Characterization of a Chemically Conjugated Lipase Bioreactor

Lipase from Candida cylindracea was immobilized on glass beads using the biospecific and high-affinity avidin−biotin interaction. Biotinylated lipase and glass beads were prepared by reactions of lipase and 3-aminopropyl glass beads with sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin). Avidin and biotinylated lipase were sequentially adsorbed to the biotinylated glass beads. Biotinylated lipase in solution retained about 63% of the hydrolytic specific activity of native lipase when an average 3 mol of biotin was incorporated/mol of lipase. Nonporous glass beads contained more biotin and protein (avidin and lipase) per unit of surface area, followed by 302 nm mean pore diameter controlled-pore glass beads (CPG-3000) and 198 nm mean pore diameter controlled-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 immobilized on CPG-2000 was about 50% less. The long spacer of NHS-LC-biotin (22.4 Å maximum length) and avidin (70 Å diameter) reduced steric hindrances with emulsified substrates on the matrix surface, resulting in a higher hydrolytic activity as compared with lipase immobilized via covalent linkages. The interesterification activity was 4-fold greater for immobilized lipase than for free lipase. Keywords: Biotinylated lipase; immobilized lipase; immobilized avidin; interesterification