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Immobilized xanthine oxidase: Kinetics, (in)stability, and stabilization by coimmobilization with superoxide dismutase and catalase
Author(s) -
Tramper Johannes,
Müller Franz,
Van Der Plas Henk C.
Publication year - 1978
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260201002
Subject(s) - xanthine oxidase , chemistry , substrate (aquarium) , xanthine , immobilized enzyme , michaelis–menten kinetics , chromatography , glutaraldehyde , adsorption , kinetics , urate oxidase , enzyme , sepharose , nuclear chemistry , biochemistry , enzyme assay , organic chemistry , physics , quantum mechanics , oceanography , geology
Abstract Milk xanthine oxidase was immobilized by covalent attachment to CNBr‐activated Sepharose 4B and by adsorption to n ‐octylamine‐substituted Sepharose 4B. The amounts of activity immobilized for the two preparations were 30 and 90%, respectively. The pH optima for free and adsorbed xanthine oxidase were at 8.6 and 8.2, respectively. Both free and immobilized xanthine oxidase show substrate inhibition. The apparent inhibition constant ( K i ′) found for adsorbed xanthine oxidase with xanthine as substrate was higher than the K i for the free enzyme, which was shown to be due to substrate diffusion limitation in the pores of the carrier beads (internal diffusion limitation). Higher substrate concentrations, as desirable for practical application in organic synthesis, can therefore be used with the immobilized enzyme without decreasing the rate. As a result of the internal diffusion limitation the apparent Michaelis constant ( K m ′) for adsorbed xanthine oxidase was also higher than the K m for the free enzyme. Immobilized xanthine oxidase was more stable than the free enzyme during storage at 4 and 30°C. Both forms rapidly lost activity during catalysis. The loss was proportional to the amount of substrate converted. Coimmobilization of xanthine oxidase with superoxide dismutase and catalase improved the operational stability, suggesting that O 2 − and H 2 O 2 side‐products of the enzymatic reaction were involved in the inactivation. Coimmobilization with albumin also had some stabilizing effect. Complete surrounding of xanthine oxidase by protein, however, by means of etrapment in a glutaraldehyde‐crosslinked gelatin matrix, considerably enhanced the operational half‐life. This system was less efficient than the Sepharose preparations either because much activity was lost during the immobilization procedure and/or because it had poor flow properties. Xanthine (15 mg)was converted by an adsorbed xanthine oxidase preparation and product (uric acid) was isolated in high yield (84%).