Low-temperature crosslinking of proteins using non-toxic citric acid in neutral aqueous medium: Mechanism and kinetic study

For the first time, mechanism and kinetics of citric acid crosslinking of protein in neutral aqueous condition under low temperature are reported. Crosslinking was a conventional method to improve properties of protein products to replace petroleum-derived polymers. However, current crosslinking methods were toxic, expensive or required harsh conditions. Non-toxic carboxylic acid could effectively enhance wet performance properties of multiple protein products under mild conditions, which were critical for preserving the activities during immobilization of enzymes. In this study, more than one carboxyl groups in one citric acid molecule were found capable of reacting with protein in aqueous medium at pH 6.8 under 50 and 75°C, verifying occurrence of crosslinking. The pseudo second-order reaction had activation energy as low as 24.956 kJ mol^-1, indicating its easy occurrence. Mild crosslinking using citric acid might provide alternative to accelerate low-cost and efficient enzyme immobilization as well as industrial utilization of protein-based products.