Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

Abstract

BackgroundEnzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions.ResultsImmobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts.On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 μmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone.ConclusionsActivity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.

Ultrasound-assisted biosynthesis of novel methotrexate-conjugates

“…After reaction, it was possible to observe the hydrolysis of the longer triacylglycerols and the formation of the MTXacylglycerol conjugates. Our data corroborate previous findings which highlight an high activity of this lipase for long triacylglycerols, such as triolein [24].…”

Immobilization of lipases on hydrophobic supports: immobilization mechanism, advantages, problems, and solutions

“…This makes the opening of the lipase by other external interfaces unnecessary, and it becomes a guarantee of the immobilization of the lipases in monomeric and open form. The exact structure and properties of the support may condition the final properties of the lipases (Cabrera et al, 2009;Fernandez-Lorente et al, 2008;Hernandez et al, 2011a;Tacias-Pascacio et al, 2016), and there are also some reports suggesting that the immobilization conditions may also alter the final enzyme catalytic features (Silveira et al, 2017).…”

“…(octadecyl methacrylate) (Silveira et al, 2017), magnetic copolymer styrene-divinilbezene (Silva et al, 2018), silica coated with octyl groups (Vescovi et al, 2017), polyhydroxybutyrate particles (Corradini et al, 2017), and Diatomite (Zhang et al, 2019). Table 1 shows also some of the uses of these biocatalysts.…”

Microbial lipases and their industrial applications: a comprehensive review

“…Several lipases catalyze a number of useful reactions such as esterification due to their activeness in organic solvents [ 47 , 53 ]. Lipases displayed pH dependent activities, generally at neutral pH 7.0 or up to pH 4.0 and 8.0 lipases are stable, Chromobacterium viscosum, A. niger and Rhizophus sp., produced extracellular lipases are active at acidic pH, and P. nitroaeducens produced alkaline lipase and active at pH 11.0 [ 54 ]. Under certain experimental conditions lipases have capability to reversing the reactions which leads to esterification and interesterification in the absence of water [ 55 , 56 ].…”