Modification of the enantioselectivity of two homologous thermophilic carboxylesterases from Alicyclobacillus acidocaldarius and Archaeoglobus fulgidus by random mutagenesis and screening.
The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively. The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop. In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction. The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations. Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.
Manco
Giuseppe
G
Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 10, 80125, Napoli, Italy. manco@dafne.ibpe.na.cnr.it
Carrea
Giacomo
G
Giosuè
Elena
E
Ottolina
Gianluca
G
Adamo
Giovanna
G
Rossi
Mosè
M
D019611:Archaeoglobus fulgidus / Q000201:enzymology*; D001407:Bacillus / Q000201:enzymology*; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D008958:Models, Molecular; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D013237:Stereoisomerism; D013379:Substrate Specificity
D017931:DNA Primers; D002265:Carboxylic Ester Hydrolases