Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1
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Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.
KeywordsPhytase Activity Phytase Gene Recombinant Phytase Histidine Acid Phosphatase pelB Signal Peptide
Financial support for A. S. from DAAD is gratefully acknowledged. We are especially grateful to Monika Schmid for analysis of peptide masses and Thomas Leya for his help in using the PAUP package for construction of evolutionary trees. We thank Romy Scholz and Kristin Rosner for their support in DNA sequence analysis. Dr. Steffen Porwollik is thanked for critical reading of the manuscript. The technical assistance of Christiane Müller and Sybille Striegl is gratefully acknowledged.
- Damiani G, Amedeo P, Bandi C, Fani R, Bellizi D, Sgamarella V (1996) Bacteria identification by PCR-based techniques. In: Adolph KW (ed) Microbial genome methods. CRC Press, Boca Raton, Fla., pp 167–178Google Scholar
- Greiner R, Muzquiz M, Burbano C, Cuadrado C, Pedrosa MM, Goyoaga C (2001) Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba var. Alameda). J Agric Food Chem 49:2234–2240Google Scholar
- Lassen SF, Breinholt J, Ostergaard PR, Brugger R, Bischoff A, Wyss M, Fuglsang C (2001) Expression, gene cloning, and characterization of five novel phytases from four basidiomycete fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens. Appl Environ Microbiol 67:4701–4707Google Scholar
- McClelland M, Florea L, Sanderson K, Clifton SW, Parkhill J, Churcher C, Dougan G, Wilson RK, Miller W (2000) Comparison of the Escherichia coli K-12 genome with sampled genomes of a Klebsiella pneumoniae and three Salmonella enterica serovars, Typhimurium, Typhi and Paratyphi. Nucleic Acids Res 28:4974–4986CrossRefPubMedGoogle Scholar
- Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6Google Scholar
- Reddy NR, Pierson MD, Sathe SK, Salunkhe DK (1989) Phytases in cereals and legumes. CRC Press, Boca Raton, Fla.Google Scholar
- Sajidan A (2002) PhD thesis, Humboldt University, BerlinGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
- Selle PH, Ravindran V, Caldwell RA, Bryden WL (2000) Phytate and phytase: consequences for protein utilization. Nutr Res Rev 13:255–278Google Scholar
- Skoglund E, Carlsson NG, Sandberg AS (1998) High-performance chromatographic separation of inositol phosphate isomers on strong anion exchange columns. J Agric Food Chem 46:1877–1882Google Scholar
- Swofford DL (2002) PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Mass.Google Scholar