| Asymmetric synthesis with biocatalyst has become an increasingly interesting and cost effective manufacturing process in fine chemicals, pharmaceuticals, and agrochemical intermediates. Enoate reductases from the Old Yellow Enzyme family offer high substrate efficiency, region, stereo-, and enantioselectivity in the catalyzed biotransformations. Asymmetric reduction of activated C=C bond is one of the most widely applied synthetic tools for the potential to generate up to two stereogenic centers in one step reaction. Current existing synthetic methods of asymmetric hydrogenation involve the use of precious metals in conjunction with chiral phosphines as well as organocatalytic hydrogenation of cyclic enones to allow a wider choice of substrates and products. However, the addition of biocatalytic hydrogenation of activated alkenes proved to be highly effective to produce enantiomerically pure compounds and become a precious addition tools for both the established synthetic pathways and novel methods.;The thesis contributed to the development and characterization of the Old Yellow Enzyme family with regards to addition of new chemistry such as exploitation of nitroreductase activity with NRSal from Salmonella typhimurium, where we investigated the reduction of alpha,beta-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatics substrates, and also reported the first single-isolated enzyme-catalyzed reduction of nitrobenzene to aniline through the formation of nitrosobenzene and phenylhydroxylamine as intermediates. The development and characterization of novel enzymes YersER from Yersinia bercoviei and KYE1 from Kluyveromyces lactis showed both enzymes have broad enoate reductase specificity and able to operate under broad temperature and pH optima but different specificity patterns. Both substrate- and enzyme-based stereocontrol were also observed for both YersER and KYE1 to furnish opposite stereoisomeric products. Further improvement and characterization on XenA from Pseudomonas putida showed broad catalytic activity and reduces various alpha,beta-unsaturated and nitro compounds with moderate to excellent stereoselectivity. Mutations of XenA C25G and C25V are able to reduce nitrobenzene, a non-active substrate for the wild type, to produce aniline. The combination of computational protein engineering in form of the Principal Component Analysis method together with structure-guided consensus enabled the improvement of YersER, where significant improvements of specific activity were detected. Lastly, the work also contributed towards larger-scale system preparation by looking into enzyme stability and organic solvent behavior. We studied the total turnover number (TTN) as an indicator of lifetime biocatalyst productivity for YersER, KYE1, and XenA. The TTN of XenA proved to be dominated by chemical rather than thermal instability. Also, the effects of organic solvents on enzyme activity and stereoselectivity were tested with YersER and KYE1 to show the sustainable bioreduction efficiency even at high organic solvent content. Future work will focus to enable sustainable and stable scale-up system to demonstrate the possible applications of these enzymes.;In summary, the increasing knowledge about this Old Yellow Enzyme family together with recent advances in biotechnology renders the enoate reductases a tool of choice for industrial applications. Detailed understanding of the knowledge of Old Yellow Enzyme substrate specificity and stereoselectivity, combined with structural and computational modeling techniques enable the future development and improvement of novel enoate reductase using enzyme engineering approach. |
