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.
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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.PROCESS BIOCHEMISTRY 07/2012; 47:1095-1101. · 2.52 Impact Factor
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ABSTRACT: The self assembled monolayers (SAM) have become in the most popular strategy for design and generate surfaces characterizing by specific functional organic groups. The aimed of this work is applied this SAM as optical transducer in biosensors. The techniques, Infrared (in ATR mode) and UV/Vis spectroscopy have been used to study the films generated in each step in the self assembled process. The SAM was generated as follow; first silane group was added to the glass substrate. After that, the substrates were immersed in a solution containing carbomiide group (EDC). Finally the spiropyran 1',3'-Dihydro-8-methoxy-1',3',3'-trimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indole] was attached to functionalized slides. In each process absorbance was analyzed by UV/Vis (270 to 500 nm) and FTIR (650 to 1800 cm-1). In UV, the spectra shows an absorbance band centered at 280 associated to EDC film and a lower intensity band centered at 380 nm associated to spiropyran. In FTIR spectra, the Si-Si and Si-O bond are present below the 1250 cm-1. The EDC film shows very weak bands in the region from 1300 to 1800 cm-1. For the spiropyran film the band associated to the C-N, N-O, C=C, C-H and aromatic ring have a very well defined peaks. Once the transducer bands were detected, it was immersed in glucose solution; the infrared spectral show bands are associated to glucose in the transducer.Proceedings of SPIE - The International Society for Optical Engineering 08/2011; · 0.20 Impact Factor
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ABSTRACT: Bioresponsive polymers (BRPs) allow the detection of potentially pathogenic microorganisms. Here, peptidoglycan and cellulose based hydrogels were constructed with potential for diagnosis of wound infection or, for example, Aspergillosis, respectively. These systems respond to extracellular enzymes from microbes or enzymes secreted from the human immune system in case of infection. Laccases as 'enhanzymes' were incorporated into these devices for signal and stability enhancement when compared to simple dye release based systems. To retain the enhanzymes within the BRPs, they were either PEGylated laccase (Laccase_PEG) to increase size or methacrylated laccase (Laccase_MA) to allow covalent attachment to the polysaccharide matrices. PEGylation of Trametes hirsuta laccase led to a fivefold increase in size to 270kDa according to size exclusion chromatography (SEC). Likewise, successful methacrylation of the laccase was demonstrated by using reversed phase chromatography while SEC analysis proved covalent attachment of the enzyme to the methacrylated polysaccharide matrix. Upon incubation of peptidoglycan based BRPs with fluid from infected wounds, the difference to controls was four times higher for Laccase_PEG based signalling when compared to simple dye release. Similarly, the control signals (i.e. leaching) were considerably reduced in case of Laccase_MA incorporated in crosslinked peptidoglycan (PG) and carboxymethylcellulose (CMC) hydrogels for signalling. In addition, Laccase_MA catalysed colour formation enhanced the signal dramatically with factors between 100- and 600-fold. Laccase_MA was demonstrated to oxidise silica gel immobilised ferulic acid incorporated into the BRP with clearly visible colour changes of 4.5 ΔE units according the CIELab concept upon incubation by trigger enzymes as well as infected wound fluids.New Biotechnology 03/2012; 29(4):502-9. · 2.11 Impact Factor