Purification and biochemical characterization of thermostable alkaline phosphatases produced by Rhizopus microsporus var. rhizopodiformis

• DOI: 10.1007/s12223-008-0080-4
• Publication Date: 2009-04-17T13:59:35Z
• Authors: A. Barbosa; L. H. S. GuimarÃes; H. F. Terenzi; J. A. Jorge; F. A. Leone; M. L. T. M. Polizeli
• Keywords: 0301 basic medicine; 0303 health sciences; Hot Temperature; Hydrogen-Ion Concentration; Alkaline Phosphatase; Chromatography, Agarose; Chromatography, DEAE-Cellulose; Fungal Proteins; Molecular Weight; 03 medical and health sciences; Cations; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Rhizopus

The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K (m) 0.28 and 0.22 mmol/L, with upsilon (lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.