The E. coli ALP was synthesized using PURExpress spiked with the disulfide bond enhancer (New England Biolabs) in a PCR tube for 3 hours at 37°C. The C. marina ALP was synthesized using PURExpress spiked with 100 μM zinc acetate but without the disulfide bond enhancer. Template DNA (150 ng) was added to 15 μl of reaction solution. The resulting solution containing ALP was diluted 106 times with a buffer (Solution I from PUREfrex kit, GeneFrontier). The diluted enzyme solution was mixed with 3 mM 4-MUP (Invitrogen) or 1.5 mM DiFMUP (Invitrogen) in an assay buffer [1 M diethanolamine, 1 mM MgCl2, and 0.05 vol % Tween-20 (pH 9.25)]. The mixture was immediately introduced and sealed into the microchamber array device, so that single enzyme molecules could be loaded stochastically into the microchambers following Poisson distribution. The turnover number, kcat (s−1), was determined with a time-course measurement at room temperature, combining with a calibration curve of 4-MU (Invitrogen) or DiFMU (Invitrogen). The time-course measurement showed a discrete distribution of the hydrolysis rate corresponding to one, two, or three enzyme molecules encapsulated in the droplet. The difference between adjacent slopes in the time-course data was used to extract the increment of the hydrolysis rate resulting from exactly one enzyme molecule.

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