mutants of micromonospora viridifaciens sialidase have highly variable activities on natural and nonnatural substrates this study aimed to improve the hydrolase activity of the wellcharacterised bacterial sialidase from micromonospora viridifaciens the enzyme and its mutated versions were produced in bacillus subtilis and secreted to the growth medium twenty amino acid positions in or near the active site were subjected to sitesaturation mutagenesis and evaluated on the artificial sialidase substrate 2opnitrophenyldnacetylneuraminic acid and on the natural substrate casein glycomacropeptide a considerably higher fraction of the mutants exhibited increased activity on the artificial substrate compared with the natural one with the most proficient mutant showing a 13fold improvement in kcatkm in contrast no mutants displayed more than a 2fold increase in activity on the natural substrate to gain further insight into this important discrepancy we analysed the stability of mutants using the popmusic software a property that also correlates with the potential for introducing chemical variation after validating the method with a set of experimental stability estimates we found a significant correlation between improved hydrolase activity on the artificial substrate and reduced apparent stability together with the minor improvement on the natural substrate this shows an important difference between naturally evolved functionality and new laboratory functionality our results suggest that when engineering sialidases and potentially other proteins towards nonnatural substrates that are not optimized by natural evolution major changes in chemical properties are advantageous and these changes tend to correlate with decreased stability partly explaining commonly observed tradeoffs between stability and proficiency