Improvement of Yarrowia lipolytica lipase enantioselectivity by using mutagenesis targeted to the substrate binding site.
Lip2p lipase from Yarrowia lipolytica was shown to be an efficient catalyst for the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. Enantioselectivity of this lipase was improved by site-directed mutagenesis targeted to the substrate binding site. To guide mutagenesis experiments, the three-dimensional model of this lipase was built by homology modelling techniques by using the structures of lipases from Rhizomucor miehei and Thermomyces lanuginosa as templates. On the basis of this structural model, five amino acid residues (T88, V94, D97, V232, V285) that form the hydrophobic substrate binding site of the lipase were selected for site-directed mutagenesis. Position 232 was identified as crucial for the discrimination between enantiomers. Variant V232A displayed an enantioselectivity enhanced by one order of magnitude, whereas variant V232L exhibited a selectivity inversion. To further explore the diversity, position 232 was systematically replaced by the 19 possible amino acids. Screening of this library led to the identification of the V232S variant, which has a tremendously increased E value compared to the parental enzyme for the resolution of 2-bromo-phenylacetic acid ethyl ester (58-fold) and 2-bromo-o-tolylacetic acid ethyl ester (16-fold). In addition to the gain in enantioselectivity, a remarkable increase in velocity was observed (eightfold increase) for both substrates.
Bordes
F
F
Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, Toulouse, France.
Cambon
E
E
Dossat-Létisse
V
V
André
I
I
Croux
C
C
Nicaud
J M
JM
Marty
A
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D025062:Yarrowia / Q000201:enzymology*
D008049:Lipase