Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes

Abstract

The fungus Candida rugosa produces multiple lipase isoenzymes (CRLs) with distinct differences in substrate specificity, in particular with regard to selectivity toward the fatty acyl chain length. Moreover, isoform CRL3 displays high activity towards cholesterol esters. Lipase isoenzymes share over 80% sequence identity but diverge in the sequence of the lid, a mobile loop that modulates access to the active site. In the active enzyme conformation, the open lid participates in the substrate-binding site and contributes to substrate recognition. To address the role of the lid in CRL activity and specificity, we substituted the lid sequences from isoenzymes CRL3 and CRL4 in recombinant rCRL1, thus obtaining enzymes differing only in this stretch of residues. Swapping the CRL3 lid was sufficient to confer to CRL1 cholesterol esterase activity. On the other hand, a specific shift in the chain-length specificity was not observed. Chimeric proteins displayed different sensitivity to detergents in the reaction medium.

A parametric study on biphasic medium conditions for the enantioselective production of naproxen by Candida rugosa lipase

“…On the other hand, the conversion and enantiomeric ratio obtained in the presence of toluene, which has the lowest log P among the organic solvents used, was higher than it was in hexane. Organic solvents facilitate the substrate to access the catalytic center of CRL by opening the polypeptide lid that covers the active site (21,22). However, the results of the present study indicate that the hydrophobicity of organic solvents is not sufficient alone to explain the enantioselective feature of CRL.…”

Kinetic study on esterification of palmitic acid catalyzed by glycine-based crosslinked protein coated microcrystalline lipase

“…This could be due to the fact that these enzymes possess a flapping lid structure at the active site that leads to decreasing enzyme accessibility to methanol binding. This structure is not found in C. antarctica lipase B (Novozyme 435) and C. rugosa [14,24].…”

Marine invertebrate lipases: Comparative and functional genomic analysis

“…Two important structural features that characterize lipases are the β9 loop (Winkler et al, 1990) and α-helix fragment or lid, a mobile amphipathic structure that covers the active catalytic site of most lipases whose length and complexity depend on the enzyme and is involved in modulating activity and selectivity of lipases (Dugi et al, 1995;Holmquist et al, 1995;Martinelle et al, 1996;Brocca et al, 2003;Secundo et al, 2004;Aoki et al, 2007). This is supported by several studies showing that exchanges of lid between two members of the lipase gene family were made and produce chimeras with altered substrate specificity (Faustinella et al, 1992;Dugi et al, 1995).…”