Elsevier

Journal of Membrane Science

Volume 268, Issue 2, 15 January 2006, Pages 198-207
Journal of Membrane Science

Lipase-immobilized biocatalytic membranes for enzymatic esterification: Comparison of various approaches to membrane preparation

https://doi.org/10.1016/j.memsci.2005.06.039Get rights and content

Abstract

In this paper, lipase-immobilized membranes were prepared by both non-covalent and covalent immobilization methods using (i) lipase adsorption on membranes, (ii) inclusion of enzyme in membrane structure by filtration and (iii) covalent attachment of lipase to membrane. The catalytic properties of these membranes have been studied in reaction of butyloleate synthesis through esterification of oleic acid with n-butanol in isooctane. Ultrafiltration membranes made of regenerated cellulose (C030F) and polyethersulphone (PM30) were used for lipase immobilization. It was found that the lipase inclusion in the wide porous supporting layer of membrane was the most efficient method in preparing highly effective biocatalytic membranes. The degree of oleic acid conversion using these membranes was about 70–72% with a reaction time of 8 h. It was shown that the distribution profile of the lipase in the membrane was important for the effective enzyme utilization.

The profile imaging atomic force microscopy (AFM) technique was used to visualize surfaces of lipase-immobilized biocatalytic membranes. AFM has also been used to directly quantify interactions between lipase-coated tip and membrane surfaces. It was concluded that the direct measurements of the interaction force between the enzyme-coated tip and the membrane surface would be a useful and practical approach for the choice of membranes as porous polymeric support for lipase immobilization through adsorption.

Introduction

Currently, lipases, also known as triacylglycerol ester hydrolases EC 3.1.1.3, have generated interest in fundamental and applied research [1]. Lipases catalyze a number of different reactions, although they were designed by nature to cleave ester bonds of triacylglycerols with the subsequent release of free fatty acids, diacylglycerols, monoacylglycerols and glycerol. Lipases are also able to catalyze reverse reactions under micro aqueous conditions, viz. the formation of ester bonds between alcohol and carboxylic acid moieties (ester synthesis). Although ester synthesis can be done chemically with acid or base catalysis, the use of enzyme technology offers the advantages of mild conditions, high specificity including stereospecificity and reduced side reactions [1].

Generally, many lipase-catalyzed reactions studied were carried out in emulsion systems [2]. Currently, attempts are being made to avoid the use of emulsion systems because of difficulties not only in controlling the reaction but also in the re-use and stability of lipases [3]. Consequently, numerous efforts have been focused on the preparation of lipases in immobilized forms involving a variety of both immobilization methods and support materials [2], [4], [5], [6], [7].

Immobilized lipases display a number of advantages over the use of soluble enzymes, e.g. the possibility of recovery and re-use, simplicity in operation, improved stability and activity [1], [2], [8], [9]. In the last decade, lipase immobilization on the surface or within semi-permeable membranes has gained a growing interest to produce immobilized forms enabling controllable transport of reaction substrates and products through catalytic support [10]. Enzyme-immobilized membrane reactors offer advantages over conventional enzyme reactors for the membrane ability to operate simultaneously as a catalytic support and selective barrier combining a chemical reaction with a selective mass transfer through the membrane [11], [12]. The attractive features of enzyme-immobilized membrane reactors are: easy control, straightforward scaling-up, high enzyme loading, prolonged enzyme activity, high flow rates and reduced costs [13].

Since lipases can be used in the wide variety of reaction systems, the preparation of immobilized lipase derivatives has to be made according to each synthetic process of interest. In this context, the combined evaluation of the complex mechanism of the biocatalytic membrane action as well as the special requirements for the membrane reactor performance should be taken into account. Thus, availability of immobilization procedure suitable for each different lipase and specific biotransformation process is of great importance in developing advanced membrane biocatalysts for organic synthesis including the esterification process i.e. immobilization of lipases on or within membranes is far from an already solved problem.

In this paper the catalytic behaviour of lipase-immobilized membranes prepared by both non-covalent and covalent immobilization methods using (i) lipase adsorption on membranes, (ii) loading of membranes with enzyme by filtration of lipase solution through active or support membrane layers and (iii) covalent attachment of lipase to activated membrane, have been studied in the reaction of butyloleate synthesis through esterification of oleic acid with n-butanol.

Section snippets

Materials

Candida rugosa lipase type VII with a ratio of 1:5.88 g proteins/g solids (raw lipase, 700–1500 units/mg solid, molecular weight 57–60 kDa), oleic acid, n-butanol, glutaric dialdehyde, isooctane, hexamethylenediamine (HMD) phosphate buffer (pH 7.0) were purchased from Sigma-Aldrich (Dorset, UK). All the chemicals were analytical grade and used without further purification. Molecular sieves (4–8 mesh) were supplied by Fisher Scientific (Loughborough, UK).

Two types of ultrafiltration membranes used: (1)

Biocatalytic membranes with non-covalent immobilized lipase

Immobilization through the adsorption is one of the most widely used methods of enzyme immobilization [2]. Physical adsorption of lipases on the membrane surface provides the simplest approach for the preparation of membrane-immobilized lipases [20]. In this way, lipases become adsorbed on the membrane surface through a combination of van der Waals, hydrophobic, electrostatic forces, hydrogen bonds and aromatic π–π binding. Immobilization of enzymes by physical adsorption has the advantages of

Conclusions

This paper has shown that effective biocatalytic membranes for synthesis of butyloleate through esterification of oleic acid with n-butanol in isooctane can be prepared using both non-covalent and covalent methods for lipase immobilization on/in cellulose and polyethersulphone ultrafiltration membranes. It was found that lipase inclusion in the wide porous support membrane layer provides highly active membranes able to convert 70–72% of oleic acid in 8 h of reaction time. On the contrary, the

Acknowledgement

We thank the Royal Society (UK) for the financial support of this work (GR 15335).

References (27)

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    Enzyme immobilization is considered to allow frequent use of the enzyme [12]. Moreover, immobilization can also improve the stability, activity, and reusability of enzymes [13]. The appropriate supports in immobilization process, being low cost and providing adequate large surface area, play a key role for the immobilization of lipase [14].

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