No abstract

The digestion of raw starch by a glucoamylase (GA MU-H) from a mutant strain of Aspergillus awamori var. kawachi was closely correlated with mannoside chains O-linked to the Gp-I region (A470-V514), but not sugar chains N-linked to catalytic GAI' domain of GA MU-H. The partial replacement of mannose residues by glucose residues led to a significant decrease raw starch digestion. By the substitution of D2O for H2O in the reaction mixture, the raw starch digestion of GA MU-H decreased to 80% of that at 30 degrees C, although the rate of hydrolysis of soluble starch by and the ability to bind beta-cyclodextrin of GA MU-H were unchanged. Glycerol, known as an antichaotropic reagent, decreased the raw starch digestion of GA MU-H significantly. However, it did not have any effect on the enzymatic activity for soluble starch when soluble starch was the substrate. The efficient digestion of raw starch with raw starch-digesting glucoamylase needed the mannoside chains O-linked to the Gp-I region, which were suggested to contribute to digestion of raw starch through the interaction with water.

Glucoamylase I (GAI) bound to β-cyclodextrin (β-CD) with the binding constants of Kd = 17.7 μm and n = 1.69. Binding resulted in formation of an inclusion complex, as indicated by the fact that organic guest compounds for β-CD inhibited the binding of GAI to β-CD. Titration of GAI (A(1)-R(615)) with β-CD or Amylose A showed critical spectral perturbations at 286 nm that reflected the aromatic side chains of Tyr and Trp, but no spectral changes were observed in GAI' (A(1)-V(469)) and Gp-I (A(470)-V(514)). Titration of GAI with derivatives of β-CD, namely, 6-hydroxypropyl-β-CD (H-CD), 2,6-O-dimethyl-β-CD, and 2,3,6-O-trimethyl-β-CD, yielded spectral changes only in the case of H-CD. Therefore, the Cp region (A(515)-R(615)) of GAI seems stereospecifically able to recognize the structure of the secondary OH-side but not the primary OH-side of β-CD. Chemical modifications specific for aromatic amino acids of the GAI-CD inclusion complexes were done with N-bromosuccinimide and tetranitromethane. In the presence of β-CD, one Trp residue was protected from oxidation but Tyr residues were not. The analysis of a Cp region/β-galactosidase fusion protein indicated that Trp(562) contributed to formation of inclusion complexes.

No abstract

The enzymatically inactive but raw-starch-adsorbable glycopeptide-I (Gp-I), which was obtained from raw-starch-adsorbable, raw-starch-digesting glucoamylase I (GAI) of Aspergillus awamori var. kawachi as a result of proteolysis with subtilisin and proved to contain the raw-starch-affinity site essential for raw-starch-digestion, was found to promote the digestion rate of raw corn starch with GA I, maximally to the extent of 2.5 times. The raw-starch-adsorbability of Gp-I was decreased by the partial removal of carbohydrate moiety from Gp-I. The carbohydrate-split GAI could hydrolyze gelatinized substrates almost at the same rate as the native one, but significantly decreased in the adsorbabih'ty and digestibility onto raw starch in comparison with the original one. From the function and the characteristic structure of the parallel short mannoside chains linked to the successive sequence of hydroxy amino acid residues of Gp-I, the "water-cluster-dissociating model" for the hypothetical mechanism of raw-starch-digestion by the adsorption of GAI at the affinity site onto starch was proposed.