Improvement of Yarrowia lipolytica Lipase Enantioselectivity by Using Mutagenesis Targeted to the Substrate Binding Site

Abstract

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.

Rationally engineered double substituted variants of Yarrowia lipolytica lipase with enhanced activity coupled with highly inverted enantioselectivity towards 2‐bromo phenyl acetic acid esters

“…1; Bordes et al, 2009). The construction of a variant (V232S) with an improved S-selectivity was the major result from this work.…”

“…; Bordes et al, 2009). Variant V232T was also found more active for the hydrolysis of the Renantiomer (2.7-fold increase in activity compared to the wild-type lipase).…”

“…Using the previously reported homology model of Lip2p from Y. lipolytica (Bordes et al, 2009), tetrahedral intermediate models were built for both (R, S)-2-bromophenylacetic acid octyl ester enantiomers. The substrates were placed in the active site so that they were covalently bound to catalytic Ser162 and fulfiled the hydrogen bonding interactions required for catalysis.…”

“…These two positions were systematically replaced by other 19 possible amino acids. The library of variants was then screened resulting in tremendous enhancements in enzyme enantioselectivity (up to 58-fold for the V232S variant) compared to the wild-type (Bordes et al, 2009;Cancino et al, 2008). In addition to the gain in enantioselectivity, a remarkable increase in velocity was also observed for some variants.…”

“…Nonetheless, this lipase showed a poor enantioselectivity (E-value ¼ 4) for the resolution of the 2-bromo-phenylacetic acid ethyl ester racemate, what has been considered insufficient to utilize the biocatalyst in the pharmaceutical industry. In order to improve the selectivity of Lip2p, a rational engineering strategy, consisting in applying site-directed mutagenesis to amino acid residues located at the substrate binding site, was adopted (Bordes et al, 2009). Given the absence of crystallographic data, a three-dimensional model of the Lip2p lipase was predicted by homology modeling using the X-ray structures of similar lipases from Rhizomucor miehei and Thermomyces lanuginosa as templates.…”