PhD Oral Defence: A cold-active transglutaminase: purification, characterization, expression & application in producing food hydrogels
PhD Oral Defence of Yi Zhang, Food Science & Agricultural Engineering
A 78 kDa Transglutaminase (TGase) was purified from Antarctic krill with specific activity of 53.5 U/mg. The enzyme was Ca2+ dependent and was most active in the temperature range of 0–10o C. Addition of 0.1 U/mg into cold-set gelatin hydrogels improved its textural properties. Thermostability and kinetics studies found the cold-active TGase was stable at 0–40o C but was readily inactivated above 60o C. The corresponding conformational states and structural changes were identified. The gene encoding krill TGase was obtained and the amino acids sequence was deduced. The conserved domains and catalytic triad were predicted from homology modeling. A gelatin high in α-chain was produced as substrate for TGase. Optimized conditions of the alcalase-assisted process produced 32% of α-chain containing 34% glycine and 16% imino acids. The amide absorption bands and thermal decomposition of gelatin were characterized. Antioxidative peptides were also produced as substrates for TGase via proteolysis by alcalase versus trypsin. The identified peptide sequences were further subjected to predict in vivo functions using molecular docking. The cold-active TGase was expressed in E. coli and the recovered enzymes were used for forming cold-set peptides/gelatin hydrogels with improved antioxidant activity.