Characteristics Of Microbial Transglutaminase Reaction

Transglutaminase transaminase (MTG) is an important enzyme that catalyzes the acyl transfer reaction between a primary amine and the r-carboxyamide group of glutamine residues in peptides and proteins. It has a very wide range of applications in the food industry and feed industry. Wheat gluten has a large number of applications in the food industry. Glutamate and glutamine content is in very high levels in gluten proteins, accounting for about 35% of the total proteins. So wheat gluten can be used as a good substrate of MTG. In this paper, wheat gluten hydrolyzed acts as a substrate to carry out MTG reaction characteristics for the studies and explorations.First, determine the method for analyzing the cross-linking reaction of MTG. And the OPA method was adopted.Second, analyzing the change of the hydrolyzes'molecular weight when MTG was added in. Obtained the change is larger in the part of that molecular weight is more than 3,000Da. And determined the conditions for Trypsin hydrolysis were as follows: 4% degree of hydrolysis, hydrolyze time: 5.5h. Under these conditions, the most part of obtained hydrolyzes'molecular weight is more than 3,000Da.Third, determine the substrate by analyzing the content ofε-amino, change of fluorescence intensity, and surface hydrophobic when the MTG was added in the two different hydrolyzes whose molecular is 3000-5000Da and 5000-30000 Da. And determined the hydrolyses whose molecular weight is 3000-5000Da was the substrate for analyzing the characteristic of MTG. Forth, analyze the differences of characteristic of MTG reaction under different reaction environmental conditions: temperatures, ionic strength and pH. It was obtained that reaction temperature has an obvious effect on MTG-catalyzed cross-linking reaction and hydrolysis reaction. When the reaction temperature is 50℃, the cross-linking reaction and hydrolysis reaction rate are larger than those in 30℃and 4℃. At the temperature of 4℃, the cross-linking reaction began to slow, the rate of cross-linking reaction and hydrolysis reaction is the minimum in the three kinds of temperature conditions.Different concentrations of salt added into the system have an impact on the MTG-catalyzed reaction. When the NaCl concentration is 0.6mol/L, the cross-linking reaction has not changed a lot but the rate hydrolysis reaction reduced. When the NaCl concentration is 0.3mol/L, the cross-linking reaction and hydrolysis reaction rate are larger than whose NaCl concentration is 0.6mol/L but smaller that whose NaCl concentration is 0mol/L.Different reaction pH also has an affect on the MTG catalytic reaction. When under the conditions of PH = 8.5,the time of complete the whole cross-linking reaction is shorter than pH = 7 and pH = 5.5. And when the pH = 5.5 , the time of complete cross-linking reaction is significantly longer and the hydrolysis reaction is also be delayed.When the substrate is the hydrolyzes whose molecular weight is 3000-5000Da, the kinetic parameters and activation energy of cross-linking and hydrolysis reaction MTG-catalyzed were determined. Obtained: cross-linking reaction of Km = 0.010372mmol/min, Vmax = 4.321521 mmol / min hydrolysis reaction Km = 0.201896mmol / L, Vmax = 0.009206 mmol / L.min. And the Ea for Cross-linking reaction is 31.316KJ/mol while the Ea for the hydrolysis reaction is 65.880 KJ / mol.