Biocatalytic performance of carbonic anhydrase immobilized within polyurethane foam in water-miscible organic solvents

Abstract

The effects of water-miscible organic solvents such as acetonitrile and ethanol on the activity of free and immobilized bovine carbonic anhydrase (CA) were investigated. The CA was covalently immobilized within polyurethane (PU) foam by cross-linking. Although PU foam holds water almost 12 times of its weight, it was found that adsorption isotherm of moisture on PU foam was a Type III indicating that water and PU foam were non-interacting to each other. The activities for the free and immobilized CA were estimated using para-nitrophenyl acetate (p-NPA) as the substrate. The enzyme activities were estimated in increasing volume percents of organic solvent in Tris buffer (10-90%). p-NP, which is one of the products of the hydrolysis reaction of p-NPA, was characterized in the presence of organic solvents and it was observed that its aborptivities were decreased significantly as the organic solvent percentages were increased indicating that p-NP and the water-miscible organic solvent form a complex through mostly a hydrogen bonding. The free CA showed decreasing activity up to critical percentages of organic solvent (40-60%), and then exhibited an increasing activity. The immobilized CA showed decreasing activity in acetonitrile at percentages up to 50%, and then lost its total activity at higher acetonitrile percentages, however, the immobilized CA exhibited no activity in ethanol at percentages above 10%. Stability tests showed that the immobilized CA was dramatically inactivated in the organic solvents at percentages above 30% in shorter times. It was concluded that the water-miscible organic solvents severely perturbed the active site of the enzyme, thus denaturating the enzyme.