Affinity of Microbial Transglutaminase to α_s1-, β-, and Acid Casein under Atmospheric and High Pressure Conditions

Abstract

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.

Influence of Microbial Transglutaminase on Physicochemical and Cross-Linking Characteristics of Individual Caseins

“…To determine the values of the kinetic parameters (V max , K m , and k ca ) , we plotted linear regressions results using the Hanes diagrams developed by Menéndez et al [30]. The MTGase-catalyzed reaction with individual caseins was investigated at various substrate concentrations (from 2 to 12 mg/mL).…”

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

“…SEC column materials differ in composition and pore volume. The column most frequently used for studying cross-linked casein is Superdex 200, e.g., [30,44,47,48,54,100,115,131,[134][135][136][137][138][139][140][141][142][143][144][145][146][147][148], which is provided by GE Healthcare (Uppsala, Sweden). The column material is based on agarose/dextran with a ratio of pore volume to void volume of V i /V o = 1.7 and exhibits a fractionation range from 10 to 600 kg/mol for globular proteins [128].…”

“…Despite the numerous reaction sides of casein, Menéndez et al [140] assumed TGase-catalysed cross-linking to be a single substrate reaction of monomers into polymers at short incubation times (<15 min) and fitted their data to Michaelis-Menten enzyme kinetic (see [151]). The kinetic data revealed that maximum reaction velocity is highest for cross-linking of β-casein, followed by non-micellar acid casein and α S1 -casein; however, the enzyme had the highest affinity to acid casein, which was explained by the presence of κ-casein.…”

Significantly Improving the Thermostability and Catalytic Efficiency of Streptomyces mobaraenesis Transglutaminase through Combined Rational Design

“…41 The catalytic activities of TGm1 variants toward proteins were examined using β-casein (Macklin, Shanghai, China) as the substrate according to a previous study. 42 β-Casein was diluted to 3 mg/mL with the Tris-acetate buffer (100 mM, pH 7) containing 10 mM NaCl. The catalytic reaction at 40 °C was initiated by adding each TGm1 variant at a final concentration of 0.005 mg/mL.…”