The artificial evolution of an enzyme by random mutagenesis: the development of formaldehyde dehydrogenase.
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 degrees C of the variant was 1.7-fold higher than that of the wild type. The K(m) values of the mutant at 37 degrees 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 degrees C, and 47 degrees C, respectively. The thermal stability of the mutant was lower than that of the wild type.
Fujii
Yasuyo
Y
Institute of Advanced Energy, Kyoto University, Uji 611-0011, Japan. yfujii@iae.kyoto-u.ac.jp
Yamasaki
Yoshiaki
Y
Matsumoto
Masahiro
M
Nishida
Hiroyuki
H
Hada
Megumi
M
Ohkubo
Katsutoshi
K
D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D005075:Biological Evolution; D005557:Formaldehyde / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics*; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D005557:Formaldehyde; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent