Expression of Hydroxynitrile Lyase from Manihot esculenta in Yeast and Its Application in (S)-Mandelonitrile Production Using an Immobilized Enzyme Reactor

• Published in: Bioscience, biotechnology, and biochemistry Vol. 72; no. 6; pp. 1457 - 1463
• Main Authors: SEMBA, Hisashi, DOBASHI, Yukio, MATSUI, Toru
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.06.2008; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Acetonitriles - metabolism; Aldehyde-Lyases - genetics; Aldehyde-Lyases - metabolism; Biological and medical sciences; Bioreactors; Biotechnology; cyanohydrin; Enzymes, Immobilized - genetics; Enzymes, Immobilized - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression; General aspects; Genetic Vectors - genetics; hydroxynitrile lyase; immobilization; Immobilization of enzymes and other molecules; Immobilization techniques; mandelonitrile; Manihot - enzymology; Manihot - genetics; Manihot esculenta; MeHNL; Methods. Procedures. Technologies; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; MeHNL; Yeast; Enzyme; Manihot esculenta; mandelonitrile; Immobilization; Lyases; Cyanohydrin; Dicotyledones; Angiospermae; Production; Euphorbiaceae; Spermatophyta; Reactor; Application; Immobilized enzyme; hydroxynitrile lyase
• ISSN: 0916-8451; 1347-6947; 1347-6947
• DOI: 10.1271/bbb.70765

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.