Substrate analysis and molecular cloning of the extracellular alkaline phosphatase of

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

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Abstract

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 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 has been cloned using as probe an oligomer based on the N-terminal sequence of the AP. encodes a protein of 62678 Da with low homology to the AP of . The gene was found to be homologous to three alkaline-phosphatase-encoding genes previously identified in the 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 AP uses a wide range of organic phosphate substrates and is different from the phosphatases of Gram-negative bacteria.

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2001-06-01
2024-03-29
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