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Substrate analysis and molecular cloning of the extracellular alkaline phosphatase of Streptomyces griseus

Instituto de Biotecnología de León INBIOTEC, Parque Científico de León, Avda del Real no. 1, 24006 León, Spain¹
Area de Microbioloía, Facultad de Ciencias Biológicas y Ambientales, Universidad de León, 24071 León, Spain²

Author for correspondence: Paloma Liras. Tel: +34 987 291504. Fax: +34 987 291506. e-mail: degplp{at}unileon.es

Streptomyces species secrete large amounts of alkaline phosphatase (AP) enzymes that have not been characterized so far. An AP has been purified to homogeneity from cultures of Streptomyces griseus IMRU 3570. The enzyme has a monomer size of 62 kDa and is processed in the culture to a 33 kDa protein as shown by immunoblotting. The enzyme was purified by ammonium sulfate precipitation, CM-Sephadex cationic exchange, chromatofocusing and HPLC Sphaerogel 3000SW filtration. The pure enzyme uses a variety of organic phosphorylated compounds as substrates. The N-terminal end of the mature protein was found to be RLREDPFTLGVASGDPHP. The gene phoA has been cloned using as probe an oligomer based on the N-terminal sequence of the S. griseus AP. phoA encodes a protein of 62678 Da with low homology to the AP of Escherichia coli. The phoA gene was found to be homologous to three alkaline-phosphatase-encoding genes previously identified in the Streptomyces coelicolor genome. On the basis of the optimal pH, substrate specificity and differences in amino acid sequence of motifs defining the active centre of APs, the S. griseus AP uses a wide range of organic phosphate substrates and is different from the phosphatases of Gram-negative bacteria.

Keywords: phytases, exocellular enzymes, protein purification, gene cloning

Abbreviations: AP, alkaline phosphatase; PNPP, p-nitrophenyl phosphate; X-phosphate, 5-bromo-4-chloro-3-indolyl phosphate

The GenBank accession number for the sequence reported in this paper is AJ278740.

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