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

摘要

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(1M 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.