Evolving a lipase for hydrolysis of natural triglycerides along with enhanced tolerance towards a protease and surfactants

Abstract

In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.

Drosophila Lipase 3 Mediates the Metabolic Response to Starvation and Aging

“…Lipase 3 Substrate Specificity Differs From Human LIPA Human lysosomal acid lipase hydrolyzes TAG and CE (Witeck et al, 2021). Bacterially expressed Lip3 has been shown to have weak TAG lipase activity on short-chain fatty acid glyceryl trioctanoate but no detectable activity on glyceryl trioleate, a more physiological substrate (Alfaro-Chávez et al, 2019). To further characterize the substrate specificity of Drosophila Lip3, we expressed LIPA, Lip3 and GFP as control in mammalian tissue culture cells.…”