pH-Stability and Thermal Properties of Microbial Transglutaminase-Treated Whey Protein Isolate

Abstract

Whey protein isolate (WPI) was treated to various extents using microbial transglutaminase (MTGase) and changes in pH-stability and thermal stability of its protein components were investigated. The MTGase treatment significantly increased the denaturation temperature (T(d)) of beta-lactoglobulin in WPI, from 71.84 degrees C in the untreated sample to 78.50 degrees C after 30 h of incubation with MTGase. The enthalpy change of denaturation of WPI did not change upon cross-linking, indicating that the increase in T(d) was primarily due to covalent cross-linking and not due to an increase in nonpolar interactions within the protein. The surface hydrophobicity (S(o)) of the protein decreased upon cross-linking; however, this decrease was not due to burial of the surface hydrophobic cavities in the protein interior, but due to occlusion of the hydrophobic cavities to the fluorescent probes. Fluorescence emission and circular dichroism spectroscopic analyses revealed no major changes in the secondary and tertiary conformations as a result of cross-linking. However, unlike native WPI, the cross-linked protein exhibited a U-shaped pH-stability profile with maximum turbidity at pH 4.0-4.5. The study revealed that even though enzymatic cross-linking significantly improved the T(d) of beta-lactoglobulin in WPI without causing major structural changes, a reduction in the hydrophilic-hydrophobic balance of the protein surface as a result of elimination of the positive charge on lysyl residues caused precipitation at pH 4.0-4.5.

Effect of microbial transglutaminase treatment on thermal stability and pH-solubility of heat-shocked whey protein isolate

“…Comparison of the results of the present study with those of the previous study (Agyare & Damodaran, 2010) indicate that conformational changes occurring in whey proteins under mild heat-shock conditions render them a much better substrate for MTGase.…”

“…Previously, it has been reported that the rate and extent of MTGase-catalyzed polymerization of a-la and b-lg in native WPI was very low over a reaction time of 44 h (Agyare & Damodaran, 2010). Comparison of the results of the present study with those of the previous study (Agyare & Damodaran, 2010) indicate that conformational changes occurring in whey proteins under mild heat-shock conditions render them a much better substrate for MTGase.…”

“…MTGase treatment was performed as outlined in our previous report (Agyare & Damodaran, 2010). Specifically, heat-shocked WPI dispersion (5% protein) was incubated with MTGase (50 U/g of protein substrate) at 37 C for various times (designated as MTGase-treated WPI).…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of protein samples under reducing conditions was performed as described previously (Agyare & Damodaran, 2010) using a 12% acrylamide separating and a 4% acrylamide stacking slab gel. Protein solutions (0.2% protein) were dissolved in an equalvolume of sample buffer (4% SDS, 20% glycerol, 10% b-mercaptoethanol, 0.125 M Tris, pH 6.8).…”

“…The inter-molecular cross-linking, which increases the weight average molecular weight of the protein, may improve the gelation and emulsifying properties (Liu & Damodaran, 1999;Wang & Damodaran, 1990) and the thermal stability of proteins. A few studies have reported that transglutaminase-mediated cross-linking of native b-lactoglobulin enhanced its thermal stability (Agyare & Damodaran, 2010;Tang & Ma, 2007;Tanimoto & Kinsella, 1988); however, it was not clear whether the improved thermal stability was due to intra-or inter-molecular cross-linking. Globular proteins in the native state are less susceptible substrates to transglutaminase action and require partial or complete unfolding to undergo enzymatic cross-linking (Han & Damodaran, 1996).…”