Studies on amyloglucosidase from rhizopus delemar
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Amyloglucosidase has been obtained from Rhizopus delemar and purified by ammonium sulphate fractionation; ethanolic precipitation; chromatography on DEAE-cellulose, DEAE-sephadex A-50 and Sephadex G-50/ Amberlite IRC-50. Ethanolic precipation and chromatography on DEAE-cellulose by stepwise elution have been proved to be the most simple and rapid procedure to remove traces of alpha-amylase from amyloglucosidase preparations. The apparent kinetic perameters of amyloglucosidase free of traces of alpha-amylase has been shown to be altered after inclusion of alpha-amylase. The increased apparent Km and V for the alpha-1,4-glucan/ amyloglucosidase/alpha-amylase system compared with those parameters for the system in the absence of alpha-amylase have been explained on the basis of the decrease in the size of the substrate molecule, which is caused by the action alpha-amylase (endo-attack pattern). The apparent kinetic perameters of amyloglucosidase oxidized traces of alpha-amylase has also substrates., Amyloglucosidase action has been established to be unable of by passing oxidation points introduced in the alpha-1,4-glucan by periodate oxidation. A theory has been developed to describe the action of amyloglucosidase on partially oxidized amylases, which predicts the decrease in the apparent Km and V with the increase in the degree of oxidation of the amylase, as it has been demonstrated experimentally. A direct recording method of assaying amyloglucosidase has been developed involving ascorbic acid as the oxygen acceptor in a glucose oxiddase/peroxidase assay system for glucose. This method permits amyloglucosidase activity to be followed by ultraviolet spectrophotometry. The merits and limitations of this procedure have been discussed. An immobilized cellulose derivative of amyloglucosidase in which the prosthetic group is involved in the covalent linkage between the enzyme end the support has been investigated and compared with the soluble enzyme. The kinetics, pH profile and the thermal stability of this immobilized enzyme and their comparison with these characteristics for the free enzyme have been investigated.
Thesis, PhD Doctor of Philosophy
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