title: Covalent immobilisation of protease and laccase substrates onto siloxanes.
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-Succinyl-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 immobilised 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 Delta E values of 7.2 and 2.3, respectively after 24h of incubation, where Delta E describes the distance between two colours. Similarly, clearly visible colour changes with a Delta E 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 consequently led to a higher colour difference of Delta E=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+1m/z601) and successfully oxidised with laccase.
authors.affiliation: Graz University of Technology, Department of Environmental Biotechnology, Petersgasse 12, 8010 Graz, Austria.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Rollett
authors.forename: Marc
authors.initials: M
authors.lastname: Schroeder
authors.forename: Konstantin P
authors.initials: KP
authors.lastname: Schneider
authors.forename: Roland
authors.initials: R
authors.lastname: Fischer
authors.forename: Franz
authors.initials: F
authors.lastname: Kaufmann
authors.forename: Rainer
authors.initials: R
authors.lastname: Schöftner
authors.forename: Georg M
authors.initials: GM
authors.lastname: Guebitz
mesh_terms: D000813:Anilides / Q000737:chemistry; D003373:Coumaric Acids / Q000737:chemistry; D004151:Dipeptides / Q000737:chemistry; D004784:Environmental Monitoring / Q000379:methods*; D007700:Kinetics; D042845:Laccase / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D011093:Polyethylene Terephthalates / Q000737:chemistry; D011437:Propylamines; D012821:Silanes / Q000737:chemistry; D012833:Siloxanes / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D000813:Anilides; D003373:Coumaric Acids; D004151:Dipeptides; D011093:Polyethylene Terephthalates; D011437:Propylamines; D012821:Silanes; D012833:Siloxanes; C001536:1-leucine-4-nitroanilide; C029660:glycylphenylalanine; C004999:ferulic acid; D042845:Laccase; D010447:Peptide Hydrolases; C477625:amino-propyl-triethoxysilane