Influence of the operating conditions on lipase-catalysed synthesis of ricinoleic acid estolides in solvent-free systems

Abstract

In this work, the synthesis of ricinoleic acid estolides, also named polyricinoleic acid (PR), in two different solvent-free reaction systems, catalysed by immobilised Candida rugosa lipase is described. First, the esterification was performed in an open air jacketed batch reactor and the influence of amount of enzyme, temperature and initial water content was tested. The best results were obtained when 5 g of soaked immobilised derivative was used as biocatalyst, and the reaction was carried out at 40 °C. It was observed that environmental relative humidity plays an important role in the enzymatic synthesis of ricinoleic acid estolides and, given that this parameter takes on a wide range of values depending on the season, it is considered the main cause of the poor reproducibility achieved in the open air reactor. To solve this problem, the ricinoleic acid estolides were synthesised under controlled atmosphere in a vacuum reactor with dry air intake. The optimum drying time of 7 h was selected. In these conditions, PR with an acid value of 57.5 mg KOH/g was synthesised in 48 h of reaction and the results were entirely reproducible.

Introduction

The use of enzymes in non-aqueous environments has increased in the past two decades as an attempt to broaden the applications of biocatalysis [1]. In the context of “green chemistry”, the technological utility of enzymes can be greatly enhanced by using them in non-conventional media. Currently, there are five main “green” solvent systems: supercritical fluids, fluorinated solvents, ionic liquids, water, and solvent-free systems (SFS) [2]. The major advantages of the latter are that the absence of solvents facilitates downstream processing, offering significant cost saving and minimizing the environmental impact [3]. The possibility of using enzymes in media containing only the reactants and the enzyme preparation without the addition of other compounds has only been considered for processes using lipase as catalyst and triglycerides as substrate [4].

Estolides are oligomeric molecules that are mostly composed of hydroxy fatty acids. The polymerisation takes place by covalently ester bonds between hydroxyl moiety of one hydroxy acid and the carboxyl moiety of another hydroxy acid molecule (Fig. 1). The estolide made from ricinoleic acid (18:1⁹—OH¹²), is a useful substance with many applications in industry. For example, it is used as a viscosity controller for chocolate and an emulsifier in margarine, as cutting oil base in metal processing and as pigment dispersant in paint, ink and cosmetics [5], [6]. The high temperature of the conventional chemical route of polyricinoleic acid synthesis, leads to a lower selectivity, undesired by-products, colouring and malodour of the product. Because of these limitations a new process for the enzyme-based production of ricinoleic acid estolides was developed using Candida rugosa lipase [5], [7].

The optimisation of some reaction conditions is especially important in an experimental system like the one described. It is known that temperature is a crucial parameter in every enzyme catalysed reactor but in our case, due to the special characteristics of the reaction medium (solvent-free), temperature greatly influences viscosity, mass transport phenomena and, as a consequence, the esterification rate [9]. While high temperature favours the medium fluidity, enzyme has to be prevented from thermal deactivation [8], [9].

Another decisive parameter in these processes is the water content. Water plays multiple roles in lipase-catalysed esterifications performed in non-conventional media. It is widely known that water is absolutely necessary for the catalytic function of enzymes because it participates, directly or indirectly, in all non-covalent interactions that maintain the conformation of the catalytic site of enzymes [10], [11], [12]. On the other hand, in esterification/hydrolysis reactions it is well-known that the water content affects the equilibrium conversion of the reactions. Particularly, in the case of estolides production, the water formed by the reaction must be removed from the reaction mixture if polyricinoleic acid with a high degree of condensation is to be obtained [5].

Usually, enzyme immobilisation improves their operational stability, while preventing contamination of the substrate being converted. In addition, immobilised enzymes can be easily separated from the reaction media for reuse or for use in continuous reactors. For these reasons, efforts have been devoted to obtaining an immobilised derivative with a high immobilised protein percentage and enzymatic activity for the present application [13].

The present study describes a detailed investigation on the synthesis of estolides of ricinoleic acid catalysed by immobilised Candida rugosa lipase in a medium containing only the reactant and the enzyme derivative without the addition of solvents not involved in the reaction. We examine the influence of several key reactions conditions, such as temperature, initial water percentage, and enzyme amount, on the enzymatic reaction rate and the equilibrium conversion. In addition several reactor configurations have been tested.