Affinity of Microbial Transglutaminase to α_(s1)-,β-, and Acid Casein under Atmospheric and High Pressure Conditions

摘要

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 x 10~(-3), 0.8 ± 0.01 x 10~(-3), and 1.3 ± 0.30 x 10~(-3) mmoI/L·min and K_m values of 59 ± 2 x 10~(-3), 64 ± 3 x 10~(-3), and 50 ± 2 x 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 x 10~(-3) mmol/L·min, but, comparable to a competitive inhibition, the K_m value increased to 144 ± 34 x 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.