Preparation and activity of carbonic anhydrase immobilized on porous silica beads and graphite rods

Abstract

Techniques for the immobilization of bovine carbonic anhydrase (BCA) on porous silica beads and graphite are presented. Surface coverage on porous silica beads was found to be 1.5 × 10⁻⁵ mmol BCA/m², and on graphite it was 1.7 × 10⁻³ mmol BCA/m² nominal surface area. Greater than 97% (silica support) and 85% (graphite support) enzyme activity was maintained upon storage of the immobilized enzyme for 50 days in pH 8 buffer at 4°C. After 500 days storage, the porous silica bead immobilized enzyme exhibited over 70% activity. Operational stability of the enzyme on silica at 23°C and pH 8 was found to be 50% after 30 days. Catalytic activity expressed as an apparent second-order rate constant K′_Enz for the hydrolysis of p-nitrophenyl acetate (p-NPA) catalyzed by BCA immobilized on silica beads and graphite at pH 8 and 25°C is 2.6 × 10² and 5.6 × 10² M⁻¹s⁻¹ respectively. The corresponding K_ENZ value for the free enzyme is 9.1 × 10² M⁻¹s⁻¹. Activity of the immobilized enzyme was found to vary with pH in such a manner that the active site pK, on the porous silica bead support is 6.75, and on graphite it is 7.41. Possible reasons for a microenvironmental influence on carbonic anhydrase pK_a, are discussed. Comparison with literature data shows that the enzyme surface coverage on silica beads reported here is superior to previously reported data on silica beads and polyacrylamide gels and is comparable to an organic matrix support. Shifts in BCA-active site pK_a values with support material, a lack of pH dependent activity studies in the literature, and differing criteria for reporting enzyme activity complicate literature comparisons of activity; however, immobilized BCA reported here generally exhibits comparable or greater activity than previous reports for immobilized BCA.