Functionalized Graphene Oxide In Enzyme Engineering

How functionalized GO interacts with bioactive proteins such as enzymes, and itspotential in enzyme engineering has been rarely explored. In this thesis, we carefullyinvestigated the interactions between serine proteases and GO functionalized withdifferent amine terminated polyethylene glycols (PEG).First，three well characterized serine proteases (trypsin, chymotrypsin, andproteinase K) with important biomedical and industrial applications were analyzed. It isfound that these PEGylated GO could selectively improve trypsin activity andthermostability (70%retained activity at80°C), while exhibiting barely any effect onchymotrypsin or proteinase K. Detailed investigation illustrated that the improvedtrypsin activity was substrate-dependent, affecting only phosphorylated proteinsubstrates. Based on the fact that addition of GO-10k-6br-PEG-NH2in trypsinproteolysis reactions can selectively accelerate the digestion of phosphoproteins， asimple and quick estimation method of phosphoprotein contents in mixed proteinsamples was explored.We then started to explore the mechanism of trypsin activity stimulation. Detailedinvestigation illustrated that the activity improvement was attributed to both theterminal amines on polymer coatings and the2-D structure of GO. Enzyme kineticsdemonstrated that trypsin-catalyzed hydrolysis of casein might fit the model of substrateinhibition，and PEGylated2-D GO nanosheets with terminal amines could facilitate theinitial recognition step likely through their interactions with both trypsin as well as thesubstrate and later intermediates.This is the first success of using functionalized GO as enzyme positive modulatorwith great selectivity, exhibiting a novel potential of GO, when appropriatelyfunctionalized, in enzyme engineering as well as enzyme-based biosensing anddetection.