Determination of Transglutaminase Activity Using Fluorescence Spectrophotometer

An improved fluorometric assay for determining the activity of the microbial transglutaminase (TGase) in the culture medium samples has been developed. The assay procedure measures the fluorescence enhancement due to the incorporation of monodansyl cadaverine (Substrate A) into pentafluorophenyleste...

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Bibliographic Details
Published in:Food biotechnology 2008-07, Vol.22 (3-4), p.297-310
Main Authors: Sokullu, Esen, Bas, Deniz, Boyacı, Ismail Hakkı, Oner, Zubeyde, Karahan, Aynur Gul, Cakir, Ibrahim, Cakmakci, M. Lutfu
Format: Article
Language:English
Subjects:
detection
enzyme activity
enzyme substrates
fluorescence
fluorescence spectrophotometer
fluorometric assay
food analysis
food composition
protein-glutamine gamma-glutamyltransferase
spectrophotometers
transglutaminase
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Summary:An improved fluorometric assay for determining the activity of the microbial transglutaminase (TGase) in the culture medium samples has been developed. The assay procedure measures the fluorescence enhancement due to the incorporation of monodansyl cadaverine (Substrate A) into pentafluorophenylester of CBZ-Gln-Gly (Substrate Q) at λexc. 260 nm and λem 538 nm. The effect of the competitive inhibitors in the culture medium samples on TGase activity was determined. The assay was combined with HPLC method for determining enzyme activity as an international unit (IU). Enzymatic reaction was monitored by HPLC and the rate of product formation was measured via amine substrate consumption rate. A conversion factor was obtained using HPLC and fluorescence spectrophotometer data together. This was formulated for quantification of TGase activity as IU using fluorometric assay reported in this study. The detection limit of the assay was determined as 0.0014 IU (0.5 mg). TGase activity remained linear upto the enzyme concentraion of 20 mg. This technique dramatically decreases the incubation time of enzyme to a few minutes of activity measurement.
ISSN:0890-5436
1532-4249
DOI:10.1080/08905430802265775