Kei Takeshita

Mass production of D-psicose from d-fructose by a continuous bioreactor system using immobilized D-tagatose 3-epimerase.

An improved process for the mass production of D-psicose from D-fructose was developed. A D-fructose solution (60%, pH 7.0) was passed at 45 degrees C through a column filled with immobilized D-tagatose 3-epimerase (D-TE) which was produced using recombinant Escherichia coli, and 25% of the substrate was converted to D-psicose. After epimerization, the substrate, D-fructose, was removed by treatment with bakers yeast. The supernatant was concentrated to a syrup by evaporation under vacuum and D-psicose was crystallized with ethanol. Approximately 20 kg of pure crystal D-psicose was obtained in 60 d.

Production of l-psicose from allitol by Gluconobacter frateurii IFO 3254

Abstract A rare ketohexose, l -psicose, was produced from allitol by Gluconobacter frateurii IFO 3254. The transformation reaction was carried out at 30°C with shaking using the washed cells. The conversion rate was about 98% when 10% allitol was used. The cells grown on tryptic soy broth containing 1% glycerol were found to have the best conversion potential. Cells stored at −20°C for 10 d showed almost the same transformation activity as intact cells.

Direct Production of Allitol from D-Fructose by a Coupling Reaction Using D-Tagatose 3-Epimerase, Ribitol Dehydrogenase and Formate Dehydrogenase.

Allitol was produced from D-fructose via a new NADH-regenerating enzymatic reaction system using D-tagatose 3-epimerase (D-TE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH). D-fructose was epimerized to D-psicose by the D-TE of Pseudomonas cichorii ST-24 and the D-psicose was subsequently reduced to allitol by the RDH of an RDH-constitutive mutant, X-22, derived from Klebsiella pneumoniae IFO 3321. NADH regeneration for the reduction of D-psicose by the RDH was achieved by the irreversible formate dehydrogenase reaction, which allowed the D-psicose produced from d-fructose to be successively transformed to allitol with a production yield from D-fructose of almost 100%. The reactions progressed without any by-product formation. After separation of the product from the reaction mixture by a simple procedure, a crystal of allitol was obtained in a yield exceeding 90%. This crystal was characterized and determined to be allitol by HPLC analysis, its IR and NMR spectra, its melting point, and optical rotation measurement.

Production of L-erythrose via L-erythrulose from erythritol using microbial and enzymatic reactions.

A rare aldotetrose, L-erythrose, was produced from erythritol via a two-step reaction. In the first step, complete oxidation of erythritol to L-erythrulose was achieved by using Gluconobacter frateurii IFO 3254. Washed cell suspension of the strain grown on tryptic soy broth (TSB) supplemented with 1% d-sorbitol was used to carry out the transformation reaction at 30 degrees C with shaking at 170 rpm. At 10% substrate concentration, 98% erythritol was converted to L-erythrulose within 48 h. The produced L-erythrulose was then used as a substrate for the production of L-erythrose. The isomerization of L-erythrulose to L-erythrose was carried out using constitutively produced L-ribose isomerase (l-RI) from the mutant strain Acinetobacter sp. DL-28 grown on D-lyxose mineral salt medium. At equilibrium, the yield of L-erythrose from L-erythrulose was 18% and finally 1.7 g L-erythrose was obtained from 10 g erythritol. After a number of simple purification steps, the product was isolated from the reaction mixture by ion-exchange column chromatography (Dowex 50W-X2, Ca2+). The structure of the product was determined after NaBH4 reduction from Infrared (IR) and 13C nuclear magnetic resonance (NMR) spectra.

Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase

Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.