ScienceAsia 30 (2004): 383-390 |doi: 10.2306/scienceasia1513-1874.2004.30.383
Purification, Characterization, Gene Cloning and
Sequence Analysis of a Phytase from Klebsiella
pneumoniae subsp. pneumoniae XY-5
Xueying Wang,a Suchart Upatham,a,e Watanalai Panbangred,b Duangnate Isarangkul,c
Pijug Summpunn,b Suthep Wiyakrutta,d and Vithaya Meevootisomd,*
ABSTRACT: A phytase produced by the soil bacterium Klebsiella pneumoniae subsp. pneumoniae strain XY-5 was
isolated and purified. The enzyme was a single chain protein with a molecular mass of 41.7 kDa, as determined
by SDS-PAGE. The isoelectric point (pI) of the native enzyme was found to be 8.7. It exhibited two pH
optima (3.7 and 5.5) when assayed both at 37oC (297 units/ mg protein) and 55oC (318 units/ mg protein)
and it was found to be stable up to 60oC for 4.0 hours. The enzyme was found to have broad substrate
specificity. It was activated by EDTA, Al3+ and Co2+, but was strongly inhibited by Hg2+. The putative gene
encoding the phytase was cloned by PCR, and DNA sequencing revealed an ORF of 1269 nucleotides
downstream from a potential ribosome-binding site. The deduced amino acid sequence of the mature protein
comprised 394 aa with a calculated molecular mass of 43.4 kDa and a signal peptide consisting of 28 aa. The
mature enzyme contained the conserved active site RHGXRXP and an HD motif that placed it in the
histidine acid phosphatase (HAPs) family. Expression of the cloned gene in an E. coli yielded active phytase.
Due to its relatively high specific activity, broad substrate specificity, good pH profile and temperature
stability, the enzyme could be a good candidate for industrial applications.
a Department of Biology, Faculty of Science, Mahidol University, Bangkok, Thailand.
b Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand.
c Center for Biotechnology, Institute of Research and Development in Science and Technology, Mahidol University, Salaya campus, Nakhonpathom, Thailand.
d Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
e Department of Medical Science, Faculty of Science, Burapha University, Chonburi, Thailand.
* Corresponding author, Email: firstname.lastname@example.org
Received 7 Jul 2004,
Accepted 13 Sep 2004