Tsuyoshi Shimonishi

Biochemical preparation of l-Ribose and l-Arabinose from ribitol: A new approach

L-ribose and L-arabinose were prepared biochemically from ribitol via a two-step reaction, by which the complete oxidation of ribitol to L-ribulose (approximately 98%) was achieved by the reaction of washed cells of Acetobacter aceti IFO 3281. The produced L-ribulose was then used as a substrate for the production of L-ribose and L-arabinose. The isomerization of L-ribulose to L-ribose and L-arabinose was carried out using L-ribose isomerase (L-RI) of Acinetobacter sp. strain DL-28 and L-arabinose isomerase (L-AI) of Mycobacterium smegmatis, respectively. At equilibrium, the ratio of L-ribose: L-ribulose was 70:30 and that of L-arabinose: L-ribulose was 90: 10. After a simple purification treatment, both pentoses could be crystallized without the use of column chromatography. The crystals were confirmed as L-ribose and L-arabinose by High-performance liquid chromatography (HPLC), Infrared (IR), Nuclear magnetic resonance (NMR) and optical rotation measurements.

A new enzyme, l-ribose isomerase from Acinetobacter sp. strain DL-28

Abstract A new enzyme, l -ribose isomerase, was found constitutively in Acinetobacter sp. strain DL-28. The enzyme was purified by polyethylene glycol precipitation, anion exchange chromatography on diethylaminoethyl (DEAE)-Toyopearl 650M and gel filtration on Sephadex G-150. The enzyme was found to be homogenous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 120,000 by Sephadex G-150 gel filtration. On the basis of sodium dodecyl sulfate (SDS) gel electrophoresis, the enzyme is composed of four identical subunits with molecular weights of about 32,000. The isoelectric point of the enzyme was estimated to be about 5.1. The enzyme was specific for l -ribose, d -lyxose and d -mannose. The Km for l -ribose was 44 mM and Vmax was 357 μmol/mg·min. The equilibrium ratio between l -ribose and l -ribulose was 70 : 30. The maximum activity at 30°C was obtained at pH 9.0, and the enzyme was stable in the pH range of 7.0–9.0. The optimum temperature was around 30°C, and the enzyme was stable up to 30°C for 10 min.

Production of L-tagatose from galactitol by Klebsiella pneumoniae strain 40b

Abstract A process for the production of l -tagatose from galactitol has been developed. An organism isolated from soil, Klebsiella pneumoniae strain 40b, was found to convert galactitol to l -tagatose at a higher rate when glycerol was added to the reaction mixture than when no glycerol was added to the reaction mixture. The cells grown on 1.0% xylitol showed maximum conversion activity. The conversion rates were about 90%, 80%, 70% and 60% with 0.5%, 1.0%, 1.5% and 2.0% galactitol concentration, respectively. Since the strain does not utilize galactitol and l -tagatose, almost 100% of galactitol consumed was converted to l -tagatose. This is the first report of l -tagatose production by microbial cells.

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.

Characterization of a solvent resistant and thermostable aminopeptidase from the hyperthermophillic bacterium, Aquifex aeolicus

A leucine aminopeptidase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli, and its expression product was purified and characterized. The expressed protein was purified to homogeneity by using heat to denature contaminating proteins followed by ion-exchange chromatography to purify the heat-stable product. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 54 kDa. Kinetic studies on the purified enzyme confirmed that it was a leucine aminopeptidase. The optimum temperature for its activity was around 80 degrees C and the optimum pH was in the range from 8.0 to 8.5. It was stable at high temperatures and 27% of its activity was retained after heating at 115 degrees C for 30 min. The purified enzyme had a pH stability range between 4.0 and 11.0. This aminopeptidase was highly resistant to organic solvents such as methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide, acetone, acetonitrile, dimethyl formamide, 1-propanol, 2-propanol, and dioxane.

Production of d-lyxose from d-glucose by microbial and enzymatic reactions

D-arabitol was first prepared from D-glucose using Candida famata R28. The reaction gave 5.0% D-arabitol from 10.0% D-glucose. D-arabitol was then almost completely converted to D-xylulose using Acetobacter aceti IFO 3281. Finally, D-lyxose was prepared from D-xylulose enzymatically using L-ribose isomerase from toluene-treated cells of Acinetobacter sp. strain DL-28. The isomerization reaction progressed steadily and the concentration of D-xylulose increased from 1.0 to 10.0%. About 70% of D-xylulose was converted to D-lyxose in all cases. Separation of residual D-xylulose from the reaction mixture is very difficult to achieve by column chromatography, but D-xylulose could be selectively degraded easily using Saccharomyces cerevisiae IFO 0841. The product was crystallized and was confirmed to be D-lyxose by HPLC, 13C-NMR spectra, IR spectra analysis, and optical rotation measurement.

Improving enzyme characteristics by gene shuffling; application to β-glucosidase

The genes of family 3 β-glucosidase enzymes consist of five distinct regions; the N-terminal residues, an N-terminal catalytic domain, a nonhomologous region, a C-terminal domain of unknown function and the C-terminal residues. The β-glucosidase genes derived from Cellvibrio gilvus (CG) and Agrobacterium tumefaciens (AT) have been subjected to gene deletion, truncation and shuffling. The folding information was found to be distributed unevenly across the different regions based on the gene manipulation results. Chimeric enzymes with improved enzyme characteristics were obtained only by gene shuffling at the C-terminal domain.

Intestinal absorption, organ distribution, and urinary excretion of the rare sugar D-psicose

Background The purpose of this study was to evaluate intestinal absorption, organ distribution, and urinary elimination of the rare sugar D-psicose, a 3-carbon stereoisomer of D-fructose that is currently being investigated and which has been found to be strongly effective against hyperglycemia and hyperlipidemia. Methods This study was performed using radioactive D-psicose, which was synthesized enzymatically from radioactive D-allose. Concentrations in whole blood, urine, and organs were measured at different time points until 2 hours after both oral and intravenous administrations and 7 days after a single oral administration (100 mg/kg body weight) to Wistar rats. Autoradiography was also performed by injecting 100 mg/kg body weight of 14C-labeled D-psicose or glucose intravenously to C3H mice. Results Following oral administration, D-psicose easily moved to blood. The maximum blood concentration (48.5±15.6 μg/g) was observed at 1 hour. Excretion to urine was 20% within 1 hour and 33% within 2 hours. Accumulation to organs was detected only in the liver. Following intravenous administration, blood concentration was decreased with the half-life=57 minutes, and the excretion to urine was up to almost 50% within 1 hour. Similarly to the results obtained with oral administration, accumulation to organs was detected only in the liver. Seven days after the single-dose oral administration, the remaining amounts in the whole body were less than 1%. Autoradiography of mice showed results similar to those in rats. High signals of 14C-labeled D-psicose were observed in liver, kidney, and bladder. Interestingly, no accumulation of D-psicose was observed in the brain. Conclusion D-psicose was absorbed well after oral administration and eliminated rapidly after both oral and intravenous administrations, with short duration of action. The study provides valuable pharmacokinetic data for further drug development of D-psicose. Because the findings were mainly based on animal study, it is necessary to implement human trials to study the metabolism pathway, which would give an important guide for human intake and food application of D-psicose.

Preparation of D -Gulose from Disaccharide Lactitol Using Microbial and Chemical Methods

When an M31 strain of Agrobacterium tumefaciens was grown in a mineral salt medium at 30 °C containing 1.0% lactitol as sole carbon source, a keto-sugar was efficiently accumulated in the supernatant. This oxidation from lactitol to the keto-sugar was caused by M31 cells grown with medium containing a disaccharide unit, including sucrose, lactitol, lactose, maltose, or maltitol, suggesting that the enzyme is inducible. M31 also demonstrated good growth characteristics in Tryptic Soy Broth (TSB) medium containing 1.0% sucrose, and cells grown under these conditions showed strong lactitol transformation activity. The keto-sugar product was reduced by chemical hydrogenation and the resulting product was hydrolyzed to D-gulose, D-galactose, and D-sorbitol by acid hydrolysis, revealing that the reduced products are lactitol and D-gulosyl-(β-1,4)-D-sorbitol. Taken together, these results indicate that M31 can convert lactitol to 3-ketolactitol and thus provide access to the rare sugar D-gulose.

Characterization of N-acetylmuramidase M-1 of Streptomyces globisporus produced by Escherichia coli BL21(DE3)pLysS.

A gene of Streptomyces globisporus encoding N-acetylmuramidase M-1 was cloned into the pET26b vector and expressed in Escherichia coli BL21(DE3)pLysS. Maximal activity for the purified enzyme was observed at 55 degrees C with an optimal pH of 5.3, and N-bromosuccinimide strongly inhibited lytic activity even at a concentration of 0.01 mM. The enzyme showed N,O-diacetylmuramidase activity.