The aldol reaction between pyruvate and formaldehyde catalyzed by three aldolases, i.e., YfaU WT, YfaU 3 and YfaU 5, was carried out in this work. These are three versions of the enzyme, the first of which is natural, and the remaining two were obtained by protein engineering with the aim of improving the stability properties. As a product, aldol sodium 4-hydroxy-2-oxobutanoate was formed. Sodium pyruvate was a donor substrate and formaldehyde was an acceptor substrate. The experiments were carried out in a batch reactor. The course of the reaction and the operational stability of the three enzymes were monitored during the process. Based on the experimental results, i.e., the change in enzyme activity over time, the enzyme deactivation constants were estimated using the software package Scientist. In addition to different deactivation rates, the results also show different reaction progressions in the reactor. It can be concluded from the results that the activity and operational stability of all three enzymes decreases over time. The most stable aldolase in the batch reactor was YfaU 5, and the least stable was YfaU WT. Thus, it can be concluded that protein engineering led to an improvement of the enzyme stability.