Studies of Activity and Stability of Immobilized ß -N-Acetylhexosaminidase
A mixture of glycosidases from the liver of sea worm T. cornutus was co-immobilized with bovine serum albumin and glutaraldehyde and then cast as membranes. When the activities of nine glycosidases were examined, it was found that only a - mannosidase was completely inactivated by this immobilization procedure. The properties of immobilized ß-N-acetylhexosaminidase were studied in some detail. The yield of immobilized ß -N-acetylhexosaminidase was unaffected by increasing the concentration of glutaraldehyde but was decreased by increasing the bovine serum albumin concentrations. While the pH optimum for the soluble enzyme was 4. 0, after insolubilization, a bimodal pH curve with optima at 3.4 and 5.0 was observed. The bimodality was abolished when the immobilized enzyme was assayed in the presence of 1M NaCl. The Km values of the soluble and immobilized ß -N-acetylhexosaminidase for p-nitrophenyl — ß -2-acetamido-2-deoxy-D-galactopyranoside were 3.6 and 10.1 mM, respectively. The immobilized enzyme showed enhanced stability towards thermal and proteolytic inactivation. No loss of activity could be detected in the membrane after using it for 24 consecutive assays or after storage at 40 for at least 50 days. This glutaraldehyde-albumin co-crosslinking technique provides a simple general method of preparing immobilized glycosidases.
KeywordsImmobilize Enzyme Enzyme Replacement Therapy Sodium Citrate Buffer Soluble Enzyme Membrane Matrix
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