Covalent immobilisation of protease and laccase substrates onto siloxanes.

Graz University of Technology, Department of Environmental Biotechnology, Petersgasse 12, 8010 Graz, Austria.
Chemosphere (Impact Factor: 3.14). 08/2010; 80(8):922-8. DOI:10.1016/j.chemosphere.2010.05.022
Source: PubMed

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.

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    ABSTRACT: The aim of this work was to assess the possibility of using beer spent grain (a byproduct of beer's brewing industry) as a carrier for laccase immobilization. Both adsorption (on spent grain – SG and on digested spent grain – DSG) and covalent binding (using glycidol and glycidol followed by ethylenediamine on DSG) were used. The effect of different immobilization conditions on the immobilization yields and recovered activities such as contact time, enzyme concentration and pH was evaluated. For the best conditions, immobilization yields, recovered activities and thermal, operational and storage stabilities were also evaluated. Finally, the Michaelis–Menten mechanism was applied and the parameter with respect to ABTS oxidation was determined. Enzyme immobilization on DSG led to the best enzyme activities (recovered activities as high as 90%) and to high storage and operational stabilities (10 cycles). Thermal stability was also improved and the half-life of immobilized laccase in SG increased from 0.64 h to 1.1 h at 70 • C.
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