Modification of Glucoamylases from Rhizopus Sp. with 1-Cyclohexyl-3-(2-Morpholinyl-(4)-Ethyl)Carbodiimide

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

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