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 β-structures and 9%-spirals. Under optimal conditions in the medium of 10 mM phosphate buffered saline (pH 7.0) at 30°C, catalases from P. piceum F-648 were characterized by the following parameters: K _M, 158.8 mM; catalytic constant, 2.83 × 10⁶ s^?1; enzyme inactivation rate constant in H₂O₂ decomposition, 3.5 × 10^?2 s^?1; and constant of the interaction between catalase complex I and second molecule of H₂O₂, 1.8 × 10⁷M^?1 s^?1.