Abstract

Alcohol dehydrogenase (ADH) from S. cerevisiae has a low stability under several conditions of temperature and pH. This represent a limitation in the industrial applications of ADH. In this line, the immobilization techniques have shown an enhancement of the stability. Particularly, silica has given good performances for analogous systems. The good mechanical properties of silica makes it suitable for carrying out repeated reaction batches. In this work we compared two different immobilization supports (i.e. agarose and silica) activated with glyoxyl groups in order to form a covalent union with ADH. The charge capacity, expressed activity and stability were evaluated. The immobilization of the enzyme was carried out for 1 hour at pH 10 and cross-linked with polyethylenimine (M.W. 25000). The activity was measured spectrophotometrically, by the formation of NADH (λ=340 nm). One UI is the amount of ADH that oxidizes 1 µmol of ethanol per minute (30ºC, pH 7). The immobilization experiments have shown that the yield in proteins for the samples with 1 mg/g were identically, whereas the yield in activity for agarose was 4.3 fold the yield for silica. Similar ratio was found for the expressed activity. The latter is related to the size of the mesopores of silica (24 nm), whereas the agarose matrix has micrometric sized pores (>100 nm). Since the diameter of ADH is 22 nm, the immobilization in mesoporous silica is limited to the external surface of the particle. The most active catalysts of each support were chosen for stability measurements, showing a 100% of residual activity after 10 days for both matrixes. Even though the activity of the catalysts with silica is lower than with agarose, they are as stable as with agarose but with better mechanical properties, which could be of great interest for applicative purposes.