The production of D-xylose by enzymatic hydrolysis of agricultural wastes.

Agricultural wastes, rich in D-xylose content, were hydrolyzed using the mixed crude enzymes produced by Penicillium sp. AHT-1 and Rhizomucor pusillus HHT-1. Shells of pistachio, peanut, walnut, chestnut, barley brans and sunflower seed peels, were used as raw or pretreated forms. Pretreatment was performed by milling or steam explosion. Enzymatic hydrolysis after steam explosion was more effective than milling processing. More than 13 g of D-xylose was produced from 100 g of milled pistachio shells, walnut shells, sunflower seed peels and peanut shells (less than 0.5 mm size) by the action of mixed enzyme solutions. A maximum of 36 g of D-xylose was produced from 100 g of milled pistachio shells when mixed enzyme solution, containing 3,000 U and 33 U per g of substrate with xylanase and beta-xyosidase activities, respectively, was applied. The ratio of the enzymatic hydrolysis as compared to acid hydrolysis in this finding was 100%.

Physicochemical properties of a novel alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1.

The zygomycete fungus Rhizomucor pusillus HHT-1, cultured on L(+)arabinose as a sole carbon source, produced extracellular alpha-L-arabinofuranosidase. The enzyme was purified by (NH4)2SO4 fractionation, 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 degrees C, and it was stable up to 70 degrees C. The Km and Vmax for p-nitrophenyl alpha-L-arabinofuranoside were 0.59 mM and 387 micromol x min(-1) x mg(-1) protein, 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 pnitrophenyl alpha-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.

Purification, cloning, and sequencing of an enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from Clostridium bifermentans DPH-1.

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 Vmax and Km of 73 nmol/mg protein and 12 microM, respectively. Maximal activity was recorded at 35 degrees 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.

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.

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).

Role of the DExH motif of the Japanese encephalitis virus and hepatitis C virus NS3 proteins in the ATPase and RNA helicase activities.

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.

Development of a xylitol biosensor composed of xylitol dehydrogenase and diaphorase.

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 degrees 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 K(m) values for xylitol and NAD+ were 49.8 mM and 38.2 microM, 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 degrees 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.

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.

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.

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.

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.

Novel compounds, 1,3-selenazine derivatives, as antibacterial agents against Escherichia coli and Staphylococcus aureus.

The antibacterial activities of several kinds of novel 4H-5,6-dihydro-1,3-selenazine derivatives against Escherichia coli and Staphylococcus aureus were investigated. 4-Hydroxy-4-methyl-2-p-tolyl-4H-5,6-dihydro-1,3-selenazine (TS-1), 4-ethyl-4-hydroxy-2-p-tolyl-4H-5,6-dihydro-1,3-selenazine (TS-2), and 4-hydroxy-4-methyl-2-pentyl-4H-5,6-dihydro-1,3-selenazine (PS-1) exhibited strong inhibitory activity against E. coli, and TS-1, TS-2, PS-1, 4-hydroxy-4-methyl-2-pentyl-6-propyl-4H-5,6-dihydro-1,3-selenazine (PS-6) and 4-hydroxy-4-methyl-2-pentyl-6-phenyl-4H-5,6-dihydro-1,3-selenazine (PS-8) also showed inhibitory activity against S. aureus. 4H-5,6-dihydro-1,3-thiazines and 1,3-selenazole had no inhibitory activities against both bacteria. TS-1, TS-2, and PS-1 exhibited marked antibacterial activities against both Gram-negative and Gram-positive bacteria.

PF1092A, B and C, new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum. I. Taxonomy of producing strain, fermentation, isolation and biological activities.

Three new nonsteroidal progesterone receptor ligands, PF1092A, B and C, have been isolated from Penicillium oblatum. They were purified from the solid cultures of rice media using ethyl acetate extraction, silica gel and Sephadex LH-20 column chromatographies, and crystallization. All three ligands competitively inhibited [3H]-progesterone binding to porcine uteri cytosol preparations with IC50 of 3.0 x 10 nM (PF1092A), 2.2 x 10(2) nM (PF1092B) and 2.2 x 10(3) nM (PF1092C).

PF1092A, B and C, new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum. II. Physico-chemical properties and structure elucidation.

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.

PCR inhibition assay for DNA-targeted antibiotics.

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.

Benarthin: a new inhibitor of pyroglutamyl peptidase. I. Taxonomy, fermentation, isolation and biological activities.

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.

Benarthin: a new inhibitor of pyroglutamyl peptidase. III. Synthesis and structure-activity relationships.

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.

Benarthin: a new inhibitor of pyroglutamyl peptidase. II. Physico-chemical properties and structure determination.

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.