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

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 diethylpyrocarbonate 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 Nacetylimidazole 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 Nbromosuccinamide 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-l,3.

Structure and stability of glucoamylase II from Aspergillus niger: A circular dichroism study.

Glucoamylase II (EC 3.2.1.3) from Aspergillus niger has 31 % α-helix, 36 % ßstructure and rest aperiodic structure at pH 4·8 as analysed by the method of Provencher and Glockner (1981, Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4·8 than at pH 7·0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30°C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.

Purification and properties of diaminopimelate decarboxylase of Micrococcus glutamicus.

Diaminopimelate decarboxylase (EC 4.1.1.20) of Micrococcus glutamicus ATCC 13059 was purified to homogeneity. The enzyme had an apparent molecular weight of 191,000 as determined by gel filtration on Sephadex G 200. At protein concentrations of 20 and 10 μg per ml and in the absence of pyridoxal 5 ' phosphate, it dissociated into a species of molecular weight 94,000. The polypeptide chain molecular weight as determined by sodium dodecyl sulphate Polyacrylamide gel electrophoresis was 100,000. The Km for meso diaminopimelate was 0.5 mM and that for pyridoxal 5' phosphate was 0.6 μΜ. Sulphydryl groups and pyridoxal 5' phosphate were essential for activity and stability. The enzyme was inhibited significantly by L lysine and DL aspartic β semialdehyde.

Aspartokinase of a lysine producing mutant of Micrococcus glutamicus.

Aspartokinase from Micrococcus glutamicus AEC RN-13-6/1 [a homoserine requiring, S-(2–aminoethyl)-L-cysteine resistant, lysine producing strain] was purified 71 fold. The partially purified enzyme was inhibited by L-lysine. L-threonine, L-methionine, Lisoleucine, L-valine and L-phenylalanine activated the enzyme and reversed the inhibition by Llysine. Aspartokinase activity was not derepressed by growth–limiting concentrations of Lthreonine and/or L–methionine. It was not repressed by an excess of L-lysine (20 mM) and/or L-isoleucine (15.3 mM). The degree of activation or inhibition by amino acids was dependant on the composition of the growth medium. This observation is in contrast with the enzyme from the original (non–lysine–producing) strain which was inhibited by lysine or threonine and in a concerted manner by threonine plus lysine.

Studies on plant gums. Role of calcium in polysaccharide-protein interaction in the neem (Azadirachta indica) gum.

The partial removal of tightly bound Ca2+ from dialysed neem (Azadirachta indica) gum, resulted in the release of a basic protein from a highly anionic polysaccharide-protein complex as evidenced by chromatographic studies on TEAEcellulose. Complete removal of Ca2+ caused, in addition, the release of a minor heteropolysaccharide which was found in association with the basic protein. These processes were reversed on the addition of Ca2+. The gum, in addition, contained a protein-rich component accounting for 35% protein and 7·5% total carbohydrate. This component behaved as a distinct entity during ion-exchange chromatography of the native gum solutions, or which were either partially or completely depleted of bound Ca2+.