Title: Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity Authors
Submitted to: Journal of Molecular Catalysis B: Enzymatic
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 24, 2010
Publication Date: February 1, 2011
Repository URL: http://hdl.handle.net/10113/48187
Citation: Laszlo, J.A., Jackson, M.A., Blanco, R.M. 2011. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity. Journal of Molecular Catalysis B: Enzymatic. 69:60-65. Interpretive Summary: Expanded use of enzymes in the manufacture of vegetable oil-derived consumer and industrial products could be achieved by developing more durable and lower cost catalysts (enzyme and its support material). We have demonstrated that a technique to count the amount of active enzyme in a catalyst preparation provides new insights into the best practices for enzyme immobilization (attachment of the enzyme to a solid support). A lipase from the yeast Candida antarctica that is broadly employed in many applications was the focus of our study. Lipase immobilization is needed to conserve expensive enzyme in commercial processes. Conventional approaches for discerning the effectiveness of a particular immobilization generally rely on measures of apparent activity of the prepared catalyst. We show that underlying differences exist in these protocols that is not evident from determinations of apparent activity. These new findings can be used by scientists to produce improved support materials for enzyme applications.
Technical Abstract: Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fraction by active-site titration with methyl 4-methylumbelliferyl hexylphosphonate, were made. 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 (SBA-15 silica, octyl-modified silica, poly(methylmethacrylate) > fumed silica > unmodified amorphous silica). 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 nature conformation.