Folia Microbiol. 51 (6), 541–545 (2006) http://www.biomed.cas.cz/mbu/folia/ Aspergillus niger pH 2.1 Optimum Acid Phosphatase with High Affinity for Phytate S. GARGOVA, M. SARIYSKA, A. ANGELOV, I. STOILOVA Department of Biotechnology, University of Food Technologies, 4002 Plovdiv, Bulgaria e-mail sgargova@lycos.com Received 12 September 2005 Revised version 30 January 2006 ABSTRACT. An extracellular acid phosphatase isolated from the culture of a wild strain Aspergillus niger, producing the dephosphorylating 3-phytase, was obtained in a homogeneous form by sequential application of ultrafiltration through PS 50 membrane, gel filtration on Sephadex G-100 and ion exchange chromatogra- phy on DEAE-Sepharose CL 6B and CM-Sepharose CL 6B. The enzyme showed a maximum catalytic value in a strongly acidic range (pH 2.0–2.4) with pH opt 2.1 and t opt 66 °C. The acid phosphatase showed a wide substrate specificity and a high affinity for sodium phytate, 2.5 × higher than with 4-nitrophenyl phosphate. This property of the acid phosphatase demonstrated that it is a potent 3-phytase at pH 2.1 and is of great sig- nificance for a practical application of the dephosphorylating complex – its addition to the diets of mono- gastric animals in view of the low pH values in the digestive tract. Acid phosphatases (EC 3.1.3.2) catalyze the hydrolysis of various phosphorylated substrates and are common in nature. These enzymes normally accompany the 3-phytase of mold origin (Żyła 1990; Shi- mizu 1993; Ullah and Phillippy 1994; Gargova et al. 1997; DvoĜáková 1998; VoĜíšek and Kalachová 2003). They can be used to increase the amount of free phosphorus in cereal and legume forage, as well as in pro- tein soybean isolates. In combination with 3-phytase (EC 3.1.3.8) their action is much more efficient than with a separate application (Żyła 1993; Wyss et al. 1998; Näsi et al. 1999). Depending on their pH optimum these enzymes are divided into 3 groups: (a) strongly acid with pH optimum 2.2–2.5, (b) moderately acid with pH optimum 5.0–5.5, and (c) weakly acid with pH optimum ≈6.0 (Ullah et al. 1994). We examined the strain A. niger 307, selected for the biosynthesis of 3-phytase, which we have reported to be accompanied by acid phosphatase (Gargova et al. 1997). This 3-phytase was obtained in a homogeneous form, had 2 pH optima (at 2.6 and 5.0), temperature optimum at 55–58 °C, and a narrow substrate specificity with a maximum enzyme activity with dodecasodium phytate, while with regard to other phosphorylated substrates its activity was only 2–3 % of the maximum (Sariyska et al. 2005). Here we present the results of the purification and report on the properties of acid phosphatase isolated from the culture of a natural strain A. niger 307. MATERIALS AND METHODS Medium and cultivation. Acid phosphatase was produced in a culture medium containing (in g/L): corn starch 50, glucose 50, NaNO 3 8.6, MgSO 4 ·7H 2 O 0.5, KCl 0.5, FeSO 4 ·7H 2 O 0.1, K 2 HPO 4 0.1 g P; pH 5. Erlenmeyer flasks (300 mL), with 30 mL medium were inoculated with 50-d conidia (final concentration 20–30/nL, i.e. 2–3 × 10 7 /mL) and shaken (30 °C, 3.7 Hz, 7 d; Gargova and Sariyska 2003). Steps of purification. The filtrate of the culture medium was concentrated by using a PS 50 mem- brane (Membrane Technologies Ltd., Bulgaria) at room temperature and pressure of 0.3 MPa (achieved with nitrogen). Portions of the concentrate containing 8 mg protein per mL were run through 12/600 Sephadex G-100 column (Pharmacia), equilibrated with 200 mmol/L sodium acetate buffer (pH 5.0). The active fract- ions were eluted with the same buffer (0.15 mL/min), pooled and applied on a 14/180 DEAE-Sepharose CL 6B (Fluka) column, equilibrated with 50 mmol/L Tris-HCl buffer (pH 8.0). The active fractions were pooled and chromatographed on a 14/180 CM-Sepharose CL 6B (Fluka) column, equilibrated with 200 mmol/L glycine (Merck) buffer (pH 2.6). From both ion exchange columns the proteins were eluted with a stepwise gradient of pH.