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

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...

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Bibliographic Details
Published in:Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2008-06, Vol.72 (6), p.1457-1463
Main Authors: SEMBA, Hisashi, DOBASHI, Yukio, MATSUI, Toru
Format: Article
Language:English
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
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Summary: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.
ISSN:0916-8451
1347-6947
1347-6947
DOI:10.1271/bbb.70765