Isolation, Purification, and Characterization of Catalase from the Methylotrophic Yeast Pichia pastoris

M. V. Potapovich¹, A. N. Eryomin¹, I. M. Artzukevich², I. P. Chernikevich², and D. I. Metelitza^1*

¹Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, Minsk, 220141 Belarus; fax: (375)-(172) 63-7274; E-mail: enzyme@ns.iboch.ac.by

²Grodno State Medical University, ul. Gor'kogo 80, Grodno, 230015 Belarus; fax: (375)-(152) 33-5341; E-mail: artsuk@grsmi.unibel.by

^* To whom correspondence should be addressed.

Received October 26, 2000

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Catalase (CAT_pp) 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 CAT_pp solutions and of its membrane-concentrated form (CAT_pp-conc) were studied. Rates of H₂O₂ decomposition and kinetic characteristics K_m and k_cat of CAT_pp and CAT_pp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30°C, as well as the effective constant k_in 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 CAT_pp in solutions at 45°C was characterized by the effective rate constant k_in*, and the low-frequency (27 kHz) ultrasonic inactivation of CAT_pp at 20°C was characterized by the first-order rate constant k_in(US). All spectral and kinetic characteristics of CAT_pp and CAT_pp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CAT_cb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CAT_pp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of k_cat/K_m, thermal stability comparable with the thermal stability of CAT in terms of k_in*, the minimal k_in, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm².

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KEY WORDS: catalase from the yeast Pichia pastoris, bovine liver catalase, absorption spectra, CD spectra, kinetics, operational stability, inactivation by cyclic process, thermal inactivation, ultrasonic inactivation

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