Enzymes are biocatalysts that catalyze reactions in living cells. Their usage provides a number of advantages, such as conducting a reaction in milder conditions and a reduction in the occurrence of harmful by-products. The natural role of halohydrin-dehalogenases is the catalysis of halohydrins to the corresponding epoxides (dehalogenation). Halohydrin-dehalogenase catalyzes the epoxy ring closure in which halogen ions are released. In this work, the reaction of biocatalytic synthesis of (R)-4-chloro-3-hydroxybutyronitrile with type B halohydrin-dehalogenase was investigated. The synthesis of optically pure substances is an extremely important part of the pharmaceutical industry since two enantiomers can have completely different properties and very often one of them can show harmful and toxic biological effects. Epoxides are highly valuable compounds in organic synthesis because the epoxy ring can be chemically transformed into a number of synthetic beneficial intermediates. Reactions involving enzymes need to be carefully investigated and characterized in order to be carried out on an industrial scale. The biocatalytic synthesis of (R)-4-chloro-3-hydroxybutyronitrile was carried out in a batch reactor with sodium cyanide as a nucleophile. The samples were analyzed using gas chromatography. The influence of the concentration of the reaction components on the rate of the enzymatic reaction was tested. Low enzyme affinity for the substrate and nucleophile was observed in reactions with both enzymes. A significant inhibitory effect of the product was also shown. The activity of the enzyme was monitored by spectrophotometer and it was observed that in both enzymes there is a decrease in activity over time.