Effect of chemical modification on struture and activity of glucoamylase from Aspergillus candidus and Rhizopus species

Abstract

The histidine, tyrosine, tryptophan and carboxyl groups in the enzyme glucoamylase from Aspergillus Candidus and Rhizopus species were modified using group specific reagents. Treatment of the enzyme with diethyl pyrocarbonate resulted in the modification of 0.3 and 1 histidine residues with only a slight loss in activity (10% and 35%) of glucoamylase from Aspergillus candidus and Rhizopus species respectively. Modification of tyrosine either by N-acetylimidazole or [I¹²⁵]-leads to a partial loss of activity. Under denaturing conditions, maltose did not help in protecting the enzyme against tyrosine modification or inactivation. Treatment with 2-Hydroxy-5-nitro benzyl bromide in the presence of urea, Photooxidation at PH 9.0, N-bromosuccinamide at PH 4.8 resulted in a complete loss of activity. However, the results of experiments in the presence of maltose and at PH 4.8 photooxidation and N-bromosuccinamide treatment suggested the presence of two tryptophan residues at the active site. There was a complete loss of enzyme activity when 10 and 28 carboxyl groups from Aspergillus candidus and Rhizopus, respectively were modified. Modification in the presence of substrate maltose, showed at least two carboxyl groups were present at the active site of enzyme and that only one active center seems to be involved in breaking ally 3 types of α-glucosidic linkages namely α-1, 4, α-1, 6 and α-1, 3.