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 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.
Semba
Hisashi
H
Strategic Technology Research Center, Nippon Shokubai Co., Ltd., Tsukuba, Ibaraki 305-0856, Japan. hisashi_semba@shokubai.co.jp
Dobashi
Yukio
Y
Matsui
Toru
T
D000097:Acetonitriles / Q000378:metabolism*; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism*; D019149:Bioreactors; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D002365:Manihot / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D000097:Acetonitriles; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; C045516:mandelonitrile; D000446:Aldehyde-Lyases; C079359:hydroxymandelonitrile lyase