Properties and action of glucoamylase from an Aspergillus species

Abstract

Different techniques of enzyme active site labelling have been classified and discussed with particular reference to the glycoside hydrolases and glucoamylase. Glucoamylase from Aspergillus niger has been purified and fractionated into 2 major forms. The physical properties of the forms have been measured and compared. Both purified enzyme forms have similar sedimentation coefficients, behave similarly on disc-gel electrophoresis and have similar molecular weights (80,000). The purified enzyme forms showed distinct amino acid compositions, but both had carbohydrate contents of about 15%. The specific activities of the enzyme fractions toward wheat amylopectin were similar and about four times greater than those towards maltose. Both purified enzyme forms converted wheat amylopectin to glucose to the extent of virtually 100%. The phenomenon of reversion has been discussed and analysed from a thermodynamic point of view. Extents of reversion theoretically expected have been calculated from available thermodynamic data and compared with experiment. Both purified enzyme forms have a pH optima of 4*6 and are devoid of "endo"-activity. The chemical modification of protein carboxyl groups has been reviewed, and the technique of differential modification has been applied to the two purified glucoamylases. Carboxyl groups were Deactivated by reaction with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride and labelled with glycine methyl ester, glycine ethyl ester and 2-amino ethane sulphonic acid (taurine). It was found that deactivation was most rapid in the case of taurine, and the deactivation is restricted to ca. 20% in the presence of maltose. The use of taurine also facilitates deter-mination of the extent of modification by amino acid analysis. The partially deactivated enzyme was stable towards subsequent purification procedures and could be fully deactivated by a second treatment in the absence of maltose. The use of radio-labelled taurine in the second stage showed that maltose protected an enzyme substrate site which contained 6 and 5 modifiable groups in glucoamylase I and II respectively. Peptide maps of chemically modified glucoamylase I and glucoamylase II showed distinct differences.