Literature on the effects of microbial transglutaminase on various dairy‐based systems is discussed. Beginning with a short synopsis on the development of microbial transglutaminase as a functional tool for modifying foods, the principles of reactions catalyzed by transglutaminase and their structural implications, as well as the mechanisms of formation and cleavage of isopeptide bonds are reviewed. After summarizing the present knowledge on the specificity of microbial transglutaminase towards milk proteins, including reactions determined by individual lysine and glutamine residues, emphasis is placed on the effects of enzymatic cross‐linking on physicochemical properties in foods and, particularly, dairy‐based systems. Discussed are implications of cross‐linking on acidified milk gels including yogurt and effects on single milk protein fractions, with respect to several physicochemical properties including rheology and mechanical properties of these systems, but also syneresis, and emulsification behaviour.

The influence of enzymatic crosslinking by microbial transglutaminase (mTG) on the stability of casein micelles of ultrahigh temperature (UHT)-treated milk in the presence of EDTA (0-0.45 mM) or ethanol (0-74 vol%) as well as under high hydrostatic pressures up to 400 MPa was investigated. Disintegration of micelles and changes in micelle size were monitored by the measurement of turbidity as well as by dynamic light scattering. The results show that the incubation of UHTtreated milk with mTG resulted in an improved micelle stability toward disintegration on addition of EDTA, ethanol, or pressure treatment. Intramicellar formed isopetides significantly enhanced the stability of casein micelles. It is supposed that net-like crosslinks are formed within the external region of the micelles and they adopt the stabilizing role of colloidal calcium phosphate within the micelles, thus making the micelles less contestable for disrupting influences.

Microbial transglutaminase (mTG) mediated modification of bovine beta-lactoglobulin (bLG) at ambient and high hydrostatic pressure was investigated in order to characterize preferred sites of the crosslinking reaction by identifying reactive glutamine residues. bLG was labeled with triglycine (GGG) by incubation with mTG at ambient pressure or at 400 MPa, respectively, and was subjected to an enzymatic digestion with trypsin. The resulting peptides were separated and those containing glutamine residues modified with GGG were unambiguously identified using RP-HPLC with ESI-TOF-MS. For bLG treated with mTG at ambient pressure for 1 h at 40 degrees C, no labeling was observed, thus confirming that the native protein is no substrate for mTG. After incubation of the protein with mTG at 400 MPa for 1 h at 40 degrees C, four out of nine glutamine residues, namely at positions 5, 13, 35, and 59 were identified as accessible for the mTG catalyzed reaction, indicating partial unfolding of bLG under pressure and exposure of previously unaccesible glutamine residues. Thus, only a limited number of glutamine residues were substrates for mTG, which points to a pronounced substrate specificity of mTG toward individual glutamine residues within a protein.

Kinetics for the reaction of microbial transglutaminase (MTG) with individual caseins in a TRIS-acetate buffer at pH 6.0 was evaluated under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40 °C. The reaction was monitored under the following limitations: The kinetics from the initial velocities was obtained from nonprogressive enzymatic reactions assuming that the individual catalytic constants of reactive glutamine residues are represented by the reaction between MTG and casein monomers. Enzyme reaction kinetics carried out at 0.1 MPa at 40 °C showed Henri-Michaelis-Menten behavior with maximal velocities of 2.7 ± 0.02 × 10(-3), 0.8 ± 0.01 × 10(-3), and 1.3 ± 0.30 × 10(-3) mmol/L · min and K(m) values of 59 ± 2 × 10(-3), 64 ± 3 × 10(-3), and 50 ± 2 × 10(-3) mmol/L for β-, α(s1)-, and acid casein, respectively. Enzyme reaction kinetics of β-casein carried out at 400 MPa and 40 °C also showed a Henri-Michaelis-Menten behavior with a similar maximal velocity of 2.5 ± 0.33 × 10(-3) mmol/L · min, but, comparable to a competitive inhibition, the K(m) value increased to 144 ± 34 × 10(-3) mmol/L. The reaction of MTG with α(s1)-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics, indicating the complex influence of pressure on protein-enzyme interactions.

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