Use of cadmium hydroxide gel for isolation of extracellular catalases from Penicillium piceum and characterization of purified enzymes

We optimized the conditions for isolation of extracellular catalases from Penicillium piceum F-648 and P. piceum A3 by means of volume chromatography with cadmium hydroxide gel. Our study showed that 55-57 mg wet gel are sufficient for the maximum sorption of catalase from 1 ml of culture fluid. This gel was formed in 1 ml 70 mM Cd(NO3)2 after addition of NaOH (Cd(NO3)2/NaOH molar ratio 1:2.2). The eluting solution contained 50 mM NaH2PO4 (pH 7.0), 5.0 mM dithiothreitol, and 0.3% sodium cholate and was potent in desorbing catalase from the gel. Subsequent ultrafiltration of the eluate on the membrane with a retention limit of 50 kDa allowed us to concentrate and purify the sample from low-molecular-weight protein impurities. NH4Cl (1.0 M) containing 0.3% sodium cholate was used to wash the sample from low-molecular-weight aromatic metabolites. Purified catalases included 33-34% antiparallel beta-structures and 9% alpha-spirals. Under optimal conditions in the medium of 10 mM phosphate buffered saline (pH 7.0) at 30 degrees C, catalases from P. piceum F-648 were characterized by the following parameters: K(M), 158.8 mM; catalytic constant, 2.83 x 10(6) s(-1); enzyme inactivation rate constant in H2O2 decomposition, 3.5 x 10(-2) s(-1); and constant of the interaction between catalase complex I and second molecule of H2O2, 1.8 x 10(7) M(-1) s(-1).