Journal of Molecular Catalysis B: Enzymatic 48 (2007) 84–89 Influence of self-assembled monolayer surface chemistry on Candida antarctica lipase B adsorption and specific activity  Joseph A. Laszlo ∗ , Kervin O. Evans New Crops and Processing Technology Research, USDA-Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, IL 61604, USA Received 24 May 2007; received in revised form 10 June 2007; accepted 22 June 2007 Available online 1 July 2007 Abstract Immobilization of Candida antarctica B lipase was examined on gold surfaces modified with either methyl- or hydroxyl-terminated self- assembled alkylthiol monolayers (SAMs), representing hydrophobic and hydrophilic surfaces, respectively. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance. Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrol- ysis. Adsorbed lipase topography was examined by atomic force microscopy. The extent of lipase adsorption was nearly identical on either surface (approximately 240 ng cm −2 ), but its specific activity was sixfold higher on the methyl-terminated SAM, showing no activity loss upon immo- bilization. A uniform, 5.5 nm high, highly packed monolayer of CALB formed on the methyl-terminated SAM, while the adsorbed protein was disordered on the hydroxyl-terminated SAM. Hydrophobic surfaces thus may specifically orient the lipase in a highly active state. Published by Elsevier B.V. Keywords: Protein adsorption; Lipase; Immobilization 1. Introduction Nonaqueous biocatalysis is finding commercial utility in the production of fine and specialty chemicals [1–3]. Lipases are among the most broadly deployed biocatalysts because of their ability to produce chiral chemicals with high enantiomeric purity [4]. Lipases catalyze hydrolysis, alcoholysis, esterifica- tion and transesterification of carboxylic acids or esters. The B lipase from Candida antarctica (CALB) has been a synthesis workhorse, as well as the subject of numerous fundamental stud- ies regarding nonaqueous enzymology [5–7]. CALB typically is used in an immobilized form, such as the commercial product Novozym 435. Enzyme immobilization offers many potential benefits. Immobilization can improve enzyme operational performance and stability, as well as provide for ready separation of bio-  Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable. ∗ Corresponding author. Tel.: +1 309 681 6322; fax: +1 309 681 6686. E-mail address: Joe.Laszlo@ars.usda.gov (J.A. Laszlo). catalyst from the reaction medium [8,9]. CALB has been immobilized on various support materials such as porous resins and silicas [5,10–15]. Polypropylene and acrylic resins, regarded as hydrophobic supports, are particularly efficacious. These meso- and macroporous polymeric materials accommodate high CALB loadings (up to 20% w/w protein) and good retention of enzyme activity. It is not understood how the support matrix influences CALB activity. The support matrix can impose activ- ity limitations by altering the lipase’s native conformation or by diminishing substrate diffusivity. Distinguishing between these influences can be difficult. Infrared detection of CALB on var- ious supports indicates a very heterogenous distribution within the polymer matrix [12,16]. Potentially, there are substantial amounts of lipase that do not interact directly with the poly- mer surface. Thus, conventional immobilization materials do not provide a sufficiently uniform enzyme–support interface for study of support surface influences on CALB activity and topography. The current study examines the impact of support sur- face properties (hydrophobicity) on CALB activity, in an arrangement that does not impose substrate internal transport limitations, through the use of flat, self-assembled alkylthiol- modified gold surfaces [17]. A correlation between surface 1381-1177/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.molcatb.2007.06.010