Altering the Activation Mechanism in Thermomyces lanuginosus Lipase

Abstract

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

Probing role of key residues in the divergent evolution of Yarrowia lipolytica lipase 2 and Aspergillus niger eruloyl esterase A

“…It showed that the mutant enzymes exhibited enhanced esterase characteristics. These results were similar to those reported by Skjold-Jørgensen et al (2014). Hermosa pointed out that the substrate specificity and lipase/esterase preference of AnFaeA were due to local or minor structural variations (Hermoso et al, 2004).…”

“…For WT, the activity increased more sharply than mutants. Once the substrates concentrations exceed CMC, interfacial behavior of the mutants is close to esterases rather than true lipases (Verma et al, 2008;Reis et al, 2009;Skjold-Jørgensen et al, 2014). All the results mentioned above indicate that the residues we've selected are directly related to interfacial activation.…”

“…For WT, the activity of hydrolyzing pNPB is low when the concentration is below CMC. When the substrates concentrations are lower than CMC, they are in soluble forms (Martinelle et al, 1995;Verma et al, 2008;Reis et al, 2009;Skjold-Jørgensen et al, 2014). This is a homogeneous system where there is no oil-water interface and most of the lipases are in their closed conformations, thus their activities are low.…”

Engineering and application of enzymes for lipid modification, an update

“…This behaviour of the lid is supported by molecular dynamic studies [54]. To understand this fact, Skjold-Jørgensen et al generated TLL variants with changed behaviour on lipidwater interfaces by altering the lid domain [55]. Owing to the structural similarity of TLL with a ferulic acid esterase from Aspergillus niger (ANFAE), this enzyme was used to generate TLL variants containing the lid region of ANFAE or hybrids of both of them.…”

Characterization of a feruloyl esterase B from Talaromyces cellulolyticus

“…The activity of FAE was evaluated as reported previously, 22,23) with MFA, pNF, and pNA. As concerns the pNF and pNA assays, each experiment was performed under various buffer conditions from pH 3.0 to 11.0.…”