Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

Authors: Laszlo, Joseph; Evans, Kervin

Submitted to: Journal of Molecular Catalysis B: Enzymatic
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2008
Publication Date: 1/4/2009
Citation: Laszlo, J.A., Evans, K.O. 2009. Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B. Journal of Molecular Catalysis B: Enzymatic. 58:169-174.

Interpretive Summary: Enzymes are particularly useful for the production of high-value biobased materials such as skincare products from vegetable oils. Enzymes in such application areas must be utilized under rather harsh conditions (from the enzyme’s perspective). It is known that capturing an enzyme such as a lipase on a carefully crafted inert support greatly benefits the long-term usage of the enzyme. In this work, we have examined specific conditions for applying a commercially important lipase to a model support surface to find the best conditions. We found that, unexpectedly, inclusion of ethanol during the application process is detrimental. Biotechnology companies will be able to use this information to produce faster acting enzyme forms for their reactions.

Technical Abstract: The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., gold modified with a methyl-terminated, self-assembled alkylthiol layer. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance (QCM). Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy (AFM). Lipase adsorption from low ionic strength aqueous buffer produced a uniform confluent protein monolayer. Inclusion of 10% (v/v) ethanol in the buffer during immobilization resulted in a 33% adsorbed mass increase. Chemically similar cosolvents, all at 10% (v/v) in buffer, were also individually examined for their influence on CALB adsorption. Glycerol or 1-propanol increased mass adsorption by 10%, while 2-propanol increased mass adsorption by 33%. QCM dissipation values increased three-fold with the inclusion of either ethanol or 2-propanol in the medium during lipase adsorption, indicating formation of multilayers of CALB. Partial multilayer formation using 10% ethanol was confirmed by AFM. Inclusion of 10% ethanol in the CALB immobilization buffer decreased the specific activity of the immobilized lipase. The formation of lipase multilayers in the presence of certain cosolvents thus results in lower specific activity, which might be due to either influences on lipase conformation or substrate active site accessibility.