Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity.
A lipase from Pseudomonas aeruginosa was subjected to directed molecular evolution for increased amide-hydrolyzing (amidase) activity. A single round of random mutagenesis followed by screening for hydrolytic activity for oleoyl 2-naphthylamide as compared with that for oleoyl 2-naphthyl ester identified five mutants with 1.7-2.0-fold increased relative amidase activities. Three mutational sites (F207S, A213D and F265L) were found to affect the amidase/esterase activity ratios. The combination of these mutations further improved the amidase activity. Active-site titration using a fluorescent phosphonic acid ester allowed the molecular activities for the amide and the ester to be determined for each mutant without purification of the lipase. A double mutant F207S/A213D gave the highest molecular activity of 1.1 min(-1) for the amide, corresponding to a 2-fold increase compared with that of the wild-type lipase. A structural model of the lipase indicated that the mutations occurred at the sites near the surface and remote from the catalytic triad, but close to the calcium binding site. This study is a first step towards understanding why lipases do not hydrolyze amides despite the similarities to serine proteases in the active site structure and the reaction mechanism and towards the preparation of a general acyl transfer catalyst for the biotransformation of amides.
Fujii
Ryota
R
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan and Tsuruga Institute of Biotechnology, Toyobo Co., Ltd, 10-24 Toyo-Cho, Tsuruga, Fukui 914-0047, Japan.
Nakagawa
Yuichi
Y
Hiratake
Jun
J
Sogabe
Atsushi
A
Sakata
Kanzo
K
D000577:Amides / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D019020:Directed Molecular Evolution; D006868:Hydrolysis; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D000577:Amides; D017931:DNA Primers; D008049:Lipase