotovota ACCC 10276

A phytase with high activity at low temperatures has great potential for feed applications, especially in aquaculture. Therefore, this study used a degenerate PCR and TAIL PCR to clone a phytase gene from Erwinia carotovora var. carotovota, the cause of soft rot of vegetables in the ground or during cold storage. The full-length 2.5-kb fragment included an open reading frame of 1,302 bp and encoded a putative phytase of 45.3 kDa with a 50% amino acid identity to the Klebsiella pneumoniae phytase. The phytase contained the active site RHGXRXP and HD sequence motifs that are typical of histidine acid phosphatases. The enzyme was expressed in Escherichia coli, purified, and displayed the following characteristics: a high catalytic activity at low temperatures (retaining over 24% activity at $5^{\circ}C$) and remarkably thermal lability (losing >96% activity after incubation at $60^{\circ}C$ for 2 min). The optimal phytase activity occurred at pH 5.5 and ${\sim}49^{\circ}C$, and the enzyme activity rapidly decreased above $40^{\circ}C$. When compared with mesophilic counterparts, the phytase not only exhibited a high activity at a low temperature, but also had a low $K_m$ and high $k_{cat}$. These temperature characteristics and kinetic parameters are consistent with low-temperature-active enzymes. To our knowledge, this would appear to be the first report of a low-temperature-active phytase and its heterogeneous expression.