Expression of hydroxynitrile lyase from Manihot esculenta in yeast and its application in (S)-mandelonitrile production using an immobilized enzyme reactor

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Abstract

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.

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Semba, H., Dobashi, Y., & Matsui, T. (2008). Expression of hydroxynitrile lyase from Manihot esculenta in yeast and its application in (S)-mandelonitrile production using an immobilized enzyme reactor. Bioscience, Biotechnology and Biochemistry, 72(6), 1457–1463. https://doi.org/10.1271/bbb.70765

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