Immobilization of HLADH on polymer materials for reduction of cyclohexanone with NADH regeneration under two-phase conditions

Abstract

Horse liver alcohol dehydrogenase (HLADH) was effectively immobilized by adsorption to poly(vinyl alcohol) (PVA), cross-linked polyacrylamide (PAA), or cross-linked chitosan beads (CP). The activity of the immobilized HLADH was estimated from the initial rate of the reduction of cyclohexanone coupled with NADH regeneration via oxidation of alcohols under aqueous/ n-heptane two-phase conditions. Both the activity and the specificity for alcohols of PAA-immobilized HLADH were almost the same as those of free HLADH. The activity of HLADH decreased upon immobilization on PVA or CP, but the specificity for alcohols was not altered markedly. For all the immobilized HLADH's, the apparent activity was dependent on water content and the amount of support material. More than 2.5% of water content in reaction solution was required for activation of the enzyme, and large amounts of support material led to a decrease in the reaction rate. The activity of immobilized HLADH decreased during repeated batch reactions, and the residual activity after 15 12-h reactions was 25–55% depending on the type of support material.