We compare aequorin, alkaline phosphatase and horseradish peroxidase as reporters for luminescent immunoassays using α-fetoprotein (AFP) as a model analyte. Biotinylated aequorin was prepared from mutated aequorin containing a reactive cysteine residue by chemical conjugation. The measurable range of AFP using this biotinylated aequorin was 0.02–200 ng/ml, with a lower background level than the other biotinylated enzymes tested.

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Aqueous solutions of several new amide compounds for use as simple thermal history indicators in the low-temperature transport of food and other products were synthesized. The phase transition temperatures of the aqueous solutions can be freely adjusted by changing the amide-water ratio in solution, the sodium chloride concentration of the water, and the type of amide compound. It is expected that these aqueous solutions can be applied as new thermal history indicators.

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Three seaweed chitinase isozymes (Chi-A, B, and C) were purified from a red algae, Chondrus verrucosus. The molecular weights and isoelectric points were 24.5 kDa and 3.5 for Chi-A, 25.5 kDa and 4.6 for Chi-B, and 24.5 kDa and <3.5 for Chi-C. Optimum pH and temperature were observed at pH 2.0 at 80 °C for Chi-A and Chi-C, and at pH 1.0 and 70 °C for Chi-B. Toward N-acetylchitooligosaccharide (GlcNAc_n) (n=2 to 6), Chi-A, B, and C hydrolyzed GlcNAc₅ and GlcNAc₆ and produced GlcNAc_n (n=2 to 4). GlcNAc_n (n=3, 4) with the reducing end-side of β anomer was detected in the hydrolysis products. These results indicate that the reactions of Chi-A, B, and C for GlcNAc_n were a retaining mechanism similar to that of family 18 chitinase. Toward crystalline chitins, Chi-A, B, and C degraded squid pen β-chitin more than crab shell or shrimp shell α-chitin.

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In order to determine the allelopathic nature of lichen acids produced by lichen mycobionts, we compared lichen photobionts with other photosynthetic organisms in terms of inhibition of photosynthetic electron transport around photosystem II (PSII) by representative lichen acids. Whereas at the thylakoid level we found no clear difference in tolerance against lichen acids between lichen photobionts and other species, at the cellular level lichen photobionts showed strong tolerance as compared with other species. These findings suggest the presence of a lichen acid-specific exclusion or detoxification mechanism in lichen photobiont cells.

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Most members of the type-2 phosphatidic acid phosphatase (PAP2) superfamily are integral membrane phophatases involved in lipid-related signal transduction and metabolism. Here we describe the cloning of a novel gene from Geobacillus toebii T-85, encoding a PAP2-like protein, Gtb PAP2L2, which contains 212 amino acids and shows a limited homology to other known PAP2s, especially at conserved phosphatase motifs, and a similar six-transmembrane topology structure. This enzyme was expressed, and purified in Escherichia coli. Recombinant Gtb PAP2L2s from the membrane fractions were solublized with 0.3% (v/v) Triton X-100 and purified by Ni²⁺ affinity chromatography. The purified enzyme showed broad substrate specificity to phosphatidic acid, diacylglycerol pyrophosphate, and lysophosphatidic, but preferred phosphatidic acid and diacylglycerol pyrophosphate in vitro. Gtb PAP2L2 is a thermal stable enzyme with a half-life of 30 min at 60 °C. The enzyme was strongly inhibited by 1% SDS, 10 mM veranda, and Zn²⁺, whereas it was independent of the Mg²⁺ ion, and insensitive to N-ethylmaleimide. The purified recombinant Gtb PAP2L2 was catalytically active and highly stable, making it ideal as a candidate on which to base further PAP2 structure/function studies.

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In conducting an in vitro screening of ethanol extracts from various natural foods using a human colon cancer cell line (CoLoTC cells), an extract of buckwheat sprouts (ExtBS) was found to express significant anti-inflammatory activity. The anti-inflammatory activity of ExtBS was confirmed by oral administration of lipopolysaccharide (LPS) to mice. Inflammatory cytokines (interleukin 6 and tumor necrosis factor alpha) were markedly up-regulated in the spleen and liver from LPS-administrated mice, and combinatory treatment with LPS and ExtBS decreased up-regulation of them in both cytokines. Both serum cytokine levels corresponded to their gene expressions in tissues, but no anti-inflammatry effect in mice was observed when ExtBS was treated intraperitoneally. ExtBS oral administration also showed protective activity as to hepatic injury induced by galactosamine/LPS treatment. Based on these data, we suggest that ExtBS contains anti-inflammatory compounds.

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Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.

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Δ12 and ω3 fatty acid desaturases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs), which are important constituents of membrane glycerolipids and also precursors to signaling molecules in many organisms. In this study, we determined the substrate specificity and regioselectivity of the Δ12 and ω3 fatty acid desaturases from Saccharomyces kluyveri (Sk-FAD2 and Sk-FAD3). Based on heterologous expression in Saccharomyces cerevisiae, it was found that Sk-FAD2 converted C16–20 monounsaturated fatty acids to diunsaturated fatty acids by the introduction of a second double bond at the ν+3 position, while Sk-FAD3 recognized the ω3 position of C18 and C20. Furthermore, fatty acid analysis of major phospholipids suggested that Sk-FAD2 and Sk-FAD3 have no strong substrate specificity toward the lipid polar head group or the sn-positions of fatty acyl groups in phospholipids.

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Cyclic electron transport and NADH and/or NADPH (NAD(P)H)-oxidizing activities were investigated in Synechocystis sp. PCC6803 grown under various stressed conditions and in ndhB-less (M55) and ycf33-deletion mutants. Activity staining and inhibitor data suggested that the ferredoxin-quinone reductase (FQR) route is the main pathway in ycf33-deletion and high-light (300 μE m⁻² s⁻¹)-grown cells as well as in M55 cells. The FQR route was highly sensitive to HgCl₂, but not to diphenyleneiodonium (DPI). On the other hand, cells grown under low CO₂ (0.03%) or normal (100 μE m⁻² s⁻¹, 3% CO₂) conditions were found perhaps to use the complex I-type NAD(P)H dehydrogenase route, which was found to be highly sensitive to DPI but not to HgCl₂. In high-salt (0.55 M NaCl)-grown cells, the amount of ferredoxin-NADP⁺ oxidoreductase (FNR) increased, and the main cyclic electron flow was perhaps the FNR route. Both DPI and HgCl₂ were strong inhibitors of the FNR route.

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Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable β-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing.

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Deoxyribonuclease I (DNase I) is known to be a glycoprotein, and two potential N-linked glycosylation sites (N18 and N106) are known for mammalian enzymes. In the present study, N18 and N106 were mutated in order to investigate the biological role of N-linked glycosylation in three mammalian (human, bovine, and equine) DNases I. The enzyme activities of N18Q and N106Q were lower than that of the wild type, and that of the double mutant (N18Q/N106Q) was lower than those of the single mutants, in accord with the sugar moiety contents in the three mammals. In addition, all mutant enzymes were unstable to heat, suggesting that both sites are required for heat stability. Moreover, in human and equine enzymes, the N18Q and N106Q mutant enzymes were less resistant to trypsin, while N18Q/N106Q was the most sensitive to trypsin. As for bovine DNase I, the trypsin resistance of N18Q and N106Q was similar to that of the wild type, but that of N18Q/N106Q decreased in a time-dependent manner. On the other hand, N-linked glycosylation was not related to pH sensitivity. The results of the present study suggest that N18 and N106 are both necessary for (i) full enzymatic activity, (ii) heat-stability, and (iii) trypsin resistance.

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Redox factor-1 (Ref-1) is a multifunctional protein involved in DNA base excision repair (BER) and transcription factor modification. By the use of retrovirus-delivered shRNA, we effectively suppressed endogenous Ref-1 expression in human embryonic kidney (HEK) 293 cells. Our results showed that downregulation of Ref-1 rendered cells more sensitive to H₂O₂-induced apoptosis. In this process, the absence of Ref-1 decreased the ratio of Bcl-2/Bax protein expression, which resulted in cytochrome c release and caspase-3 activation. These data indicate that constitutive Ref-1 is required for cellular defense and that this protective function is dependent on its role in the regulation of Bcl-2 family proteins.

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The brackish water bivalve mollusk Corbicula japonica was exposed to brackish water containing approximately 9 μg/l [¹⁴C]bisphenol A (BPA) for 168 h (the uptake phase), and subsequently transferred to clean brackish water for 144 h (the depuration phase) under semi-static conditions. Mono and disulfate conjugates of BPA were detected in the bivalves as major metabolites. At the end of the uptake phase, the visceral mass contained the highest ¹⁴C-concentration, and the monosulfate conjugate of BPA was a major metabolite in the visceral mass. These data suggest that the visceral mass is the major tissue/organ to take up and metabolize BPA in these bivalves. The BPA concentration in the bivalves readily reached steady state during the uptake phase and immediately decreased in the depuration phase. The accumulation and elimination rates of the mono and disulfate conjugates of BPA were slower than those of BPA.

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We found human renin inhibitory activity in soybean and isolated the active compound, soybean renin inhibitor (SRI). The physico-chemical data on the isolated SRI were identical with those of soyasaponin I. SRI showed significant inhibition against recombinant human renin, with an IC₅₀ value of 30 μg/ml. Kinetic studies with SRI indicated partial noncompetitive inhibition, with a K_i value of 37.5 μM. On the other hand, SRI weakly inhibited pepsin, papain, and bromeline activities, but did not inhibit other proteinases, such as trypsin, kallikrein, angiotensin converting enzyme, and aminopeptidase M. Moreover, a significant (p<0.05) decrease in the systolic blood pressure of spontaneously hypertensive rats was observed when partially purified SRI was orally administrated at 40 mg/kg/d for 7 weeks. This is the first demonstration of a renin inhibitor from soybean, soyasaponin I.

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Recently, an anionic proregion was found to be conserved at the C terminus of the antimicrobial peptide, nematode cecropin. Our results suggest that the antimicrobial activity of mature peptide is suppressed by the proregion in its precursor and is released from inhibition after processing. Inhibition is not likely to be due to direct suppression of membrane disruption.

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Rice ent-kaurene oxidase 2 (OsKO2) perhaps functions in the early steps of gibberellin biosynthesis. We found that microsomes from the methylotropic yeast Pichia pastoris expressing both OsKO2 and a fungal cytochrome P450 monooxygenase (P450) reductase converted ent-kaurene to ent-kaurenoic acid. This is direct evidence that OsKO2 is involved in the sequential oxidation of ent-kaurene to ent-kaurenoic acid in gibberellin biosynthesis in rice.

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Phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins (PCs), which play a detoxification role in higher plants. Heterologous expression of CmPCS, a product of a PCS-like gene from the genomic DNA of the red alga Cyanidioschyzon merolae, rescued Cd²⁺-sensitive yeast from Cd²⁺ toxicity. The fact that these transformed cells synthesized PCs demonstrates that CmPCS is functional.

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Kothala himbutu is a traditional Ayurvedic medicinal plant used to treat diabetes. We aimed to evaluate the safety of an aqueous extract of Kothala himbutu stem (KTE) in normal mice. The mice were divided into two groups: one was administered KTE and the other distilled water for 3 weeks. During the test period, the groups showed no significant differences in body weight gain or plasma parameters, such as fasting blood glucose level, oral glucose tolerance test, or aspartate transaminase (AST) or alanine transaminase (ALT) activity. DNA microarray analysis revealed that expression of genes of known function, such as those for the stress response, ribosomal proteins, transcription, cell function, the inflammatory/immune response, and metabolism (xenobiotic, glutathione, etc.) remained largely unaffected by KTE. However some genes such as catechol-o-methyltransferase and succinyl-CoA synthetase were regulated by KTE, indicating that KTE is not toxic to normal mice and might be effective as a functional food.

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The effects of poly-γ-glutamic acid (γ-PGA) on calcium (Ca) bioavailability and Ca content in bone were examined in female rats. Increases in in vitro Ca solubility was observed with increases in the amount of γ-PGA. Acute studies showed that γ-PGA increased the amount of soluble Ca in the small intestine. In a plasma-Ca kinetic experiment, γ-PGA stimulated Ca absorption 20 min after administration, and the duration of absorption was approximately 2 h. In an acute intestinal transit experiment, γ-PGA markedly increased intestinal transit in mice. In a chronic Ca-balance experiment, the results showed that γ-PGA significantly increased the apparent Ca absorption, apparent Ca balance, bone density, and bone Ca content in rats. In the same experiment, γ-PGA was also found to decrease the amount of soluble Ca in the small intestine, especially in the proximal small intestine, and to increase calbindin-D9k mRNA expression in the proximal small intestine in rats. In a chronic intestinal transit experiment, γ-PGA decreased intestinal transit in mice. These results suggest that γ-PGA increased Ca solubility, thereby enhancing Ca absorption in rats in the acute phase. In addition, chronic administration of γ-PGA to rats increased the apparent Ca balance and bone Ca content. The active transcellular pathway in the proximal small intestine was the major mechanism by which these effects were exerted.

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We examined the preventive activity of naturally occurring antioxidants against three reactive oxygen species using a protein degradation assay. The hydroxyl, hypochlorite, and peroxynitrite radicals are typical reactive oxygen species generated in human body. Previously, we found that hydrophobic botanical antioxidants exhibited specific antioxidant activity against hydroxyl radicals, whereas anserine and carnosine mixture, purified from chicken extract and vitamin C, exhibited antioxidant activities against hypochlorite and peroxynitrite radicals respectively. Since ethanol, used as a solvent in the experiments, also showed an antioxidant action against the hydroxyl radical, we re-assessed antioxidant activities using aqueous solutions of botanical antioxidants. Among the seven hydrophobic antioxidants examined, ferulic acid exhibited the strongest antioxidant activity against the hydroxyl radical. An antioxidant preparation of anserine-carnosine mixture, vitamin C, and ferulic acid prevented oxidative stress by reactive oxygen species. Loss of deformability in human erythrocytes and protein degradation caused by reactive oxygen species were completely inhibited.

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Protein from white lupin is capable of lowering plasma lipids. We investigated in this study the effect of total protein extracts (TPEs) from different cultivars of blue lupin (Probor, Vitabor and Boregine) and α-/β-conglutin from Boregine on the plasma lipids of rats. Rats were fed on a hypercholesterolemic diet containing either lupin protein (50 g/kg) or casein (50 g/kg) for 17 d. The rats fed with TPE from Vitabor and α-/β-conglutin had lower triglyceride concentrations in the plasma (−24% and −21%, respectively) and very-low-density lipoprotein (VLDL; −40% and −29%, respectively) than the rats fed with casein. TPE from Vitabor was also capable of lowering low-density lipoprotein (LDL) cholesterol (−37%). In the liver of the rats fed with TPE from Vitabor, the expression of the genes involved in triglyceride and cholesterol synthesis was down-regulated. This study shows that the Vitabor cultivar of blue lupin had the most beneficial effect on plasma lipids which was presumed to have been caused by the down-regulation of genes involved in lipid synthesis.

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Sterol oxidation products derived from cholesterol and phytosterol are formed during the processing and storage of foods. The objective of the present study was to assess the potential unfavorable effects of oxysterols in mice. C57BL/6J mice were fed an AIN-93G-based diet containing 0.2 g/kg of oxycholesterol or oxyphytosterol for 4 weeks. The most abundant oxysterol in the diet was 7-ketosterol, but α-epoxycholesterol, β-epoxycholesterol, or 7α-hydroxyphytosterol, and 7β-hydroxyphytosterol were more prominent than 7-ketosterol in the serum and liver respectively. Consumption of both oxysterols resulted in an increased in 4β-hydroxycholesterol and total oxycholesterol in the liver, but the oxycholesterol-fed mice had a lower level of cerebral 24S-hydroxycholesterol and a higher level of the serum triacylglycerols than the control and oxyphytosterol groups. These results indicate that both oxysterols in the diet are accumulated in the body, but that the biological effect of oxycholesterol is different from that of oxyphytosterol.

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To investigate the physiological functions of polyphenols from acerola (Malpighia emarginata DC.) fruit, the effects on melanogenesis were studied. The crude polyphenol concentrated extract from acerola (C-AP) was used to examine the skin-lightening effect on brownish guinea pigs which had been subjected to controlled UVB irradiation. The results show that C-AP significantly lightened the UVB-irradiated skin pigmentation. Furthermore, treatment with C-AP reduced the content of melanin in B16 melanoma cells, suggesting that the in vivo skin-lightening effect of C-AP was due to the suppression of melanin biosynthesis in melanocytes. In addition, we found that C-AP could effectively inhibit mushroom tyrosinase activity, the main constituents responsible for this effect being thought to be such anthocyanins as cyanidin-3-α-O-rhamnoside (C3R) and pelargonidin-3-α-O-rhamnoside (P3R). This result indicates that the skin-lightening effect of C-AP can be partly attributed to the suppression of melanogenesis through the inhibition of tyrosinase activity in melanocytes. An oral ingestion of C-AP may therefore be efficacious for reducing UVB-induced hyper-pigmentation by inhibiting the tyrosinase in melanocytes.

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Licorice flavonoid oil (LFO), which contains hydrophobic flavonoids from Glycyrrhiza glabra LINNE, is a new ingredient for functional foods. In this study, we investigated the anti-obesity action of LFO in diet-induced obese rats. The addition of 2% LFO in a high-fat diet significantly decreased the weight of abdominal adipose tissue and the levels of hepatic and plasma triglycerides. We found that the enzymatic activities of acetyl-CoA carboxylase and fatty acid synthase, the rate-limiting enzymes in the fatty acid synthetic pathway, were significantly decreased by LFO, whereas the enzymatic activity of acyl-CoA dehydrogenase, the rate-limiting enzyme in the fatty acid oxidative pathway, was significantly increased. All our findings suggest that the anti-obesity action of LFO is controlled by regulation of the rate-limiting enzymes in the fatty acid synthetic and oxidative pathways in the liver.

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To determine the effect of dietary protein level on experimental hyperhomocysteinemia, rats were fed 10% casein (10C) and 40% casein (40C) diets with or without 0.5% guanidinoacetic acid (GAA) for 14 d. In addition, rats were fed 10C + 0.75% methionine (10CM) and 40C + 0.75% methionine (40CM) diets with or without 2.5% serine for 14 d to determine the relationship between the dietary protein level and intensity of the hypohomocysteinemic effect of serine. GAA supplementation markedly increased the plasma homocysteine concentration in rats fed with the 10C diet, whereas it did not increase the plasma homocysteine concentration in rats fed with the 40C diet. Although serine supplementation significantly suppressed the methionine-induced enhancement of plasma homocysteine concentration, the decreased plasma homocysteine concentration was significantly lower in rats fed with the 40CM diet than in rats fed with the 10CM diet. The hepatic cystathionine β-synthase and betaine-homocysteine S-methyltransferase activities were significantly higher in rats fed with the 40C or 40CM diet than in rats fed with the 10C or 10CM diet, irrespective of supplementation with GAA and serine. These results indicate that the high-casein diet was effective for both suppressing GAA-induced hyperhomocysteinemia and potentiating the hypohomocysteinemic effect of serine, probably through the enhanced activity of homocysteine-metabolizing enzymes.

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The biological activity of polyphenols in vitro reflects their affinity for cell membranes and the amount of cellular incorporation. The interaction of polyphenols with phosphatidylcholine was investigated by HPLC with an immobilized artificial membrane (IAM) column. The IAM partition coefficients (K_IAM) of the polyphenols, calculated by retention times, correlated well with the amounts of polyphenols incorporation into the liposomes.

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The antioxidant properties of hot-water extracts from a dried chili pepper were maintained through the postharvest ripening process at 10 °C for 18 months. In order to isolate the antioxidant from the ripe pepper, we fractionated hot-water extracts by size-exclusion gel chromatography. A certain fraction showed antioxidative activity via the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity assay. Structural analysis by reversed-phase high performance liquid chromatography (HPLC), LC-MS, and nuclear magnetic resonance (NMR) revealed that the antioxidant was a known compound, p-coumaryl alcohol. This study indicates that an effective antioxidant in chili pepper sustains its antioxidative effects during the postharvest ripening process.

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Novokinin (RPLKPW), LPYPR, and rubiscolin (YPLDLF) are bioactive peptides with respective hypotensive, hypocholesterolemic, and memory-enhancing activities. We generated transgenic soybean lines that expressed modified forms of the α′ subunit of seed storage protein β-conglycinin containing tandem repeats of these bioactive peptides. The modified α′ subunits constituted up to 0.2% of extracted proteins from the transgenic seeds.

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To elucidate the role of β-glucanases in the cell-wall degradation involved in morphogenesis, an exo-β-1,3-1,6-glucanase (FvBGL1) was purified from fruiting bodies of the edible mushroom Enoki (Flammulina velutipes), and its enzymatic properties were studied. At least three β-glucanases were detected in the crude extract by zymogram assay when 1% laminarin was used as substrate. The molecular mass of FvBGL1 was estimated by SDS–PAGE to be 80 kDa. The optimum pH and temperature for the action of FvBGL1 were 6.1 and 60 °C respectively. FvBGL1 was completely inactivated by 1 mM mercuric ions. FvBGL1 hydrolyzed F. velutipes cell-wall β-glucan as well as β-1,3- and β-1,6-glucans from various sources with glucose as the only reaction product. Transglucosylation was observed when the enzyme acted on laminarinonaose. FvBGL1 can be assumed to degrade F. velutipes cell-wall β-1,3-glucan, but most probably acts more efficiently in concert with other endogenous β-glucan degrading enzymes.

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A gene (cel4) coding for a cellobiohydrolase II (Ex-4) was isolated from the white rot basidiomycete, Irpex lacteus strain MC-2. The cel4 ORF was composed of 452 amino acid residues and was interrupted by eight introns. Its deduced amino acid sequence revealed a multi domain structure composed of a cellulose-binding domain, a linker, and a catalytic domain belonging to family 6 of glycosyl hydrolases, from the N-terminus. cel4 cDNA was successfully expressed in the yeast Pichia pastoris. Recombinant Ex-4 showed endo-processive degrading activity towards cellulosic substrates, and a synergistic effect in the degradation of Avicel was observed when the enzyme acted together with either cellobiohydrolase I (Ex-1) or endoglucanase (En-1) produced by I. lacteus MC-2.

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Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.

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Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (CARDO-O) and electron transport components. CARDO can catalyze specific oxygenation for various substrates: angular dioxygenation for carbazole and dibenzo-p-dioxin, lateral dioxygenation for anthracene, and monooxygenation for methylene carbon of fluorene and sulfide sulfur of dibenzothiophene. To elucidate the molecular mechanism determining its unique substrate specificity, 17 CARDO-O site-directed mutants at amino acid residues I262, F275, Q282, and F329, which form the substrate-interacting wall around the iron active site by CARDO-O crystal structure, were generated and characterized. F329 replacement dramatically reduced oxygenation activity. However, several mutants produced different products from the wild-type enzyme to a large extent: I262V and Q282Y (1-hydroxycarbazole), F275W (4-hydroxyfluorene), F275A (unidentified cis-dihydrodiol of fluoranthene), and I262A and I262W (monohydroxydibenzothiophenes). These results suggest the possibility that the respective substrates bind to the active sites of CARDO-O mutants in a different orientation from that of the wild-type enzyme.

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We achieved the purification of three α-keto ester reductases (Streptomyces avermitilis keto ester reductase, SAKERs-I, -II, and -III) from Streptomyces avermitilis NBRC14893 whole cells. The molecular masses of the native SAKERs-I, -II, and -III were estimated to be 72, 38, and 36 kDa, respectively, by gel filtration chromatography. The subunit molecular masses of SAKERs-I, -II, and -III were also estimated to be 32, 32, and 34 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The purified SAKERs-II and -III showed a reducing activity for α-keto esters (in particular, for ethyl pyruvate). SAKER-I showed a high reducing activity not only toward the α- and β-keto esters, but also toward α-keto acid. The N-terminal region amino acid sequences of SAKERs-I, -II, and -III were identical to that of a putative oxidoreductase, SAV2750, a putative oxidoreductase, SAV1849, and a putative oxidoreductase, SAV4117, respectively, hypothetical proteins coded on the S. avermitilis genome.

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Marine yeasts were collected from coastal and deep sea areas in the Pacific Ocean and the Sea of Japan around central and northern Japan to prepare a novel type of natural seasoning. It was found that one of the marine yeasts collected from the Pacific Ocean off Hachinohe showed a high concentration of γ-aminobutyric acid (GABA) in its extract, about 7–10 times higher than those of commercially available bread yeast and other marine yeasts. The marine yeast isolated and named Hachinohe No. 6 catalyzed the reaction from monosodium glutamate to GABA only in the presence of glucose. Subsequently, several marine yeasts belonging to the genera Pichia and Candida were found to have such catalytic activities, but not those belonging to the genus Saccharomyces. Isolate Hachinohe No. 6 was found to have the highest catalytic activity among the yeasts examined in this study.

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A clone library of the partial formyltetrahydrofolate synthetase gene (fhs), a key enzyme in reductive acetogenesis, was constructed from the DNA of bovine rumen contents. Diverse sequences were recovered, the majority of which were clustered with the fhs of authentic acetogens. Low similarity values to known fhs were observed in all sequences, suggesting the presence of unknown acetogens.

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Cyclic isomaltooligosaccharides (CIs) usually consist of 7 to 12 glucose units, although only CI-10 has strong inclusion complex-forming ability. Four Bacillus strains and two Paenibacillus strains were isolated as novel CI-producing bacteria. Among these, five strains produced small amounts of CI-7 to CI-9, but mainly produced CI-10 to CI-12. Larger CIs, up to CI-17, were also identified.

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We investigated, to determine whether disruption of lipid rafts would influence the effect of three selected strains of Lactobacillus on TNF-alpha production by peripheral blood mononuclear cells. Two strains increased TNF-alpha production; and the third one reduced cytokine levels. Disruption of rafts changed the immunomodulatory effect of the three strains. This is the first report on a potential role of rafts in lactobacilli-cell interaction.

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