Hydroxynitrile lyase from cassava, Manihot esculenta (MeHNL), catalyzes the formation of (S)-cyanohydrins from HCN and aldehydes or ketones. (S)-Mandelonitrile was produced on a bench scale with immobilized MeHNL, after optimizing the enzyme expression system using recombinant technology. MeHNL was cloned from a cDNA library prepared from a leaf of Manihot esculenta, and then expressed in a multi-auxotrophic mutant of Saccharomyces cerevisiae cells. The maximum yield of active MeHNL was obtained by integrating transformation 4 times with a tandemly repeated expression cassette. Silica gel was the most suitable support for immobilization of the prepared enzyme from the recombinant yeast. Using this immobilized enzyme, 22 batches of (S)-mandelonitrile synthesis were performed in a 20 liters bioreactor (1 M benzaldehyde and 1.5 M HCN). During this operation, about 29 kg of (S)-mandelonitrile was produced from 23.3 kg of benzaldehyde, giving 98 mol % yield and a mean enantio excess of 98.9% ee.