PURIFICATION AND STABILITY OF GLUTARYL-7-ACA ACYLASE FROM PSEUDOMONASSP

The enzyme glutaryl-7-ACA acylase from Pseudomonas sp. NCIMB 40474, pr oduced by a recombinant Escherichia coli host, was purified to homogen eity. The enzyme is a tetramer composed of two couples of asymmetric d imers, each of them constituted of two subunits of mol wt 18 and 52 kD a, respectively. It was found that glutaric acid, one of the products of the substrate hydrolysis, is an effective acylase inhibitor. Betwee n pH 6.0 and pH 10.0, the enzymatic activity is almost constant, but b elow pH 6.0 it progressively declines. The acylase activity decreased sharply as a function of guanidine HCl concentration. The loss is sign ificant even at concentrations of denaturant lower than those causing unfolding, as suggested by UV spectroscopy and fluorescence emission s tudies. In these conditions (low denaturant concentration and low pH) the inactivation of the enzyme is caused by the tetramer dissociation into dimers. The lability of the quaternary structure of the enzyme is a key feature that must be taken into account for the improvement of the catalyst stability.