Oxidation of Methanol by the Yeast,Pichia pastoris. Purification and Properties of the Alcohol Oxidase

The enzymes of methanol oxidation were investigated in a new yeast strain, Pichia pastoris IFP 206, with high yield (0.42 g cell per g of methanol). The following enzymes were detected in cell free extracts of P. pastoris: alcohol oxidase, catalase, formaldehyde and formate dehydrogenases. The alcohol oxidase was purified from cell free extracts of P. pastoris containing high amount of the enzyme (33%) with a good yield (55%). The preparation was homogenous by immunochemical methods. The enzyme had a molecular weight of 675,000 and was composed of eight identical subunits of M.W. 80,000. Each subunit contained one FAD. The N-terminal sequence was found to be: Ala-Ile-Pro-Glu-Glu-Phe-Asp-Ile-Leu-Val-Leu-Gly-The protein had 65 free ?SH groups per molecule. The optimum temperature for the enzyme activity was 37°C and the activation energy was 11.1 kcal/mol. Optimum pH was 7.5 and the enzyme activity was unstable at acidic pH. The apparent Km for methanol were 1.4 and 3.1 mm at oxygen concentrations of 0.19 and 0.93 mm. Similarly, the apparent Kms for oxygen were 0.40 and 1.0 mm at methanol concentrations of 1, 10 and 100 mm. The enzyme oxidized primary alcohols with short carbon chains like ethanol and propanol. Inhibition of enzyme activity by hydrogen peroxide was a consequence of the oxidation of essential ?SH groups. The inhibition was reversed by reducing agents.