Masahiro Hatsu

Isolation and characterization of a tetrachloroethylene dechlorinating bacterium, Clostridium bifermentans DPH-1

A tetrachloroethylene (PCE)-degrading gram-positive, endospore forming, anaerobic bacterium, strain DPH-1, was isolated from a contaminated site. The organism was identified as Clostridium bifermentans by 16S rRNA gene sequence analysis and based on its physiological characteristics. Strain DPH-1 could dechlorinate high concentrations of PCE (0.9 mM), via trichloroethylene (TCE) to cis-1,2-dichloroethylene (cDCE) at a rate of 0.43 micromol/h.mg protein, as well as a number of other halogenated aliphatic compounds.

Methanocalculus pumilus sp. nov., a heavy-metal-tolerant methanogen isolated from a waste-disposal site

A mesophilic hydrogenotrophic methanogen, strain MHT-1T, was isolated from the leachate of a sea-based site for solid waste disposal (the port of Osaka, Japan). Strain MHT-1T was found to be an irregular coccus and was able to use H2/CO2 and formate as energy sources. Acetate was required for growth. The optimum temperature and pH for growth were 35 degrees C and 6.5-7.5, respectively. Strain MHT-1T was resistant to high concentrations of several heavy metals such as CdCl2 and CuSO4. The G+C content of the DNA was 51.9 mol%. Analysis of the 16S rRNA gene revealed that the isolate was a member of the genus Methanocalculus but distinct from its nearest neighbour, Methanocalculus halotolerans, there being a sequence similarity of 98.9%. DNA-DNA hybridization analysis revealed 51% relatedness with the DNA of M. halotolerans strain SEBR 4845T. The optimum NaCl concentration was 1.0%, whereas the optimum in M. halotolerans was 5.0%. A new species, Methanocalculus pumilus, is proposed for strain MHT-1T. The type strain is MHT-1T (= DSM 12632T = JCM 10627T).

Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein

The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His‐tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp‐285‐to‐Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C‐terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.

d-Glucose feeding for improvement of xylitol productivity from d-xylose using Candida tropicalis immobilized on a non-woven fabric

SummaryA non-woven fabric was successfully applied for immobilization of Candida tropicalis to produce xylitol from d-xylose. Xylitol productivity was enhanced by feeding of d-glucose (50 g/l·d); 87 g xylitol/L was produced after 64 h cultivation. Non-woven fabric could be used five times for fed-batch cultivation.

Effect of heavy metals on the growth of a methanogen in pure culture and coculture with a sulfate-reducing bacterium

The sensitivity of a methanogen and sulfate-reducing bacterium isolated from a sea-based landfill site to Cd2+ and Cu2+ was studied. Methanogens and sulfate-reducing bacteria in leachates of the waste disposal site were enumerated using the MPN method. Methanobacterium thermoautotrophicum KHT-2, isolated from the leachate, could not grow at 0.5 mM Cd2+ or 1.0 mM Cu2+. Desulfotomaculum sp. RHT-3, isolated from the same leachate, was able to insolubilize 3.0 mM Cd2+ or 2.0 mM Cu2+ by production of hydrogen sulfide. When strains KHT-2 and RHT-3 were cultured together in the presence of the heavy metals, strain KHT-2 could grow at high heavy metal concentrations after insolubilization of the metals by strain RHT-3.

In vitro dehalogenation of tetrachloroethylene (PCE) by cell-free extracts of Clostridium bifermentans DPH-1

Cell-free extracts of Clostridium bifermentans DPH-1 catalyzed tetrachloroethylene (PCE) dechlorination. PCE degradation was stimulated by addition of a variety of electron donors. Ethanol (0.61 mM) was the most effective electron donor for PCE dechlorination. Maximum activity was recorded at 30 degrees C and pH 7.5. Addition of NADH as a cofactor stimulated enzymatic activity but the activity was not stimulated by addition of metal ions. When the cell-free enzyme extract was incubated in the presence of titanium citrate as a reducing agent, the dehalogenase was rapidly inactivated by propyl iodide (0.5 mM). The activity of propyliodide-reacted enzyme was restored by illumination with a 250 W lamp. The dehalogenase activity was also inhibited by cyanide. The substrate spectrum of activity included trichloroethylene (TCE), cis-1,2-dichloroethylene (cDCE), trans-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloroethane, and 1,1,2-trichloroethane. The highest rate of degradation of the chlorinated aliphatic compounds was achieved with PCE, and PCE was principally degraded via TCE to cDCE. Results indicate that the dehalogenase could play a vital role in the breakdown of PCE as well as a variety of other chlorinated aliphatic compounds.

Cloning and expression of a 58-kDa xylanase VI gene (xynD) of Aeromonas caviae ME-1 in Escherichia coli which is not categorized as a family F or family G xylanase

An endo-xylanase gene (xynD) encoding xylanase VI (58 kDa) of Aeromonas caviae ME-1 was cloned and expressed in Escherichia coli. The xynD gene encodes a 564 amino acid precursor (62,083 Da) and 531 amino acid mature enzyme (58,469 Da). The enzyme consists of two domains bridged by a glycine-rich linker. The N-terminal catalytic domain (392 amino acids) sequence is homologous neither to family F nor family G (families 10, 11) enzymes, into which almost all known xylanases are categorized. However, it has 41% identity with XynA of Erwinia chrysanthemi. The C-terminal domain (125 amino acids) shows homology to conserved domains in xylanase (XYLB), arabino-furanosidase (XYLC), and acetylxylan-esterase (XYLD) of Pseudomonas fluorescens subsp. cellulosa, but its function is unknown. The E. coli transformant produced 58-, 48-, 45-, and 43-kDa active xylanases as the result of C-terminal proteolytic truncation.

Expression of xyrA gene encoding for D-Xylose reductase of Candida tropicalis and production of xylitol in Escherichia coli.

The D-Xylose reductase (XR) gene (xyrA) of Candida tropicalis IFO 0618 was expressed in Escherichia coli JM109. The enzymatic properties of each recombinant XR such as the Km value for D-xylose and NADPH, the substrate specificity for other sugars and the optimal pH were essentially the same as those of the corresponding enzyme of C. tropicalis. The recombinant XR was more heat-stable than C. tropicalis XR at 60 degrees C. E. coli, expressing the xyrA gene, successfully converted D-xylose to xylitol. When D-xylose (50 g/l) and D-glucose (5 g/l) were added to IPTG-induced cells, 13.3 g/l of xylitol was produced during 20 h of cultivation.

Degradation of a variety of halogenated aliphatic compounds by an anaerobic mixed culture

Abstract We investigated the biodegradation of tetrachloroethylene (perchloroethylene, PCE) and halogenated aliphatic compounds by using anaerobic mixed cultivation. The mixed culture degraded PCE at concentrations of up to 150 mg/ l in 40 d via trichloroethylene (TCE) to cis -1,2-dichloroethylene (cDCE). Small amounts of vinyl chloride (VC) and CO 2 were also detected. The same culture degraded various halogenated aliphatic compounds such as cDCE, VC, 1,2-dichloroethane (DE), 1,3-dichloropropene (DP), dichloromethane (DM), 1,1,2-trichloroethane (TE), and chloroform (CF). Acetate was the most effective electron donor for dechlorination, although formate, glucose and lactate were also effective, but to a lesser extent. The mixed culture degraded PCE in the temperature range 25 to 43°C, with an optimum between 30 and 37°C, and the pH range of 6.2 to 11.0.

Bispolides, novel 20-membered ring macrodiolide antibiotics from microbispora.

Seven new related compounds named bispolides A1, A2, A3, B1, B2a, B2b and B3, have been found in a culture of Microbispora sp. A34030, isolated from a Malaysian soil sample. The planar structures were elucidated to be new 20-membered ring macrodiolide antibiotics on the basis of MS and NMR spectroscopic analyses. These antibiotics showed a good anti-MRSA activity in vitro.