Katsushi Nishimura

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue.

One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.

Alanine racemase from the green alga Chlamydomonas reinhardtii

Summary.Chlamydomonas reinhardtii, a unicellular green microalga, could grow to a stationary phase having optical density of 2.0–2.5 at 750 nm in Tris-acetate-phosphate (TAP) medium containing 0.1% D-alanine. D-alanine has no inhibitory effect on growth and induced alanine racemase activity 130-fold more than without D-alanine in the green alga. Although C. reinhardtii cultured in the TAP medium showed alanine racemase activity, the content of free D-alanine was only 0.14%. The enzyme was partially purified by ammonium sulfate fractionation followed by three kinds of liquid chromatography using DEAE Toyopearl, Phenyl Sepharose, and TSK G3000 SWXL columns. The specific activity for L-alanine of the partially purified alanine racemase was 3.8 µmol/min/mg. The molecular weight of the enzyme was determined to be approximately 72,000 by gel filtration. The enzyme showed a maximum activity at 45 °C and pH 8.4 and requires pyridoxal 5′-phosphate as a coenzyme.

An Improved Method for Isolating Yeasts in the Genus Lipomyces and Related Genera from Soil

An improved method for isolating soil yeasts in the genus Lipomyces and related genera was developed on the basis of their physiological properties. Liberation of yeast cells from soil, capture of the yeast cells by a membrane filter, and colony formation on an acidic (pH 3) nitrogen-free agar plate containing 0.1% cycloheximide resulted in success.

Purification of branched-chain amino acid aminotransferase from Helicobacter pylori NCTC 11637

Summary.Branched-chain amino acid aminotransferase was purified by several column chromatographies from Helicobacter pylori NCTC 11637, and the N-terminal amino acid sequence was analyzed. The enzyme gene was sequenced based on a putative branched-chain amino acid aminotransferase gene, ilvE of H. pylori 26695, and the whole amino acid sequence was deduced from the nucleotide sequence. The enzyme existed in a homodimer with a calculated subunit molecular weight (MW) of 37,539 and an isoelectric point (pI) of 6.47. The enzyme showed high affinity to 2-oxoglutarate (Km = 0.085 mM) and L-isoleucine (Km = 0.34 mM), and Vmax was 27.3 µmol/min/mg. The best substrate was found to be L-isoleucine followed by L-leucine and L-valine. No activity was shown toward the D-enantiomers of these amino acids. The optimal pH and temperature were pH 8.0 and 37 °C, respectively.

Purification of Helicobacter pylori NCTC 11637 Cytochrome bc1 and Respiration with d-Proline as a Substrate

Helicobacter pylori is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found d-amino acid dehydrogenase-mediated electron transport from d-proline or d-alanine to oxygen via the respiratory chain in H. pylori. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the d-amino acids to oxygen using the recombinant cytochrome bc(1) complex, cytochrome c-553, and the terminal oxidase cytochrome cbb(3) complex. Upon addition of the recombinant d-amino acid dehydrogenase and d-proline or d-alanine to the reconstituted electron transport system, reduction of cytochrome cbb(3) and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of H. pyloris electron transport chain, only the cytochrome bc(1) complex had been remained unpurified. Therefore, we cloned and sequenced the H. pylori NCTC 11637 cytochrome bc(1) gene clusters encoding Rieske Fe-S protein, cytochrome b, and cytochrome c(1), with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome bc(1) complex showed an alpha peak at 561 nm with a shoulder at 552 nm.

Total DNA Preparation Excluding Extracellular Acidic Polysaccharide from Lipomyces Yeasts and its Application to Taxonomic Studies

A preparation method of total DNA from Lipomyces yeasts was improved in order to exclude extracellular acidic polysaccharide thoroughly. The method combined an ultracentrifuge and polyethylene glycol precipitation with the usual method. The total DNAs obtained were analyzed for G+C content and by DNA-DNA hybridization. The results all agreed almost completely with literature data. All the DNA samples prepared using this method were pure enough for these taxonomic analyses and could also be used as templates of PCR for amplification of small subunit ribosomal DNA and the internal transcribed spacer region.

Purification of Pyridylaminated Oligosaccharides Using 1,2-Dichloroethane Extraction

Fluorescence derivatization of the oligosaccharides released from glycoconjugates is widely used for precise structural characterization. To ensure labeling of the oligosaccharides, a large excess of fluorescence reagents is usually added to the reaction tube. Therefore, any excess reagents and by-products of the labeling reaction should be removed by several column chromatographies, including using a cellulose cartridge or spin columns. However, these purification steps are often time-consuming, expensive, and laborious. In this study, we found that 1,2-dichloroethane extraction could effectively and easily purify pyridylaminated oligosaccharides with a high recovery rate.

Micro-Inactivation of Coliforms by Low-Temperature and Atmospheric-Pressure Plasma Irradiation

In this study, we report on the inactivation technique to apply the oral surgery by micro low-temperature and atmospheric-pressure plasma irradiation utilizing a capillary nozzle. Emission spectrum of the micro-plasma showed OH and O radical, which were affected inactivation of coliform. Vibration temperature of the micro-plasma with 7998 K was calculated from peak-intensity at emission due to N2 2nd positive system. Inactivation region was larger than plasma irradiation spot, and it was increasing with plasma irradiation time.