Abstract

Alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae is a highly active enzyme but present the limitation of being poorly stable at operational conditions [1]. An interesting reaction catalyzed by ADH is the oxidation of aliphatic alcohols. In particular, long chain aliphatic alcohols are not soluble in aqueous solutions, thus, the addition of an organic co-solvent is needed. [2]. The use of organic reaction media represent then an additional drawback for the soluble enzyme. In this work, ADH immobilization was performed as a tool of stabilization in order to make a comparison between the reaction rates and the conversion reached at 1h for a list of 10 aliphatic alcohols, with a chain length that varies from 2 to 24 carbons. The stabilization of the tetrameric configuration of alcohol dehydrogenase from S. cerevisiae was achieved by the immobilization of the enzyme on a support of glioxil agarose and a post-immobilization crosslinking with polyethilenimine (PEI, MW=25.000 g mol-1). This immobilization occurs due to a covalent bond between the aldehyde groups of the support and the Lysine residues of the enzyme. Even if the covalent bond helps to improve the stabilization respect to the soluble enzyme, it was the additional step of crosslinking with PEI which yield to a 10-fold more stable biocatalyst with respect to the only-covalent-bonded biocatalyst. This catalyst was now suitable to be used for reaction tests. The reaction were performed with soluble and immobilized ADH at 30 ºC and pH 7. The diverse alcohols (0.2 mM) were oxidized to their respective carboxylic acid by means of the soluble and immobilized ADH (2 UI /mlreaction). Surprisingly, ADH showed to be catalytically active towards long chain (C22 and C24) aliphatic alcohols. In addition, when the soluble and immobilized catalyst are compared two different regions were observed. First, for short chains alcohols (< C6) the reaction rates decreases with the chain length. Second, for higher alcohol length (C7 to C24), the reaction rates increases again. The biocatalyst (ADH-Agarose-PEI) showed for all cases faster reaction rates (50 – 100%) than soluble ADH. The only exception was observed with ethanol, which values were identically. As far as the reaction conversion regards, the soluble ADH showed lower values (<30%) with respect to the biocatalyst (75%), probably due to the lower thermal stability (t1/2 at 30 ºC = 7 h). By conclusion, this work demonstrate that ADH from S. cerevisiae is active for a wide range of aliphatic alcohols (C2 to C24) and that the immobilization of the enzyme duplicates or more the reaction rates for all the new studied substrates.