Isolation, purification, and characterization of catalase from the methylotrophic yeast Pichia pastoris.
Catalase (CATpp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CATpp solutions and of its membrane-concentrated form (CATpp-conc) were studied. Rates of H2O2 decomposition and kinetic characteristics Km and kcat of CATpp and CATpp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30 degrees C, as well as the effective constant kin of the enzyme inactivation rate during the catalysis and the constant k2 of the interaction rate of the Complex I catalases with H2O2. Thermal inactivation of CATpp in solutions at 45 degrees C was characterized by the effective rate constant kin*, and the low-frequency (27 kHz) ultrasonic inactivation of CATpp at 20 degrees C was characterized by the first-order rate constant kin(US). All spectral and kinetic characteristics of CATpp and CATpp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CATcb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CATpp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of kcat/Km, thermal stability comparable with the thermal stability of CAT in terms of kin*, the minimal kin, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm2.
Potapovich
M V
MV
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
Eryomin
A N
AN
Artzukevich
I M
IM
Chernikevich
I P
IP
Metelitza
D I
DI
D000818:Animals; D002021:Buffers; D002374:Catalase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002845:Chromatography; D002942:Circular Dichroism; D004795:Enzyme Stability; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D020675:Peroxisomes / Q000737:chemistry; D010843:Pichia / Q000201:enzymology*; D013696:Temperature
D002021:Buffers; D006861:Hydrogen Peroxide; D002374:Catalase