Improvement of Lipase Catalytic Properties by Immobilization in Hybrid Matrices

Abstract

Lipases are enzymes particularly amenable for immobilization by entrapment methods, as they can work equally well in aqueous or non-conventional media and long-time stability of enzyme activity and enantioselectivity is needed to elaborate more efficient bioprocesses. The improvement of Pseudomonas fluorescens (Amano AK) lipase characteristics was investigated by optimizing the immobilization procedure in hybrid organic-inorganic matrices using ionic liquids as additives. Ionic liquids containing a more hydrophobic alkyl group in the cationic moiety are beneficial for the activity of immobilized lipase. Silanes with alkyl- or aryl nonhydrolizable groups used as precursors in combination with tetramethoxysilane could generate composites with higher enantioselectivity compared to the native enzyme in acylation reactions of secondary alcohols. The optimal effect on both activity and enantioselectivity was achieved for the composite made from octyltrimethoxysilane and tetramethoxysilane at 1:1 molar ratio (60% increase of total activity following immobilization and enantiomeric ratio of 30). Ionic liquids also demonstrated valuable properties as reaction media for the studied reactions, comparable with the usual organic solvent, hexane. Optically active intermediates: alcohols, amines, and carboxylic acids are important building blocks for the synthesis of pharmaceutical and agrochemical products. Around 80% of the active compounds manufactured by pharmaceutical companies are chiral, and this percentage is estimated to increase in the next period (2). Using a pure enantiomer instead of racemic mixture allows lower dosage and improved efficacy of the product, but the availability of these compounds at large scale was limited. New technologies were needed and biocatalytic processes started to play an emerging role in this context. The excellent specificity and selectivity (including enantioselectivity) properties of enzymes have been employed to carry out processes of high complexity in less harmful experimental and environmental conditions (3). There are several tools, as protein engineering, available to improve the enzyme characteristics, but some drawbacks associated with use of soluble enzymes like high price, product contamination, difficulty of separation from the reaction mixture, and insufficient operational stability may reduce the industrial application possibilities. In these circumstances, is not astonishing to see a revival of immobilization techniques, as they could enhance the stability, activity, specificity or selectivity of enzymes, if properly