Immobilization and characterization of D–amino acid oxidase

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Abstract
Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer → monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate >borate > Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50°C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.
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