Folia Microbiol. 48 (5), 627–632 (2003) http://www.biomed.cas.cz/mbu/folia/ Extracellular Alkaline Phosphatase from the Filamentous Fungus Aspergillus caespitosus: Purification and Biochemical Characterization L.H.S. GUIMARÃES, H.F. TERENZI, J.A. JORGE, F.A. LEONE, M.L.T.M. POLIZELI* Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040 901 Ribeirão Preto, São Paulo, Brazil e-mail polizeli@ffclrp.usp.br fax +55 16 602 3666 Received 2 December 2002 Revised version 13 May 2003 ABSTRACT. Among 30 species of filamentous fungi isolated from Brazilian soil, Aspergillus caespitosus produced and secreted the highest levels of alkaline phosphatase in culture medium supplemented with xylan. The extracellular alkaline phosphatase was purified by DEAE-cellulose and concanavalin A-sepha- rose chromatography. The enzyme was a glycoprotein containing up to 56 % sugar with molar mass of 134.8 kDa, according to gel filtration in Sepharose CL-6B, and 57 kDa according to SDS-PAGE. Non- denaturing electrophoresis (6 % PAGE) of the purified enzyme produced a single band, suggesting that the native enzyme was a homodimer. Optima of temperature and pH were 75 °C and 8.5, respectively. The enzyme was stable at 50 °C and its activity was enhanced by 95 % in the presence of Mg 2+ (1 mmol/L). 4-Nitrophenyl phosphate was the preferentially hydrolyzed substrate with K m and v lim values of 74 mol/L and 285 mol/s, in the absence, and 90 mol/L and 418 mol/s, in the presence of Mg 2+ , respectively. The enzyme also hydrolyzed other phosphorylated amino acids (O-phosphothreonine, O-phosphotyrosine, O-phosphoserine). Activity of phosphatases (phosphoric monoester hydrolases, EC 3.1.3) is widely distributed in nature (McComb et al. 1979). These enzymes have been conventionally referred to as alkaline phosphatases (EC 3.1.3.1) with optimum pH above 8.0, and acid phosphatases (EC 3.1.3.2) with optimum pH below 6.0. The phosphatases have been classified into five well characterized families, viz. (i) alkaline phosphatases, (ii) purple acid phosphatases, (iii) low-molar-mass acid phosphatases, (iv) high-molar-mass acid phosphat- ases, and (v) protein phosphatases (Vincent et al. 1992). Altogether, phosphatases are able to participate in the most diverse cell functions, including the acquisition of phosphorus for nutritional purposes, carried out by enzymes of wide specificity for substrates (Oshima 1997), and to modulate networks of cell signaling, performed by specifically targeted phosphoprotein phosphatases (EC 3.1.3.16; Dickman and Yarden 1999). Our knowledge about the phosphatases of filamentous fungi is not very extensive. Phosphatase activity has been described in Neurospora crassa (Say et al. 1996; Morales et al. 2000), Humicola grisea (Buainain et al. 1998), Scytalidium thermophilum (Guimarães et al. 2001), Aspergillus niger (Elzainy and Ali 1995) and Aspergillus nidulans (Negrete-Urtasun et al. 1999), among others. It is demonstrated that in A. nidulans the expression of these enzymes depends on ambient pH (Negrete-Urtasun et al. 1999; Denison 2000). Microbial alkaline phosphatases have been used for various practical applications, such as cloning experiments, enzyme-linked immunosorbent assays (ELISA), gene sequencing, blotting, and nonisotopic probing (Dong and Zeikus 1997). Alkaline phosphatase can be also used to reveal the presence of target- detector enzyme complexes using chromogenic or chemiluminescent compounds. Some microbial alkaline phosphatases may have convenient properties, suitable for biotechnological applications, such as thermo- stability and high catalytic activity. In this context, a screening for alkaline phosphatase was carried out in approximately 30 species of filamentous fungi isolated from Brazilian soil. A high level of extracellular alkaline phosphatase production was detected in a thermotolerant Aspergillus caespitosus isolate, when cul- tivated in medium supplemented with xylan or agricultural residues (Guimarães et al. 2003). The aim of this work was to describe some biochemical and kinetic properties of this enzyme. *Corresponding author.