Identification of two novel bacterial phosphatase‐encoding genes in Pseudomonas putida strain P13

AIMS: Isolation and identification of genes encoding putative phosphatases from Pseudomonas putida strain P13 DSM 23335. METHODS AND RESULTS: By functional screening of a P. putida P13 genomic library, a number of Pho+ clones were identified. Two genes were identified that encoded proteins exhibiting both phytase and sugar phosphatase activities. The proteins were 249 and 462 amino acids, with molecular masses of 26 and 50 kDa respectively. Sequence alignments revealed no significant similarities to representatives of known phosphatase or phytase gene families. However, the genes were found to have a high similarity to members of the major facilitator superfamily (MFS). Both genes were overexpressed in Escherichia coli and the corresponding partially purified recombinant enzymes were found to have significant phytate-dephosphorylating activity. The protein designated P. putida phytase 1 (Ppp1) displayed the highest activity among potential substrates studied on Na phytate, whereas Ppp2 more likely represents a sugar phosphatase than a phytase. The optimal conditions for phytate dephosphorylation were determined as 60°C and pH 4·5 (Ppp1) or pH 5·0 (Ppp2). CONCLUSIONS: Two novel bacterial phosphatase-encoding genes, named ppp1 and ppp2, were isolated from P. putida P13 DSM 23335 by a functional screening procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: Phosphatase-encoding genes are of great importance for industrial applications, particularly in agriculture. The identified phosphatase genes represent a new class of acid phosphatases.