The involvement of salt links in the stabilization of Baker's yeast invertase Evidence from immobilization and chemical modification studies

Abstract

Listen

Translate article

Baker's yeast invertase was immobilized onto microcrystalline cellulose DEAE- and CM-Sephadex and insolubilized concanavalin A, with 46, 41, 70 and 73% recovery of activity, respectively. The concanavalin A and microcrystalline cellulose invertase complexes exhibited spreading of their pH optimum curves, possessed similar K m and V and were more thermally stable in the absence and presence of 0.12 M sucrose, than the soluble enzyme. DEAE- and CM-Sephadex · invertase complexes were extremely unstable towards heat, compared to the soluble enzyme, losing all activity within 1 min of heating to 65°C. However, normal thermal stability of these complexes was observed in the presence of 20 mM succinic acid and 20 mM ethylenediamine, respectively. The treatment of purified invertase with citraconic anhydride, which converts the amino group function to carboxyl groups, resulted in the loss of enzyme activity. The enzyme, with 50% activity after such tretments, was less thermally stable than the untreated enzyme. Similar results were obtained using ethylene diamine and a water-soluble carbodiimide that converts carboxyl group function to amino group function. Treatment of invertase with methylacetimidate (which modifies amino groups of proteins but retains the positive charge) resulted in no loss in activity and very little effect on thermal stability. It is suggested that salt linkages may be important in maintaining the conformational stability of invertase. Modification of either amin or carboxyl groups chemically or by immobilization on to charged supports, would result in the loss of conformational stability, unless these stabilizing salt linkages were preserved.