RESUMEN
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.
RESUMEN
The purification and properties of glucoamylase (α-l,4-glucan glucohydrolase, EC 3.2.1.3) from different fungal sources have been compared. The studies on the conformation and activity of the native enzyme at a function of pH, temperature, substrate concentration and the effect of denaturants and on the role of carbohydrate moiety on structure and stability have been reviewed. The chemical modification of the active centre, binding kinetics of the substrate and active site and the mechanism of action have been summarized. They differ in their fine structure as revealed by their near ultra-violet circular dichroism spectra and contain 30–35 % α-helix, 24–36 % β-structure and the rest aperiodic structure. The activity of the enzyme is very sensitive to the environment around aromatic aminoacid residues. The glucoamylases are glycoprotein in nature, differ in their content and nature of carbohydrate from different sources. The carbohydrate moiety plays an important role in stabilising the native conformation of the enzyme and is not involved in activity and antigenecity. At the active site of the enzyme, two tryptophan and two carboxyl (glutamate or aspartate) groups are present. It is likely that the histidine and tyrosine residues which are present away from the active site are involved in binding of the substrate. There seems to be seven subsites which are involved in binding of the substrate and the catalytic site is situated in between 1 and 2 subsites. In breaking of α-1,4-, α-1,3-, and α-l,6-bonds only one active centre is involved. Studies on the immobilization of either glucoamylase alone or as a part of a multienzyme system have been reviewed briefly.
RESUMEN
Rabbit antisera were prepared against the purified glucoamylases I and II of Aspergillus niger. Relationships between the two enzyme forms were investigated by using the antisera in immunodiffusion and immunoinhibition experiments. Both the forms of glucoamylase gave a single continuous precipitin band demonstrating very close structural resemblance. They gave almost identical immunoprecipitation patterns and had the same equivalence points indicating that the two forms of A. niger gluoamylases were immunologically identical. The enzyme treated with periodate was immunologically identical with the controls and had slightly less enzyme activity but showed greatly reduced stability on storage at 4° C.
RESUMEN
Five commercial preparations of glucoamylases (three from Aspergillus niger, one each from Aspergillus foetidus and Aspergillus candidus) were purified by ultrafiltration, Sepharose gel filtration and DEAE sephadex chromatography. Two forms of the enzyme, namely glucoamylase I and glucoamylase II were obtained from the fungi except from one strain of A. niger. All the enzymes appeared homogeneous by electrophoresis and ultracentrifugation. The specific activities varied between 85 and 142 units. The pH and temperature optima were between 4 and 5, and 60° C respectively. The molecular weight as determined by the sodium dodecyl sulphate polyacrylamide gel electrophoresis ranged from 75,000 to 79,000 for glucoamylase I and 60,000 to 72,000 for glucoamylase II. Only A. niger gluco amylases contained phenylalanine at the N terminal end. The amino acid compo sition of the enzymes was generally similar. However, A. niger and A. foetidus glucoamylases, in contrast to A. candidus enzymes, contained greater percentage of acidic than of basic amino acids. The enzymes contained 15 to 30% carbohydrate and 49 to 57 residues of monosaccharides per mol. A. niger enzymes contained mannose, glucose, galactose, xylose and glucosamine but the A. candidus enzyme lacked xylose and glucose and only xylose was absent in A, foetidus enzymes.. Majo rity of the carbohydrate moieties were O glycosidically linked through mannose to the hydroxyl groups of seline and threonine of the polypeptide chain.