CHARACTERIZATION OF A CHEMICALLY CONJUGATED LIPASE BIOREACTOR

Lipase from Candida cylindracea was immobilized on glass beads using t he biospecific and high-affinity avidin-biotin interaction. Biotinylat ed lipase and glass beads were prepared by reactions of lipase and 3-a minopropyl glass beads with sulfosuccinimidyl-6-(biotinamido)hexanote (NHSLC-biotin). Avidin and biotinylated lipase were sequentially adsor bed to the biotinylated glass beads. Biotinylated lipase in solution r etained about 63% of the hydrolytic specific activity of native lipase when an average 3 mol of biotin was incorporated/mol of lipase. Nonpo rous glass beads contained more biotin and protein (avidin and lipase) per unit of surface area, followed by 302 nm mean pore diameter contr olled-pore glass beads (CPG-3000) and 198 nm mean pore diameter contro lled-pore glass beads (CPG-2000). The hydrolytic specific activity of lipase immobilized on CPG-3000 and on nonporous beads was essentially the same as that for the biotinylated free enzyme, whereas that immobi lized on CPG-2000 was about 50% less. The long spacer of NHS-LC-biotin (22.4 Angstrom maximum length) and avidin (70 Angstrom diameter) redu ced steric hindrances with emulsified substrates on the matrix surface , resulting in a higher hydrolytic activity as compared with lipase im mobilized via covalent linkages. The interesterification activity was 4-fold greater for immobilized lipase than for free lipase.