Although the original function of HbHNL is to produce HCN from acetone cyanohydrins for plant defense purpose, its reversible reaction to produce enantiopure cyanohydrins makes it an industrial relevant enzyme (Fig. 1.6a). Besides acetone, it is also able to take a bigger substrate such as benzaldehyde and turn it into mandelonitrile with optical purity up to 99%.15 HbHNL is known to turn different aliphatic or aromatic aldehydes and methyl ketones into different cyanohydrins.15 Cyanohydrins are useful precursors to agrochemicals and pharmaceutical products. For example, (R)-2-chlorobenzaldehyde cyanohydrin is the precursor of Clopidogel (Plavix), which is a blood clot inhibitor.16 Pyrethroids, made from (S)-phenoxybenzaldehyde cyanohydrins, are an important synthesis route to insecticides.16 HbHNL catalyzes an unnatural reaction, nitro aldol reaction (Fig. 1.6b).17,18 The nitro-aldol reaction is an important industrial reaction as the enantiopure products could potentially be the precursors to useful intermediate such as 1,2-aminoalcohols and α-hydroxycarboxylic acids17. My overarching goals of this project is to: 1) improve the nitro-aldol activity of HbHNL and HNL1, a reconstructed ancestral enzyme of HbHNL by using directed evolution; 2) determine which enzyme is more capable of producing better nitro-aldolase by using suitable statistical analysis.
HbHNL and MeHNL share 77% sequence identity. HNL1 was reconstructed from these two enzymes along with other modern