Modification of glucoamylases from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide

To investigate the role of carboxyl groups of glucoamylases [EC 3.2.1.3] from a Rhizopus sp. (Gluc1 and Gluc2), the modification of Gluc1 and Gluc2 with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), was studied. The inactivation of Gluc1 proceeded with the incorporation of about 3 CMC moieties. In the presence of maltose, the modification of about 2.2 carboxyl groups of Gluc1 proceeded with a slight loss of enzymatic activity. In the re-modification of Gluc1 modified in the presence of maltose, Gluc1 was inactivated by further modification of about 1.3 carboxyl groups. Therefore, one carboxyl group, which was protected by maltose, was thought to be a crucial one. The inactivation of Gluc2 proceeded similarly to that of Gluc1, but the number of CMC moieties incorporated was about one less than in the case of Gluc1. Thus, it was suggested that one of the reactive carboxyl groups of Gluc1 was located in the N-terminal part of Gluc1, which is deficient in Gluc2. From the results of kinetic studies on CMC-modified Gluc1, it was suggested that the hydrolysis mechanism of malto-oligomers differs somewhat from that of PNPG.