Different methods of preparing lipases for use in organic media are critically reviewed. Solid lipase preparations can be made by typical immobilisation methods such as adsorption, entrapment, covalent coupling or cross-linking. Immobilisation is especially attractive for lipases because, in addition to the normal benefits of enzyme immobilisation, it can also lead to a considerable increase in catalytic activity, probably caused by conformational changes in the lipase molecules. Activation can be achieved, for example, using hydrophobic support materials or surfactants during the immobilisation procedure. Surfactants can also be used to solubilise lipases in organic media via the formation of hydrophobic ion pairs, surfactant-coated lipase or reversed micelles. Lipase preparation methods are discussed with regard to potential lipase inactivation and activation effects, mass transfer limitations, lipase stability and other features important for applications. The practical applications of lipases in organic media reviewed include ester synthesis, modification of triacylglycerols and phospholipids, fatty acid enrichment, enantiomer resolution, biodiesel production and acylation of carbohydrates and bioactive compounds.

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Immobilisation and application of lipases in organic media.

Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol. Besides this, they are also efficient in various reactions such as esterification, transesterification and aminolysis in organic solvents. Therefore, those enzymes are nowadays extensively studied for their potential industrial applications. Examples in the literature are numerous concerning their use in different fields such as resolution of racemic mixtures, synthesis of new surfactants and pharmaceuticals, oils and fats bioconversion and detergency applications. However, the drawbacks of the extensive use of lipases (and biocatalysts in general) compared to classical chemical catalysts can be found in the relatively low stability of enzyme in their native state as well as their prohibitive cost. Consequently, there is a great interest in methods trying to develop competitive biocatalysts for industrial applications by improvement of their catalytic properties such as activity, stability (pH or temperature range) or recycling capacity. Such improvement can be carried out by chemical, physical or genetical modifications of the native enzyme. The present review will survey the different procedures that have been developed to enhance the properties of lipases. It will first focus on the physical modifications of the biocatalysts by adsorption on a carrier material, entrapment or microencapsulation. Chemical modifications and methods such as modification of amino acids residues, covalent coupling to a water-insoluble material, or formation of cross-linked lipase matrix, will also be reviewed. Finally, new and promising methods of lipases modifications by genetic engineering will be discussed.

Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches

The continuous flow synthesis of active pharmaceutical ingredients, value-added chemicals, and materials has grown tremendously over the past ten years. This revolution in chemical manufacturing has resulted from innovations in both new methodology and technology. This field, however, has been predominantly focused on synthetic organic chemistry, and the use of biocatalysts in continuous flow systems is only now becoming popular. Although immobilized enzymes and whole cells in batch systems are common, their continuous flow counterparts have grown rapidly over the past two years. With continuous flow systems offering improved mixing, mass transfer, thermal control, pressurized processing, decreased variation, automation, process analytical technology, and in-line purification, the combination of biocatalysis and flow chemistry opens powerful new process windows. This Review explores continuous flow biocatalysts with emphasis on new technology, enzymes, whole cells, co-factor recycling, and immobilization methods for the synthesis of pharmaceuticals, value-added chemicals, and materials.

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Continuous flow biocatalysis.

A support for enzyme immobilization was prepared by functionalization of mesoporous silica with octyltriethoxysilane. The features of the surface enables the adsorption of lipase from Candida antarctica B via strong hydrophobic interactions, enhancing the stability of the adsorbed enzyme molecules. Derivatives with a high enzyme loading (200 mg protein/g of silica) can be obtained due to the high porosity and surface properties of the support while the immobilization occurs in a monolayer fashion. The lack of inactive enzyme aggregates, together with the high enzyme loading, are responsible for the high catalytic activity achieved by these species. Derivatives were prepared with different lipase loading, and the activities were tested and compared to the commercial derivative Novozym 435. The stability of the catalyst and hence its industrial applicability were tested by performing subsequent reaction cycles of acylation of ethanolamine with lauric acid in acetonitrile. Conversion was quantitative even after 15 reaction cycles.

Michael Bauer

Papers

Adsorption of lipase on polypropylene powder