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 5oC) and remarkably thermal lability (losing >96%
activity after incubation at 60oC for 2 min). The optimal
phytase activity occurred at pH 5.5 and ~40oC, and the
enzyme activity rapidly decreased above 40oC. When
compared with mesophilic counterparts, the phytase not
only exhibited a high activity at a low temperature, but
also had a low Km and high kcat. 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-temperatureactive
phytase and its heterogeneous expression.

Keywords: Aquaculture, low-temperature-active enzyme, Erwinia carotovora, phytase