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 Meevootisom^d,*

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 37^oC (297 units/ mg protein) and 55^oC (318 units/ mg protein) and it was found to be stable up to 60^oC 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.

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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: scvmv@mahidol.ac.th

Received 7 Jul 2004, Accepted 13 Sep 2004

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,*

Xueying Wang,^a Suchart Upatham,^a,e Watanalai Panbangred,^b Duangnate Isarangkul,^c Pijug Summpunn,^b Suthep Wiyakrutta,^d and Vithaya Meevootisom^d,*