Title : Catalase enhanced using two green processes
Abstract:
Catalase (CAT) is a very strong natural antioxidant that metabolizes hydrogen peroxide, an important reactive oxygen species. CAT is used to treat some medical conditions, is used in the food and the textile industries to remove peroxide, and in cosmetic formulations for the skin targeting increased cellular oxygenation and mitigation of ageing effects. Catalase is a tetramer, each unit containing over 500 amino acids and a heme (prosthetic group). Because it is inactivated at high, toxic, non-physiological levels of peroxide, it is difficult to study this enzyme, due to low sensitivity of assays and/or enzyme inactivation. We report here 2 green methods to enhance and stabilize CAT. Being an oxidoreductase, CAT is involved in redox reactions with its prosthetic groups being the centers where electron transfer takes place. Both methods reported herein target the enhancement of the kinetics of electron transfer by increasing the number of effective redox centers within the 3-D structure of CAT. They are both green methods. In the first method, high hydraulic pressure was used to unfold the enzyme in the presence of different modifiers. Upon removal of the high pressure, modifier molecules were entrapped in the renatured structure of catalase. In the second procedure an ionic liquid (IL, green reagent) was used as a partially reversible denaturant of CAT with/without other redox molecules present. After the removal of the IL by dialysis, CAT refolded, entrapping some of the redox units present within its 3-D structure. In both procedures species relevant to CAT were chosen as redox modifiers: Fe2+, NADH, heme/modified hemes, lipoic acid making the green procedures reported herein enzyme-friendly as well. All modified CAT retained activity and were characterized by FTIR, modifier entrapment, and antioxidant activity. The most active samples were tested in biosensors with potential applications in the food, pharmaceutical and cosmetic industries. The reported procedures afforded self-mediated CAT which displayed catalytic effect and linearity in peroxide concentration. The performance of the modified catalases recommends these environmentally and enzyme-friendly procedures for applications to the enhancement of other redox enzymes.
Audience take away:
- This research could be included in several courses: green chemistry, protein chemistry, analytical chemistry
- For researchers in the field of amperometric biosensors, the methods presented herein offer green alternatives to enzyme “wiring” (for which toxic, expensive reagents are used at the present)
- These procedures replace labor-intensive enzyme conjugation with simple environmentand enzyme-friendly procedures.
