Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99–92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4–C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12–C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.     

Enzymatic modification of triolein: Incorporation of caproic and butyric acids to produce reduced-calorie structured lipids

Lipase-catalyzed acidolysis of triolein with caproic and butyric acids was performed to produce reduced-calorie structured lipids (SL). The SL were obtained by incubating a 1:4:4 mole ratio of triolein, caproic acid, and butyric acid, respectively, with 10% of lipase (w/w of total substrates) in 1.5 mL hexane at 55°C for 24 h. Of nine commercially avaialble lipases screened, IM60, which contains the lipase from Rhizomucor miehei, was the most effective and produced 13 mol% unreacted triolein, 49% disubstituted, and 38% monosubstituted triacylglycerols that contained short-chain fatty acids. The products were analyzed by reverse-phase high performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters studied included time course, temperature, enzyme load, and substrate mole ratio. The yields obtained demonstrate that a structured lipid with long-chain and short-chain fatty acids can be synthesized by using IM60 lipase in organic medium.