The Artificial Evolution of an Enzyme by Random Mutagenesis: The Development of Formaldehyde Dehydrogenase

Abstract

A unique variant of glutathione independent formaldehyde dehydrogenase of Pseudomonas putida was obtained by random mutagenesis using the PCR-reaction. This YM042 mutant, S318G, was a cold-adapted formaldehyde dehyrogenase. The activity at 29 °C of the variant was 1.7-fold higher than that of the wild type. The K_m values of the mutant at 37 °C were 0.40 mM for NAD⁺ and 2.5 mM for formaldehyde, while those of the wild-type were 0.18 mM for NAD⁺ and 2.1 mM for formaldehyde. The catalytic efficiency for formaldehyde was about 1.5-fold greater in the mutant than in the wild-type enzyme. The optimum pHs and temperatures of the mutant and the wild-type enzyme were 7.5, and 8.0 and 37 °C, and 47 °C, respectively. The thermal stability of the mutant was lower than that of the wild type.