The lipase‐catalyzed hydrolysis of lutein diesters in non‐aqueous media is favored at extremely low water activities

Abstract

The enzymatic hydrolysis of a mixture of lutein diesters from Marigold flower (Tagetes erecta) was performed both in organic solvents and supercritical CO(2) (SC-CO(2)) using two commercial lipases: lipase B from Candida antarctica (Novozym 435) and the lipase from Mucor miehei (Lipozyme RM IM). Both lipases showed an unexpected dependence of initial reaction rate with the initial water activity (a(wi)) in hexane, with the highest rates of hydrolysis taking place at the lowest a(wi) of the biocatalyst particles. The same result was observed using isooctane, toluene, or SC-CO(2). It is proposed that an increase in a(wi) generates a hydrophilic microenvironment that prevents efficient partitioning of the highly hydrophobic lutein diesters to the enzyme. The critical role of water in this system has not been reported for other hydrolytic reactions in low water media. Calculations of water available for hydrolysis from isotherm analysis, Karl-Fischer titration, and substrate conversion at a(wi) = 0.13, indicate that the extent of reaction is not limited by the amount of available water. Accordingly, the enzyme that holds the largest amount of water after prehydration at the same a(wi) (0.13) will yield the greatest substrate conversion and concentration of the free lutein product. The highest conversion occurred in SC-CO(2), which opens up new opportunities to develop a combined extraction-reaction process for the environmentally benign synthesis of lutein, an important nutraceutical compound.

A Study on the Different Methods of Preparation of Lutein from Supercritical Fluid Processed Lutein Esters

“…It was found that the highest yield was achieved at an initial water activity of 0.33 in conventional medium, whereas in the case of hexane medium it was 0.22. The increased rate of hydrolysis of a highly hydrophobic substrate like lutein esters at low water activities were consistent with the results of the study which established the fact that in non-aqueous media the hydrolysis of lutein esters is favored at lower water activities [11]. Therefore, high levels of biocatalyst hydration result in a hydrophilic microenvironment around the biocatalyst particles that suppresses the hydrolysis reaction.…”

“…The traditional process of lutein production consists of solvent extraction of lutein esters and saponification which gives free lutein [11]. Khachik F [12] showed a method of isolating, purifying and recrystallizing lutein from saponified marigold oleoresin.…”

Effect of Water Clustering on the Activity of Candida antarctica Lipase B in Organic Medium

“…The water molecules can form large clusters and bind to certain preferred sites or regions on the enzyme surface [19]. Alternatively, the water molecules can form a monolayer surrounding the biocatalyst particles that can prevent substrate molecules from coming into close contact with the enzyme, resulting in low enzymatic activity [9]. To understand the present scenario at a molecular level, we carried out MD simulations, and the results are described in the section below.…”

“…The optimal water activity for esterification reactions of lipases from Candida rugosa and Candida antarctica in hexane medium (0.53 and 0.75; respectively) differs due to the structural differences between these two enzymes [8]. Hydrolysis of lutein diesters catalyzed by lipase B from Candida antarctica (CALB) and lipase from Mucor miehei is favored at low water activity (<0.2) due to the accumulation of water molecules at the enzyme surface which is independent of the nature of solvent considered in the study(isooctane, toluene, supercritical CO 2 ) [9]. The role of water molecules becomes crucial for the hydrolysis at high water activity, where water molecules adsorbed to the protein surface create hydrophilic microenvironments, which prevent the diffusion of substrate (i.e., ester) molecules to the catalyst resulting in a decrease in enzyme activity [9].…”

“…Hydrolysis of lutein diesters catalyzed by lipase B from Candida antarctica (CALB) and lipase from Mucor miehei is favored at low water activity (<0.2) due to the accumulation of water molecules at the enzyme surface which is independent of the nature of solvent considered in the study(isooctane, toluene, supercritical CO 2 ) [9]. The role of water molecules becomes crucial for the hydrolysis at high water activity, where water molecules adsorbed to the protein surface create hydrophilic microenvironments, which prevent the diffusion of substrate (i.e., ester) molecules to the catalyst resulting in a decrease in enzyme activity [9]. For example, Watanabe and co-workers showed that the equilibrium conversion for the CALB-catalyzed condensation of mannose and lauric acid in four different organic solvents (acetonitrile, acetone, 2-methyl-2-propanol, 2-methyl-2butanol) is higher at lower initial water content [10].…”