Active-site titration analysis of surface influences on immobilized Candida antarctica lipase B activity

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 69; no. 1-2; pp. 60 - 65
• Main Authors: Laszlo, Joseph A., Jackson, Michael, Blanco, Rosa M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.04.2011; Elsevier
• Subjects: Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Enzyme immobilization; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Silica; Specific activity; Suicide inhibitor; Specific activity; Suicide inhibitor; Enzyme immobilization; Silica; candida antarctica; Enzyme; Active site; Immobilization; Triacylglycerol lipase; Esterases; Carboxylic ester hydrolases; Fungi; Hydrolases; Inhibitor; Fungi Imperfecti
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2010.12.011

[Display omitted] ▶ SBA-15 silica adsorbes Candida antarctica lipase B with high retention of native state structure. ▶ Octyl modified silica adsorbes large quantities of the enzyme, but some fraction denatures. ▶ Fumed silica retains some native state enzyme. Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements were made of the amount of lipase immobilized and the catalyst's tributyrin hydrolysis activity, along with a determination of the lipase's functional fraction by active-site titration. Soluble, purified lipase had an active fraction of 84%. Immobilization on the hydrophobic surface of macroporous poly(methylmethacrylate) resin resulted in the full retention the lipase active fraction, while immobilization on the hydrophobic surface of mesoporous, amorphous, octyl-modified silica allowed retention of just half of the lipase active fraction. The polar surface of unmodified mesoporous, amorphous silica bound the lipase in such a manner that all of the immobilized enzyme was active. Mesoporous, crystalline SBA-15 silica also bound lipase so that it all was active. The polar, non-porous surface of fumed silica retained only a small fraction (28%) of active lipase. Substantial differences were found among the various supports in their ability to preserve catalytic activity upon vacuum drying. These findings demonstrate that surface polarity alone is not the only determinant for immobilization, as hydrophobic poly(methylmethacrylate) and hydrophilic SBA-15 were equally competent as lipase supports. The ordered-channel mesostructure of SBA-15 may provide a critical balance of interactions with the enzyme to preserve its native conformation.