Sol-Gel Entrapment of Enzymes
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Sol-Gel Entrapment of Enzymes Laurie Bergogné, Souad Fennouh, Stéphanie Guyon, Cécile Roux, Jacques Livage Chimie de la Matière Condensée (UMR CNRS 7574) - Université Pierre et Marie Curie 75252 Paris, FRANCE ABSTRACT Two enzymes, lipase and β-galactosidase, have been encapsulated within sol-gel matrices. Enzymatic activity of encapsulated lipase for hydrolysis and trans-esterification reactions is maintained. Encapsulation yields depend not only on the sol-gel porous texture but also on the water amount added for the sol-gel synthesis and the hydratation history of the enzyme. When the water amount is low, the highly active enzyme conformation generated by the phase separation is frozen during gelation. Escherichia Coli have been also encapsulated. The cellular organization appears to be well preserved. Their β-galactosidase activity seems to be better in wet gels but decreases dramatically upon drying. INTRODUCTION For ten years now, the sol-gel process has been used to encapsulate biological species such as enzymes, antibodies or cells [1-3]. This process is mild enough to avoid enzyme denaturation during synthesis and thus to maintain the biological properties (enzymatic, immunological) of the encapsulated species. The porous sol-gel matrices may even offer protection against external denaturating factors (temperature, pH). Lipase encapsulation is perhaps the most beautiful example previously reported [4]. As these enzymes work at the water-lipid interface, the synthesis of a hydrophobic matrix from RSi(OMe)3 precursors allows to improve by a factor thousand the enzymatic activity in organic medium. To our knowledge, enzymatic properties of encapsulated lipase in aqueous medium have never reported before. This subject is of importance from an “economic” point of view since it is related to environmental challenges such as the treatment of used waters. In the first part of this work, we present the enzymatic properties of encapsulated lipase in both hydrophobic and hydrophilic media. Characteristic parameters (Vmax and KM) have been determined offering basis for comparison of the influence of local environment on lipase activity. Moreover, performances of these biogels either dried or in continuous use have been less studied and seem to be very low because of enzyme denaturation [5]. It could also result from limited diffusion through the denser silica matrices and from enzymes leaching. A promising alternative is to pre-immobilize the enzymes before their encapsulation within sol-gel matrices. In this frame, we present, in a second example, the results obtained for encapsulated bacteria. Enzymes (β-galactosidase) are first induced within the bacteria before encapsulation. In this case, bacteria are used as large (c.a. 1µm) enzyme bags thus limiting enzymes leaching. EXPERIMENTAL DETAILS All reactants were purchased from Fluka, including the purified Lipase from Candida cylindracea and the lipase substrate (triolein). Escherichia Coli K12 bacteria were kept on plates
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