Towards heterogeneous biocatalysis of glutathione production by selective conjugation of enzymes

Many value-added specialty chemicals are produced using biological processes. While these processes are efficient, safe and environmentally friendly; they are fragile and sensitive to changes in environmental parameters such as pH and temperature. Enzyme immobilization may alleviate these concerns by stabilizing the proteins and allow for their convenient recycling. However, immobilization by conventional methods risks enzyme inactivation due to lack of control over the coupling chemistry. Creation of catalytic biomaterials / particles by precise conjugation may solve the mentioned issues. In this project, significant progress was made toward such a proof-of-concept catalytic biomaterial. The concept comprises a PEG-based scaffold, to which biosynthetic enzymes would be coupled that contain uniquely reactive non-natural amino acids (nnAAs). For this purpose, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) enzymes were used, which catalyze the biosynthesis of the high-value antioxidant glutathione (GSH). GCL and GS enzymes were produced by cell-free protein synthesis (CFPS), and modified by site-specific incorporation of the Click-compatible nnAA para-propargyloxy-phenylalanine (pPaF). Various parameters related to CFPS and the addition of exogenous proteins such as chaperones were studied to improve modified protein yields. The activities of the modified enzymes compared to those of their wild-type counterparts, and the modified enzymes retained 80% of their activity. Finally, modified green fluorescent protein (GFP) was used to study PEG coupling via Click chemistry. In the second part of the thesis, production of the biological anti-inflammatory drug anakinra was studied towards use in treating Covid-19-related cytokine storm. Anakinra is a licensed biological drug used in the treatment of various inflammatory diseases such as Behçet's disease and familial Mediterranean fever (FMF). It is obtained by recombinant production of human interleukin 1 receptor antagonist (IL-1Ra) protein in bacterial cultures. In a part of the study, expression of anakinra under different promoters (T7, tac and cspA) in Escherichia coli was tested. After production and purification, the pure and intact protein was shown to have comparable biological affinity, activity, purity and safety as the commercially available drug. Optimal process conditions were identified for anakinra production to be transferred to large-scale manufacturing.