Improving the thermal stability of lactate oxidase by directed evolution.
Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 degrees C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution.
Minagawa
H
H
Fundamental and Environmental Research Laboratories, NEC Corp., Miyukigaoka 34, Tsukuba 305-8501, Japan. h-minagawa@ab.jp.nec.com
Yoshida
Y
Y
Kenmochi
N
N
Furuichi
M
M
Shimada
J
J
Kaneko
H
H
D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013289:Streptococcaceae / Q000201:enzymology*; D013696:Temperature
D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase