Yoshiyuki Kumazawa

Enhanced susceptibility to transglutaminase reaction of α-lactalbumin in the molten globule state

The susceptibility of alpha-lactalbumin to transglutaminase reactions was studied using an enzyme from Streptoverticillium which can catalyze the reactions irrespective of the presence or absence of Ca2+. Transglutaminase-catalyzed polymerization of alpha-lactalbumin in the native state occurred to a very limited extent. Transformation from the native state to the molten globule state brought about by Ca(2+)-removal from holo-alpha-lactalbumin enhanced the polymerization of the protein catalyzed by transglutaminase. The incorporation of Carbobenzoxy-Gln-Gly into alpha-lactalbumin through the enzyme reaction was investigated to determine the amounts of lysine residues which are present at molecular surface and available to the enzyme. There was no significant difference in the amount of available lysine residues between the native and the molten globule molecule. However, the amount of surface glutamine residues incorporated with monodansylcadaverine by transglutaminase was remarkably higher in the molten globule state than that in the native state. The monodansylcadaverine-incorporated site of alpha-lactalbumin in the molten globule state was identified as Gln-54 by amino-acid sequence analysis of fluorescence-labeled peptides separated from chymotryptic digests of the protein. Possible reason for selective labeling of Gln-54 in molten globule alpha-lactalbumin was proposed.

Microbial transglutaminase treatment in pasta-production does not affect the immunoreactivity of gliadin with celiac disease patients' sera.

The effect of microbial transglutaminase (MTG)-treatment of pasta-dough on the immunoreactivity with celiac disease patients sera has been investigated. Modification by MTG has been proven by determination of the MTG reaction product ε-(γ-glutamyl)lysine (3.63 μmol/g protein), which was not detectable in non-MTG-treated pasta. Antigenicity has been analyzed by immunoblotting and ELISA using gliadin-extracts from pasta and MTG-treated pasta. Immunoblotting showed that the antibody-population (antigliadin antibodies and antideamidated gliadin antibodies) of the sera is specific for every individual patient. Immunoblotting and ELISA showed that there is no difference in immunoreactivity of gliadin extracted from pasta and MTG-pasta. Recognition pattern and intensity in Western blot as well as antibody titer has also been identical even for sera with a high antideamidated gliadin antibody titer. These results indicate no difference between pasta-gliadin and MTG-pasta-gliadin and especially no increased deamidation in pasta-gliadin by MTG-treatment.

Controlled Cross-Linking with Glucose Oxidase for the Enhancement of Gelling Potential of Pork Myofibrillar Protein

Differential oxidative modifications of myofibrillar protein (MP) by hydroxyl radicals generated in an enzymatic system with glucose oxidase (GluOx) in the presence of glucose/FeSO4 versus a Fenton system (H2O2/FeSO4) were investigated. Pork MP was modified at 4 °C and pH 6.25 with hydroxyl radicals produced from 1 mg/mL glucose in the presence of 80, 160, or 320 μg/mL GluOx and 10 μM FeSO4. Total sulfhydryl content, solubility, cross-linking pattern, and gelation properties of MP were measured. H2O2 production proceeded linearly with the concentration of GluOx and increased with reaction time. GluOx- and H2O2-dose-dependent protein polymerization, evidenced by faded myosin heavy chain and actin in SDS-PAGE as well as significant decreases in sulfhydryls, coincided with protein solubility loss. Firmer and more elastic MP gels were produced by GluOx than by the Fenton system at comparable H2O2 levels due to an altered radical reaction pathway.

Microbial Transglutaminase Used in Bread Preparation at Standard Bakery Concentrations Does Not Increase Immunodetectable Amounts of Deamidated Gliadin

The effect of standard bakery concentrations of microbial transglutaminase (MTG) in wheat bread preparation on the immunoreactivity of sera of celiac disease (CD) patients was investigated. Immunoblotting using monoclonal antibodies specific to unmodified and/or deamidated gliadin showed no differences between control bread and MTG bread. Deamidation of gliadin could not be detected at standard MTG concentrations. Sera of CD patients were characterized using anti-gliadin and anti-deamidated gliadin peptide (DGP) enzyme-linked immunosorbent assay and grouped into DGP high- and low-titer pools. The recognition pattern obtained after using both CD sera pools for immunoblotting did not reveal differences between control and MTG-treated bread protein extracts. Our results indicate that MTG treatment of wheat bread prepared with typical MTG concentrations used in standard bakery processes does not lead to immunodetectable amounts of CD immunotoxic deamidated gliadins.