Immobilization of Lipase on Inert Carriers

The use of macroscopic inert carrier materials to immobilize lipase is important, as it allows easy catalyst handling and reuse after separation of the catalyst by sedimentation or filtration. A vast amount of literature has been published on the immobilization of lipases on solid surfaces or inert carrier materials. The methods described cover covalent coupling, precipitation, entrapment, and adsorption. Moreover, many different carrier materials have been used over the years such as hydrophilic powders (celite), ion exchange resins, and hydrophobic polymers, either in the form of particles or as membranes. Extensive overviews on the details of these techniques can be found elsewhere (8).

The most widely used technique is the adsorption of lipases onto porous carrier materials. Such materials provide a huge internal surface area to immobilize individual lipase molecules from solution with high efficiency. The carrier materials should fulfill a number of criteria to deliver a proper biocatalyst (51, 52). Obviously the material should allow efficient lipase immobilization and an appropriate pore size to allow diffusion of the lipase (> 50 nm) (52). Furthermore, particle size is important in order to minimize the risk of substrate mass transfer limitations (40). However, to be able to use the biocatalyst in a packed-bed reactor, a minimum particle size (> 500 ^m) should be accounted for to keep the pressure drop over the packed bed within reasonable limits.

Carrier materials such as ion exchange resins (e.g., Duolite ex Rohm-Haas, Germany (53)) and polypropylene particles (Accurel EP-100, ex Akzo-Nobel, Germany) (54, 55) are most widely used for lipase adsorption. Especially the latter material has proven to be a suitable carrier, as the immobilization of a wide variety of lipases has been shown to be very efficient and reproducible (54). Though easily immobilized on the carrier, it was reported that certain lipases gave activities much lower than could be expected based on their loading. This phenomenon is attributed to conformational changes in the structure of the lipase and hence a loss of lipase activity upon adsorption of lipase molecules onto the carrier surface. Precoating of the carrier with dummy, nonlipase protein before adsorption of the lipase has proven a reliable technique to overcome the reduction of the activity of the immobilized lipase (54, 56).

It should be noted that immobilization is mentioned as a technique to enhance the stability of lipases. Fundamental studies have revealed that indeed the flexibility of enzymes immobilized onto solid surfaces is considerably reduced (24). However, data actually comparing one and the same type of reaction with both the free and immobilized versions of one lipase are rather scarce (57). Often research measures the temperature optima of the free and immobilized enzymes in different reaction systems; for example, hydrolysis in an emulsion system for the free lipase, and an (inter)esterification in a solvent system for the immobilized lipase. However, considering effects of mass transfer, substrate solubility, and water activity as described above, comparison of results from different assays can be rather tricky, and hence the impact of immobilization itself should be evaluated with care.

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