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Immobilization of biotinylated transglutaminase by bioselective adsorption to immobilized avidin and characterization of the immobilized activity



Immobilization of biotinylated transglutaminase by bioselective adsorption to immobilized avidin and characterization of the immobilized activity



Journal of Agricultural & Food Chemistry 43(4): 895-901



Transglutaminase was immobilized on a porous glass support by biotinylation followed by adsorption to avidin that had been immobilized by adsorption to the biotinylated aminopropyl glass. Thus, avidin served as a protein spacer between the support and the enzyme. Both the biotinylation of enzyme amino groups and the association of the biotinylated enzyme with soluble avidin caused some loss of enzyme activity. This loss could account for the 3-fold reduction in the specific activity of the immobilized enzyme with carbobenzoxyglutaminylglycine and hydroxylamine as substrates. However, with alpha-s-casein as a substrate, a 24-fold reduction in the k-cat value was observed, implying that the rate of reaction with this large substrate molecule was limited by mass transfer. The pH optima and temperature dependences of enzyme catalysis were similar for both the soluble and immobilized enzyme, although the slight differences observed for the immobilized form were also indicative of mass transfer effects. A bimodal pH activity profile with optima at pH 6.5 and 7.5 was obtained with both enzyme forms. Treatment of alpha-s-casein with immobilized enzyme caused a rapid disappearance of the monomeric protein with concomitant appearance of dimers and higher cross-linked polymers.

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Accession: 002634497

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DOI: 10.1021/jf00052a009


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