Technical Note Covalent immobilisation of protease and laccase substrates onto siloxanes Alexandra Rollett a , Marc Schroeder a , Konstantin P. Schneider b , Roland Fischer c , Franz Kaufmann d , Rainer Schöftner e , Georg M. Guebitz a,b, * a Graz University of Technology, Dept. of Environmental Biotechnology, Petersgasse 12, 8010 Graz, Austria b Research Centre Applied Biocatalysis, Petersgasse 14, 8010 Graz, Austria c Graz University of Technology, Institute of Inorganic Chemistry, Stremayrgasse 16/IV, 8010 Graz, Austria d BASF, R-1059.6.07, Klybeckstrasse, 4002 Basel, Switzerland e PROFACTOR GmbH, Functional Surfaces & Nanostructures, Im Stadtgut A2, 4407 Steyr-Gleink, Austria article info Article history: Received 26 February 2010 Received in revised form 18 May 2010 Accepted 19 May 2010 Available online 12 June 2010 Keywords: Alkoxysilane Activated silica gel Cross-linking Laccase substrate Protease substrate abstract Immobilisation of enzyme substrates is a powerful tool in the detection of enzymes in the chemosphere and the environment. A siloxane based strategy for the covalent immobilisation of oxidoreductase and protease substrates was developed involving activation of silica gel and polyethylene terephthalate (PET) as model carriers with (3-aminopropyl)-triethoxysilane or (3-mercaptopropyl)-trimethoxysilane (APTS, MPTS). Ferulic acid and L-Leucine-p-nitroanilide, Gly-Phe p-nitroanilide (GPpNA) and N-Succi- nyl-Ala-Ala-Pro-Leu p-nitroanilide (SAAPLpNA) as laccase and protein substrates, respectively, were covalently attached using glutaraldehyde or carbodiimide based cross-linking strategies. In contrast to conversion in solution, immobilised SAAPLpNA was hydrolysed much faster by protease than immobi- lised GPpNA indicating steric hindrance with decreasing chain length between point of attachment and site of enzyme attack. Immobilised ferulic acid was oxidised by laccase both in case of MPTS and APTS-modified silica gel giving clearly visible colour changes with DE values of 7.2 and 2.3, respectively after 24 h of incubation, where DE describes the distance between two colours. Similarly, clearly visible colour changes with a DE value of 8.6 were seen after laccase treatment of ferulic acid immobilised on APTS activated PET as carrier. Limited surface hydrolysis of PET with a cutinase enhanced coupling of APTS and ferulic acid due to a larger number of hydroxyl groups available on the surface and conse- quently led to a higher colour difference of DE = 12.2 after laccase oxidation. The covalent coupling product between ferulic acid and 1,3-bis(3-aminopropyl)-1,1,3,3-tetramethyldisiloxane was identified by LC–MS (M + 1 m/z 601) and successfully oxidised with laccase. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Biosensors based on enzymes immobilised on carrier materi- als are widely used. Laccase and tyrosinase were immobilised on carbon-fiber electrodes or chitosan films to detect phenolic compounds in agricultural or industrial waters (Freire et al., 2001, 2002; Portaccio et al., 2006; Abdullah et al., 2007). On the other hand, immobilisation of enzyme substrates is a power- ful tool in the detection of enzymes in the chemosphere. Adsorp- tive immobilisation is not suitable for this purpose since the changing environment in samples (e.g. low/high pH, unknown ion concentration) often leads to uncontrolled desorption and additionally has an influence on enzyme activity. Therefore, cova- lent immobilisation of enzyme substrates is necessary. Protease substrates were covalently immobilised for the real-time detec- tion of airborne protease in the detergent industry (Tang et al., 1995). This biosensor is based on immobilisation of fluorescein on porcine thyroglobulin which is itself immobilised onto glass beads. Upon contact with proteases, the fluorescent substrate is released to the solution which is continuously pumped through a spectrophotometer equipped with a flow cell. Such methods do not only allow simple and automated handling but also save costs of expensive protease substrates in long term continuous operation, in contrast to the use of soluble substrates (i.e. in the absence of protease no substrate is released). Similarly the detection of protease in human lung mast cells with the use of benzoyl-DL-arginine-p-nitroaniline as a substrate was described by Lavens et al. (1993). The activity of various proteases includ- ing elastase and cathepsin increases upon infection of wounds (Weckroth et al., 1996). Thus, a simple test for these activities suitable for home-care use (e.g. integrated in bandages) could indicate the need for medical treatment (antibiotics). Again immobilisation of specific enzyme substrates would be required. 0045-6535/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2010.05.022 * Corresponding author at: Graz University of Technology, Dept. of Environmen- tal Biotechnology, Petersgasse 12, 8010 Graz, Austria. Tel.: +43 3168738312; fax: +43 3168738815. E-mail address: guebitz@tugraz.at (G.M. Guebitz). Chemosphere 80 (2010) 922–928 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere