Hisashi Semba

Stereospecific degradation of phenylsuccinate by actinomycetes.

Racemic phenylsuccinate was stereospecifically degraded by the actinomycetes PS9 and PS17 isolated from soil obtained from Okinawa Island, Japan. Strain PS9, identified as a Citricoccus sp., preferentially degraded the R-form, while strain PS17, identified as a Microbacterium sp., preferentially degraded the S-form of phenylsuccinate. Analysis of the culture broths of these species with phenylsuccinate as the sole carbon source revealed that benzoic acid was produced as a metabolic intermediate. Benzoic acid was further degraded by strain PS9 with m- and/or p-hydroxybenzoic acid but not o-hydroxybenzoic acid as possible intermediates.

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.

Characterization of a novel hydroxynitrile lyase from Nandina domestica Thunb

ABSTRACT The leaves of Nandina domestica Thunb. exhibited high hydroxynitrile lyase (HNL) activity in (R)-mandelonitrile synthesis. The specific activity of young leaves was significantly higher than that of mature leaves. We isolated two HNLs with molecular mass of 24.9 kDa (NdHNL-S) and 28.0 kDa (NdHNL-L) from the young leaves. Both NdHNLs were composed of two identical subunits, without FAD and carbohydrates. We purified NdHNL-L and revealed its enzymatic properties. The whole deduced amino acid sequence of NdHNL-L was not homologous to any other HNLs, and the specific activity for mandelonitrile synthesis by NdHNL-L was higher than that by other plant HNLs. The enzyme catalyzed enantioselective synthesis of (R)-cyanohydrins, exhibited high activity at pH 4.0, and high stability in the pH range of 3.5–8.0 and below 55°C. Thus, NdHNL-L is a novel HNL with novel amino acid sequence and has a potential for the efficient production of (R)-cyanohydrins. Graphical Abstract Leaves of Nandina domestica Thunb. showing high R-hydroxynitrile lyase (HNL) activity.

Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli

The original version of this article unfortunately contained a mistake. The positions of the asterisks of Fig. 1 were shifted. The correct version is given here.