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Cadaverine, the expected raw material of polyamides, is produced by decarboxylation of L-lysine. If we could produce cadaverine from the cheapest sugar, and as a renewable resource, it would be an effective solution against global warming, but there has been no attempt to produce cadaverine from glucose by fermentation. We focused on Corynebacterium glutamicum, whose L-lysine fermentation ability is superior, and constructed a metabolically engineered C. glutamicum in which the L-homoserine dehydrogenase gene (hom) was replaced by the L-lysine decarboxylase gene (cadA) of Escherichia coli. In this recombinant strain, cadaverine was produced at a concentration of 2.6 g/l, equivalent to up to 9.1% (molecular yield) of the glucose transformed into cadaverine in neutralizing cultivation. This is the first report of cadaverine fermentation by C. glutamicum.

Thermophilic aerobic composting is a widely practiced method for the disposal of exhaust materials. We isolated a thermophilic bacteria strain from a compost sample under aerobic conditions at 60°C. On the basis of its 16S rRNA sequence and physiological characteristics, this strain was identified as Bacillus thermodenitrificans OHT-1. An 18-subunit oligonucleotide probe for 16S rRNA, labeled with fluorescein isothiocyanate, was developed for the detection of B. thermodenitrificans. Spores and vegetative cells of B. thermodenitrificans OHT-1 were detected in liquid culture and laboratory compost by whole cell hybridization using this oligonucleotide probe. The results obtained by whole cell hybridization were evaluated in growth experiments of B. thermodenitrificans OHT-1 in laboratory compost and were used to enumerate spores and vegetative cells.Key words: compost, Bacillus thermodenitrificans, 16S rRNA, whole cell hybridization.

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular α-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4fractionation, gel filtration, and ion exchange chromatography. The molecular mass of this monomeric enzyme was 88 kDa. The native enzyme had a pI of 4.2 and displayed a pH optimum and stability of 4.0 and 7.0–10.0, respectively. The temperature optimum was 65°C, and it was stable up to 70°C. The Kmand Vmaxfor p-nitrophenyl α-L-arabinofuranoside were 0.59 mM and 387 µmol·min–1·mg–1protein, respectively. Activity was not stimulated by metal cofactors. The N-terminal amino acid sequence did not show any similarity to other arabinofuranosidases. Higher hydrolytic activity was recorded with p-nitrophenyl α-L-arabinofuranoside, arabinotriose, and sugar beet arabinan; lower hydrolytic activity was recorded with oat–spelt xylan and arabinogalactan, indicating specificity for the low molecular mass L(+)-arabinose containing oligosaccharides with furanoside configuration.Key words: α-L-arabinofuranosidase, enzyme purification, amino acid sequence, Rhizomucor pusillus.

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.

An enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from cell-free extracts of Clostridium bifermentans DPH-1 was purified, cloned, and sequenced. The enzyme catalyzed the reductive dechlorination of PCE to cis-1,2-dichloroethylene via trichloroethylene, at a Vmaxand Kmof 73 nmol/mg protein and 12 µM, respectively. Maximal activity was recorded at 35°C and pH 7.5. Enzymatic activity was independent of metal ions but was oxygen sensitive. A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor. The molecular mass of the native enzyme was estimated to be approximately 70 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI–TOF/MS) revealed molecular masses of approximately 35 kDa and 35.7 kDa, respectively. A broad spectrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropane, and 1,1,2-trichloroethane) was degraded. With degenerate primers designed from the N-terminal sequence (27 amino acid residues), a partial sequence (81 bp) of the encoding gene was amplified by polymerase chain reaction (PCR) and sequenced. Southern analysis of C. bifermentans genomic DNA using the PCR product as a probe revealed restriction fragment bands. A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined. The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase.Key words: tetrachloroethylene, Clostridium bifermentans DPH-1, PCE dehalogenase, gene cloning.

An upflow fixed-bed reactor was evaluated for the removal of tetrachloroethylene (PCE) by a strictly anaerobic, PCE dechlorinating bacterium, Clostridium bifermentans DPH-1 immobilized on sterile ceramic beads. The ceramic beads provide a large surface area for development of cell mass in the column. For kinetic experiments, the conventional first-order reaction kinetics was applied to evaluate the batch and continuous immobilized reactors. Velocity constants ( K B and K C ) under the batch culture and continuous culture were determined to be 1.9 × 10 –2 (mg protein –1 h –1 ) and 1.4 × 10 –2 (mg protein –1 h –1 ), respectively. Maximum specific degradation rate of PCE was calculated as 0.16 (10 –3 μmol mg protein –1 h –1 from average influent concentration of PCE (12 μM) in the bioreactor. Simultaneously maximum volumetric degradation rate was also computed as 10.2 μmol l –1 h –1 . The volumetric degradation rate was relatively higher than those of other reactors. In order to maintain the efficiency of PCE dechlorination, 20 h or more HRT in the reactor system was required.

A newly isolated Bacillus subtilis HHT-1 has the ability to oxidize manganese ions. We attempted to immobilize the cells on the microfiltration membrane and remove manganese ions from the raw water as manganese oxides. After subculturing, the broth was filtered with NTF-5200 (pore size 0.2 μm), Nitto, Japan. The filter was set in Nitto Membrane Master RUM-2, Nitto, Japan and used as the bioreactor. Manganese sulfate was dissolved in distilled water to become 1 mg/l or 0.2 mg/l of the final manganese ion concentration, and used as the raw water. The volume of the bioreactor was 10 ml. The flow rate, pressure, retention time of the bioreactor, and circulation frequency were, 0.08 l/min, 0.4 kgf/cm2, 7.5 sec, and 0.04 time/min, respectively. Removal rate of manganese ions rose with increasing cell mass immobilized. The bioreactor was active even at low concentration of initial manganese (0.2 mg/l) and 0.04 mg/l or less of manganese ion concentration in the treated water was obtained.

リグノセルロースは, 多様な植物系廃棄物に豊富に存在している。リグノセルロースのうちヘミセルロースは, 酸加水分解或いは酵素分解によりD-キシロースなどの単糖類が得られる。D-キシロースはキシリトール, ブタノールなどの原料になる。植物系廃棄物を有効利用する際に, 一番不足しているのは植物系廃棄物の各々に含まれている成分の情報である。そこで, 植物系廃棄物の有効利用のため, D-キシロースなどの糖質成分に関するデータベースの作成とその抽出方法に関して検討した。データベースの作成の結果, 豆科の堅い殻はD-キシロースの含有量が高く, ピスタチオ殻, クルミ殻, ヒマワリ種の皮, 麦のもみがらおよび栗の殻には100gあたり36g, 18.7g, 18.3g, 17.1gおよび12.69のD-キシロースが含まれており, D-キシロースの原料として有効であることが判った。また, Penicilliumsp.AHT-1のキシラナーゼとRhizomucor pusillusのキシロシダーゼを用いたピスタチオ殻の分解の際, キシラナーゼおよびβ-キシロシダーゼの量を各々3, 000U/gと33U/gとしたところ, ピスタチオ殻100gあたり36gのD-キシロースが生産され, 100%の生産収率を示した。

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).

農産廃棄物中に大量に含まれるヘミセルロースは, 主としてD-キシロースから成る多糖類である。したがって, ヘミセルロースを加水分解することによって, D-キシロースを得ることができる。しかし, D-キシロースの有効利用方法はあまり検討されていない。ここでは, D-キシロースから食品添加物や糖尿病治療に幅広く利用される甘味料であるキシリトールを微生物生産することを目的にして, 農産廃棄物としてピーナッツ殻を取り上げ, これを酸加水分解することによってD-キシロースを主成分とする培地を得ることを検討した。ピーナッツ殻を爆砕処理, 水酸化カルシウム処理, レジン (陰イオン交換レジンと吸着レジン) による処理および濃縮処理を行うことによって, Candida tropicalis IFO0618株によるキシリトール生産培地として利用可能な処理液を得た。この方法でのピーナッツ殻からのD-キシロース抽出率は約20%であり, 初発菌体量を5.0g/1で植菌した場合, 生産速度0.23g/1・h, 収率54.7%で8.7g/1のキシリトールが生産された。

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.

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 K m 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°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.

Major problems for hydroponics are higher operational cost and pollution due to discharge of waste nutrient solution. Besides, water culture has disadvantage for plant pathogenic distribution in the system due to shortage of buffer action in comparison with soil cultivation. To solve these problems, recycling use of nutrient solution by ultrafiltration was studied. Plants used were Lettuce (Lactuca sativa L.) and sporangia of Pythium aphanidermatum, a pathogen. Growth of lettuce was inhibited by inoculation of 2.01×104 sporangia/l or more of sporangia but they recovered viability at 22nd day after inoculation. Inoculation of 1.00×106 sporangia/l represented lethal effect on lettuce. Whether inoculation period of P. aphanidermatum to water culture was at the lst week or month, lettuce deceased. Sporangia of P. aphanidermatum were perfectly removed by untrafiltration. P. aphanidermatum produced pathogenic substance of below 20, 000 MW, which caused lettuce root rod decease. This substance seems unstable under heating condition. Experimental results suggest that ultrafiltration is effective to remove pathogenic microoganisms from nutrient solution but if pathogenic microorganisms produce some harmful substances, another/combined treatment of nutrient solution should be necessary.

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.

The structures of PF1092A (1), B (2) and C (3), new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum, were elucidated by spectroscopic analyses. These compounds possess an eremophilane-type sesquiterpene carbon skeleton and differ only in that 1 and 2 are different monoacetates of 3. The absolute configurations of 1-3 were determined by single crystal X-ray diffraction analysis of the 4-bromobenzoyl ester of PF1092A and by measuring the optical rotations of the acetylation products of these compounds.

The structures of PF1092A (1), B (2) and C (3), new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum, were elucidated by spectroscopic analyses. These compounds possess an eremophilane-type sesquiterpene carbon skeleton and differ only in that 1 and 2 are different monoacetates of 3. The absolute configurations of 1-3 were determined by single crystal X-ray diffraction analysis of the 4-bromobenzoyl ester of PF1092A and by measuring the optical rotations of the acetylation products of these compounds.

A 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.

A new antitumor antibiotic SF2575 has been isolated from a culture filtrate of Streptomyces sp. SF2575. The molecular formula was determined to be C40H43NO15 by elemental analysis, mass and 13C NMR spectral analyses. The spectral data revealed SF2575 to be a new tetracycline antibiotic. It was active against Gram-positive bacteria and exhibited antitumor activity against P388 leukemia in mice.

Benarthin, a new inhibitor of pyroglutamyl peptidase (PG-peptidase), has been isolated from the culture broth of Streptomyces xanthophaeus MJ244-SF1. The structure of benarthin was determined to be L-(2,3-dihydroxybenzoyl)argininyl-L-threonine by analysis of spectral properties and through chemical studies.

Benarthin, a new inhibitor of pyroglutamyl peptidase (PG-peptidase), has been isolated from the culture filtrate of Streptomyces xanthophaeus MJ244-SF1. The structure of benarthin has been determined to be L-(2, 3-dihydroxybenzoyl)arginyl-L-threonine. This structure was confirmed by the total synthesis of benarthin. Moreover, we synthesized benarthin derivatives to obtain information on the relationship between structure and inhibitory activity. The results indicated that the catechol group of benarthin is the essential moiety for the inhibition of PG-peptidase.

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 μmol/h · mg protein, as well as a number of other halogenated aliphatic compounds.

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.

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 CO2 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.

At a sea-based, solid waste disposal site, methanogenic organisms were quantified by molecular approaches. The samples collected for analysis were from anaerobic leachate of the landfill site. When the DNA extracted from the leachate was examined by a quantitative PCR method using domain-specific 16S rDNA primers, archaeal DNA represented 2–3% of the total extracted DNA. On the basis of cloning and sequence comparison of the archaeal PCR products, more than half of the sequences belonged to Euryarchaeota, particularly relatives of the genus Methanosaeta. The cloning analysis suggested that the majority of methane emitted from the landfill site originated from the acetate-utilizing Methanosaeta.Key words: landfill, methanogen, archaea, 16S rDNA.

In preparation for the development of a xylitol biosensor, the xylitol dehydrogenase of Candida tropicalis IFO 0618 was partially purified and characterized. The optimal pH and temperature of the xylitol dehydrogenase were pH 8.0 and 50°C, respectively. Of the various alcohols tested, xylitol was the most rapidly oxidized, with sorbitol and ribitol being reduced at 65% and 58% of the xylitol rate. The enzyme was completely inactive on arabitol, xylose, glucose, glycerol, and ethanol. The enzyme's xylitol oxidation favored the use of NAD+(7.9 U/mg) over NADP+(0.2 U/mg) as electron acceptor, while the reverse reaction, D-xylulose reduction, favored NADPH (7.7 U/mg) over NADH (0.2 U/mg) as electron donor. The Kmvalues for xylitol and NAD+were 49.8 mM and 38.2 µM, respectively. For the generation of the xylitol biosensor, the above xylitol dehydrogenase and a diaphorase were immobilized on bromocyan-activated sephallose. The gel was then attached on a dissolved oxygen electrode. In the presence of vitamin K3, NAD+and phosphate buffer, the biosensor recorded a linear response to xylitol concentration up to 3 mM. The reaction was stable after 15 min. When the biosensor was applied to a flow injection system, optimal operation pH and temperature were 8.0 and 30°C, respectively. The strengths and limitations of the xylitol biosensor are its high affinity for NAD+, slow reaction time, narrow linear range of detection, and moderate affinity for xylitol.Key words: xylitol, xylitol dehydrogenase, biosensor, Candida tropicalis.

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.

DNA amplification by polymerase chain reaction (PCR) should be inhibited if the target for amplification region in the template DNA is nicked or cut. Based on this premise, we established a sensitive and differential assay using PCR to detect antibiotics that act on DNA. After template lambda DNA (10 pg) was incubated with antibiotics (10 approximately 20 ng) at 37 degrees C for 30 minutes in a 5 microliters reaction volume, a PCR assay (10 microliters reaction volume; 25 approximately 30 cycles) was performed under the conditions we modified, resulting in amplification of a 500 bp fragment of lambda DNA which was monitored by agarose gel electrophoresis. Among the several antibiotics examined, the anthracyclines, bleomycin, D-cycloserine and mitomycin C clearly inhibited the PCR amplification reaction, whereas actinomycin D and ofloxacin did not. Preincubation of template DNA in the presence of Fe++ was necessary for bleomycin and cycloserine to exhibit marked inhibition of PCR. Mitomycin C exhibited the inhibition in the presence of DTT and Cu+. By contrast, non-DNA-acting antibiotics (200 ng) such as aminoglycosides, beta-lactams, and macrolides showed no inhibition. The PCR-amplified fragment from lambda DNA was not degraded by incubation with the antibiotics (20 ng) that inhibited PCR. Furthermore, ethylacetate extracts of the cultured broths of actinomycetes proved to be suitable as samples for this PCR inhibition assay.

We found benarthin, a new inhibitor of pyroglutamyl peptidase, in the fermentation broth of Streptomyces xanthophaeus MJ244-SF1. It was purified by column chromatography and centrifugal partition chromatography (CPC) and then was isolated as a colorless powder. The binding of benarthin was competitive with substrate and its inhibition constant (Ki) was 1.2 x 10(-6) M.

The structure of a new antitumor antibiotic SF2575, has been determined by spectroscopic analyses of the antibiotic and its alkaline degradation products. The relative stereochemistry has been confirmed by X-ray crystallographic analysis. The antibiotic has a 2-naphthacenecarboxamide carbon skelton which is structurally related to the tetracycline antibiotics and it is unique by bearing C-glycoside, salicyclic acid and angelic acid moieties.

A new antibiotic SF2487 has been isolated from the culture broth of Actinomadura sp. SF2487. The structure of antibiotic SF2487 was determined by spectroscopic analyses of the sodium salt and X-ray diffraction analysis of the silver salt. The antibiotic represents a new member of polyether group antibiotics known as the acyltetronic acid type 4. The antibiotic is weakly active against Gram-positive bacteria and exhibits antiviral activity against influenza virus in vitro.

The role of the conserved DExH motif of the Japanese encephalitis virus (JEV) NS3 protein in the ATPase and RNA helicase activities was compared with that of the hepatitis C virus (HCV) NS3 protein. In the DExH motif of JEV NS3, Asp-285 and Glu-286 were essential for both ATPase and RNA helicase activities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. A His-288-to-Ala substitution in the DExH motif of HCV NS3 resulted in an increase in ATPase activity which was suppressed by poly(U). In contrast, alanine substitution at the same site in JEV NS3 did not increase basal ATPase activity which remained to be stimulated by poly(U). Thus, the mutational effect at His in motif II was different in the HCV and JEV NS3 proteins. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gln, Ser, or Arg could partly substitute for it. However, any other mutation at His-288 completely disrupted the RNA helicase activity of JEV NS3. The results suggest that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and the ATPase and helicase activities are separable enzymatic functions.