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Issue 11, 2007
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Exploring enzymatic catalysis at a solid surface: a case study with transglutaminase-mediated protein immobilization

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Abstract

The factors affecting enzymatic protein immobilization with microbial transglutaminase (MTG) were explored. As model proteins, enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST) were chosen and tagged with a neutral Gln-donor substrate peptide for MTG (Leu-Leu-Gln-Gly, LLQG-tag) at their C-terminus. To create a specific surface, displaying reactive Lys residues, to be cross-linked with the Gln residue in the LLQG-tag of target proteins by MTG catalysis, a polystyrene surface was physically coated with β-casein. Both recombinant proteins were immobilized onto the β-casein-coated surface only in the presence of active MTG, indicating that those proteins were enzymatically immobilized to the surface. MTG-mediated protein immobilization markedly depends on the pH and ionic strength of the reaction media. The optimal pH range of MTG-mediated immobilization of both recombinant proteins was around 5, at which point the MTG-catalyzed reaction in aqueous solution is not normally preferred. By utilizing a pH-dependent change in EGFP fluorescence, we found that the apparent pH at the surface is likely to be lower than bulk pH, this difference is not attributed to an optimal pH shift in MTG-mediated immobilization. On the other hand, lower yields of protein immobilization at higher ionic strength suggest that electrostatic interaction is a key factor governing MTG catalysis at a solid surface. The results of this study indicate that, in enzymatic catalysis at a solid surface, the concentration of substrates at the surface can enhance the catalytic efficiency, and this could alter the pH dependence of enzymatic catalysis.

Graphical abstract: Exploring enzymatic catalysis at a solid surface: a case study with transglutaminase-mediated protein immobilization

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Publication details

The article was received on 01 Feb 2007, accepted on 20 Apr 2007 and first published on 09 May 2007


Article type: Paper
DOI: 10.1039/B701595J
Citation: Org. Biomol. Chem., 2007,5, 1764-1770
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    Exploring enzymatic catalysis at a solid surface: a case study with transglutaminase-mediated protein immobilization

    Y. Tanaka, Y. Tsuruda, M. Nishi, N. Kamiya and M. Goto, Org. Biomol. Chem., 2007, 5, 1764
    DOI: 10.1039/B701595J

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