NAD⁺-dependent xylitol dehydrogenase (xdhA) and L-arabitol-4-dehydrogenase (ladA) deletion mutants of Aspergillus oryzae for improved xylitol production.

Xylitol dehydrogenase (XDHA) and L-arabitol dehydrogenase (LADA) are two key enzymes in xylan metabolism catalyzing the oxidation of xylitol to D-xylulose and arabitol to L-xylulose, respectively. In Aspergillus oryzae, XDHA and LADA are encoded by xdhA and ladA. We deleted xdhA and ladA and xdhA-ladA to generate mutants with decreased dehydrogenase activities and increased xylitol production. The mutants were constructed by homologous transformation into A. oryzae P4 (∆pyrG) using pyrG as a selectable marker. The xylitol productivity of the mutants was measured using D-xylose as the sole carbohydrate source. xdhA, ladA, and the double-deletion mutant produced, respectively, 12.4 g xylitol/l with a yield of 0.24 g/g D-xylose, 12.4 g/l with a yield of 0.33 g/g D-xylose, and 8.6 g/l at a yield of 0.26 g/g D-xylose.

Complete dechlorination of tetrachloroethylene by use of an anaerobic Clostridium bifermentans DPH-1 and zero-valent iron.

A laboratory test was conducted to examine the combined effect of an anaerobic Clostridium bifermentans DPH-1 and addition of zero-valent iron (Fe0) on the reductive dechlorination of tetrachloroethylene (PCE). In addition, the dechlorination of cis-1,2-dichloroethylene (cDCE) produced from PCE was examined using Fe0. The cDCE produced was completely dechlorinated to non-toxic end products, mostly, ethylene by a subsequent chemical reductive process. Production of ethylene was dramatically increased with increase of initial cDCE concentration in the range of 10.3 microM to 928 microM (1.0-90 mg l(-1)) and the velocity constant was calculated to be 0.38 day(-1). On the other hand, the combined use of strain DPH-1 and Fe0 showed the most significant effect on the initial PCE dechlorination, but cohesion of Fe0 was found to inhibit the dechlorination rate of PCE. It is thought that phosphoric acid iron contained in a medium forms film on the surface of iron particle, so oxidation of iron is inhibited.

Transport of Clostridium bifermentans DPH-1 through the laboratory column can be explained by two-region model for bioremediation.

Transport of Clostridium bifermentans DPH-1 was characterized from laboratory-made column tests using fully saturated Toyoura sand. The conventional transport models were fitted to the column test results to investigate the applicability of prediction and assessment of bacterial transport in actual subsurface or ground water. Laboratory column tests confirmed that the transport characteristics of Clostridium bifermentans DPH-1 in activating tetrachloroethylene dechlorination could be described by mobile-immobile two-region model. The parameters of two-region model i.e. peclet number, retardation factor, fraction rate of mobile water and stanton number were characterized by fitting results. These parameters were also justified by a verification experiment. Two-region model parameters suggested that bacterial injection into the ground at a large concentration is difficult for rehabilitation of widely dispersed contaminated ground water.

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%.

Lactobacillus casei strain Shirota suppresses serum immunoglobulin E and immunoglobulin G1 responses and systemic anaphylaxis in a food allergy model.

BACKGROUND: Our previous study using allergen-sensitized murine splenocyte cultures has shown that Lactobacillus casei strain Shirota (LcS), a lactic acid bacterium widely used as a starter for fermented milk products, suppresses IgE production through promoting a dominant Th1-type response mediated by IL-12 induction. OBJECTIVE: We tried to evaluate the ability of LcS to suppress both IgE response and allergic reactions in vivo using a food allergy model with ovalbumin-specific T cell receptor transgenic (OVA-TCR-Tg) mice. METHODS: The ability of heat-killed LcS to induce IL-12 in serum was tested. OVA-TCR-Tg mice were fed a diet containing OVA for 4 weeks and injected with LcS intraperitoneally three times in the first week of this period. Cytokine and antibody secretion by splenocytes, and serum IgE and IgG1 responses were examined. The inhibitory effect of LcS on systemic anaphylaxis induced by intravenous challenge of OVA-fed OVA-TCR-Tg mice with OVA was also tested. RESULTS: Intraperitoneal injection of LcS induced an IL-12 response in the serum of OVA-TCR-Tg mice. In the food allergy model, LcS administration skewed the pattern of cytokine production by splenocytes toward Th1 dominance, and suppressed IgE and IgG1 secretion by splenocytes. The ability of LcS to modulate cytokine production was blocked by anti-IL-12 antibody treatment. LcS also inhibited serum OVA-specific IgE and IgG1 responses and diminished systemic anaphylaxis. CONCLUSION: LcS administration suppresses IgE and IgG1 responses and systemic allergic reactions in a food allergy model, suggesting a possible use of this lactic acid bacterium in preventing food allergy.

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.

Small-diameter compliant arterial graft prosthesis: Design concept of coaxial double tubular graft and its fabrication.

To minimize compliance mismatch between native artery and arterial graft prosthesis over the entire pressure regions, we proposed a coaxial double tubular artificial graft which consists of an enhanced compliant inner tube and a less compliant outer tube, both of which were fabricated using well-controlled multiply micropored segmented polyurethane (SPU) films. Double tubular grafts were coaxially assembled by inserting the inner tube into the outer tube. First, the pressure-diameter (P-D) relationship of canine common carotid arteries, which exhibited a "J" curve, was determined as a targeted artery. Two determinant variables, the pressure-induced distensibility of each tube and the intertubular space distance, were defined and formulated in several models of coaxial double tubular SPU grafts, which had various intertubular space distances, micropore densities, and wall thicknesses. The distensibility of the inner tube determined the distensibility in the low-pressure regions, which was adjusted using wall thickness and microporosity. Thinner films with higher porosities resulted in a high pressure-induced distensibility. On the other hand, a low pressure-induced distensibility in the high-pressure regions was realized using an outer tube with a thicker wall and lower microporosity. The transition point from low- to high-pressure regions was determined by the intertubular distance using the theoretical values. On the basis of these results, we presented a prototype model of a coaxial double tubular graft that exhibited well-matched compliance with canine carotid artery.

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.

Serum IgE response to orally ingested antigen: a novel IgE response model with allergen-specific T-cell receptor transgenic mice.

BACKGROUND: The mechanism by which orally ingested allergens elicit an IgE response remains unclear because there are few animal models available for investigation of this response. OBJECTIVE: We tried to develop a murine model suitable for investigation of the IgE response to orally ingested allergens, which would allow us to identify T cells that could promote IgE production. METHODS: Ovalbumin (OVA)-specific T-cell receptor transgenic mice were fed a diet containing OVA, and both the serum antibody response and cytokine production by splenocytes were examined. RESULTS: Oral administration of OVA to transgenic mice led to an increase in the levels of both antigen-specific IgE and total IgE in the sera. Subsequent intravenous challenge of OVA-fed transgenic mice with OVA resulted in anaphylactic shock. Analysis of cytokine production by splenocytes revealed that high IL-4-producing T cells appeared in the spleen 1 week after the start of feeding the OVA diet. T cells from these mice were found to promote IgE secretion by BALB/c B cells in vitro. This helper activity and the levels of IL-4 secretion were diminished after long-term feeding. These findings suggest the possibility that the orally ingested antigen elicited a response by a subpopulation of T cells that produce high levels of T(H2)-type cytokines and that promote IgE secretion, and these same T cells were tolerized by the orally ingested antigen. CONCLUSION: This experimental model with transgenic mice may be a useful tool for further studies of the cellular and molecular mechanisms of the T-cell and IgE responses to orally ingested antigens.

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.

Lactobacillus casei inhibits antigen-induced IgE secretion through regulation of cytokine production in murine splenocyte cultures.

BACKGROUND: Lactobacillus casei is a nonpathogenic gram-positive bacterium widely used in dairy products and has been shown to enhance the cellular immunity of the host. METHODS: To examine the inhibitory effect of L. casei on IgE production, splenocytes obtained from ovalbumin (OVA)-primed BALB/c mice were restimulated in vitro with the same antigen in the presence of heat-killed L. casei. The effect of this bacterium on T helper (Th) phenotype development was also examined with naive T cells from OVA-specific T cell receptor-transgenic mice. RESULTS: L. casei induced IFN-gamma, but inhibited IL-4 and IL-5 secretion, and markedly suppressed total and antigen-specific IgE secretion by OVA-stimulated splenocytes. The inhibitory effect of L. casei on IgE, IL-4, and IL-5 production was partially abrogated by addition of neutralizing antibody to IFN-gamma. Augmented IL-12 production was also observed in the cell cultures containing L. casei, and anti-IL-12 monoclonal antibody completely restored the IgE, IL-4, and IL-5 production to the control levels. The IL-12 augmentation by L. casei was macrophage-dependent. The Th cell development assay showed the ability of L. casei to induce Th1 development preferentially. This effect was also completely blocked by anti-IL-12 antibody. CONCLUSIONS: This is the first demonstration that a nonpathogenic microorganism, L. casei, can inhibit antigen-induced IgE production through induction of IL-12 secretion by macrophages. The findings suggest a potential use of this organism in preventing IgE-mediated allergy.