Lim, Huey Yee2016-09-192016-09-192016-05https://hdl.handle.net/11299/182109University of Minnesota M.S. thesis. May 2016. Major: Microbial Engineering. Advisor: Romas Kazlauskas. 1 computer file (PDF); viii, 78 pages.Enzymes in superfamilies such as the α/β-hydrolase fold superfamily have similar structures and active sites because they evolved from a common ancestor. Since enzymes within a superfamily catalyze different reactions, the common ancestor may have been a promiscuous catalyst. Our hypothesis is that promiscuous ancestral enzymes may be better starting points to obtain new catalytic activities compared to modern enzymes. To test this hypothesis, I tried to improve the promiscuous and unnatural nitroaldol activity in both a modern and an ancestral hydroxynitrile lyase using semi rational and random approaches. My results showed that the ancestral enzyme was not a better starting point compared to the modern enzyme after the modern enzyme was stabilized by a single mutation.enancestral enzymeHNL activityhydroxynitrile lyaseNitroaldol activitypromiscuous reactionProtein engineeringThesis or Dissertation