The mechanisms that drive the evolution of new enzyme activities have been investigated by comparing the kinetics of wildtype and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62 500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wildtype GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that - as so-called generalists - are characterized by their promiscuous activity with many different substrates.