Neutral and acidic polysaccharides and their protein complexes were fractionated and purified from the brown seaweed umitoranoo (Sargassum thunbergii) by fractional extraction, iron-exchange chromatography, and gel filtration. Thirty-one polysaccharide fractions were obtained and tested for antitumor activity in mice with Ehrlich carcinoma transplanted i.p. Two of the fractions, GIV-A ([α]²⁵_D -127° and mol. wt., 19, 000) and GIV-B ([α]²⁵_D -110° and mol. wt., 13, 500) had such activity. On the basis of chemical and spectral analyses, these compounds were found to be a fucoidan or L-fucan containing approx. 30% sulfate ester groups per fucose residue, about 10% uronic acid, and less than 2% protein

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Water-soluble polysaccharide FI and water-insoluble polysaccharides FII, FIII-1, and FIII-2 were obtained from fruiting bodies of Tricholoma giganteum. Polysaccharides were further fractionated by ion-exchange chromatography, gel filtration, and affinity chromatography. The 24 polysaccharide fractions obtained were examined for their antitumor effect on Sarcoma 180 implanted in mice. The following antitumor-active polysaccharides were identified : FIo-a, a mixture of α-D-glucan and xyloglucomannan with an average molecular weight of 1. 6× 10⁶ ; FA-1, a β-D-glucan containing 1% protein and with a molecular weight of 4.O×10⁴ ; FII-1, a (1→3)-β-D-glucan containing 7.8% protein, with a molecular weight of 5.2×10⁴ ; FIII-1-b, a protein-polysaccharide complex (ratio, 37.5 : 62.5, w/w), with a molecular weight of 6.8×10⁴ and with xylose, galactose, mannose, and glucose in the polysaccharide moiety (proportions of 8.9 : 14.9 : 29.3 : 46.9 by weight), and FIII-2-a, b, and c, three (1→6)-β-D-glucosyl-branched (1→3)-β-D-glucans with a molecular weight from 2.6×10⁵ to 4.1×1O⁵ and containing small amounts of xylose and galactose and 3.5-8.30% protein.

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Approximately 4600 microorganisms isolated from soil and 110 bacteria from stock cultures were screened for their ability to hydroxylate 3-cyanopyridine (3-CP). Many strains were found to be capable of hydroxylating 3-CP. One of the strains with the highest activity was selected and identified as Comamonas testosteroni (MCI 2848). The product was isolated and identified as 3-cyano-6-hydroxypyridine (6-CHP). We optimized culture and reaction conditions for the production of 6-CHP by resting cells of MCI 2848. The activity was induced by addition of nicotinic acid or pyridine-3-aldoxime to the culture medium. The optimal temperature and pH of the reaction were 28°C and 7.0, respectively. When resting cells was incubated with 3% (w/v) 3-CP, 95% of the 3-CP was converted into 6-CHP within 47h.

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A glycerol oxidase from Aspergillus japonicus oxidized ethylene glycol to glyoxal by the same reaction pathway as alcohol oxidases from methanol yeast. The optimum pH and temperature for the oxidation of ethylene glycol were around 7.0 and 40°C, respectively. Those of glycolaldehyde were similar to those of ethylene glycol. The apparent K_ms for ethylene glycol and glycolaldehyde were 195 and 48.8mM, respectively. The maximum velocities for ethylene glycol and glycolaldehyde were 89.1 and 62.2μmol/min/mg of protein, respectively. Glycerol oxidase also oxidized glycolic acid, which is not oxidized by the alcohol oxidases, to glyoxylic acid like glycolate oxidases from green plants, and the apparent K_m and V_max for glycolic acid were 114mM and 2.68μmol/min/mg of proten, respectively. The glycerol oxidase was applicable to the production of glyoxal and glyoxylic acid.

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A 3.2-kb DNA fragment of the carbomycin biosynthetic region including the 3-O-acyltransferase gene (acyA) from Streptomyces thermotolerans was sequenced, and four ORFs were found in the fragment. The second ORF, designated ORF-A, was transcribed in the opposite direction to the other three ORFs. The first ORF was identified as carA, a gene for carbomycin resistance. The amino acid sequence of ORF-A was homologous to proteins of the cytochrome P-450 family. Streptomyces lividans transformed with pCB20, in which ORF-A was subcloned, epoxidized carbomycin B at its C-12, 13 positions, thus producing carbomycin A. The third ORF, the amino acid sequence of which showed a homology to macrolide antibiotics O-acyltransferases was identified as acyA. The last ORF (ORF-B), which starts just 3 bp downstream from the TGA termination codon of acyA, was thought to be a carbomycin 4-O-methyltransferase gene, because the amino acid sequence deduced from ORF-B showed high homology to a putative midecamycin 4-O-methyltransferase encoded on mdmC.

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When Bacillus sp. K40T was cultured in the presence of L-fucose, 1, 2-α-L-fucosidase was found to be produced specifically in the culture fluid. The enzyme was purified to homogeneity from a culture containing only L-fucose by chromatography on hydroxylapatite and chromatofocusing. The molecular weight of the enzyme was estimated to be 200, 000 by gel filtration on Sephadex G-200. The enzyme was optimal at pH 5. 5-7.0 and was stable at pH 6.0-9.0. The enzyme hydrolyzed the α-(1→2)-L-fucosidic linkages in various oligosaccharides and glycoproteins such as lacto-N-fucopentaose (LNF)-I <O-α-L-fucose-(1→2)-O-β-D-galactose-(1→3)-N-acetyl-O-β-D-glucosamine-(1→3)-O-β-D-galactose-(1→4)-D-glucose>, porcine gastric mucin, and porcine submaxillary mucin. The enzyme also acted on human erythrocytes, which was confirmed by the hemagglutination test using Ulex anti-H lectin. The enzyme did not hydrolyze α-(1→3)-, α-(1→4)- and α-(1→6)-L-fucosidic linkages in LNF-III <O-β-D-galactose-(1→4)[O-α-L-fucose-(1→3)-]-N-acetyl-O-β-D-glucosamine-(1→3)-O-β-D-galactose-(1→4)-D-glucose>, LNF-II <O-β-D-galactose-(1→3)[O-α-L-fucose-(1→4)-]-N-acetyl-O-β-D-glucosamine-(1→3)-O-β-D-galactose-(1→4)-D-glucose> or 6-O-α-L-fucoPyrano-syl-N-acetylglucosamine.

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The effects of isorhamnetin, rhamnetin and quercetin on the serum and liver cholesterol concentrations, liver lipoperoxide (thiobarbituric acid-reactive substances : TBARS) content, and antioxidative enzyme activities were examined with rats fed on cholesterol-enriched and cholesteero-free diets. The total serum cholesterol of those rats fed with the cholesterol-enriched diet was decreased by feeding each all these flavonoids. The total liver cholesterol concentration and TBARS content in the rats fed with the cholesterol-free diet were decreased by feeding isorhamnetin, rhamnetin and quercetin. The activities of liver superoxide dismutase and catalase were almost unaffected by feeding these flavonoids. These results, the in vitro antioxidative activities of isorhamnetin, rhamnetin and quercetin, and the activities of these flavonoids in suppressing the generation of the superoxide anion in vitro suggest the possibility that the lower liver TBARS content in those rats fed on the cholesterol-free diet with added flavonoids is ascribable in part to the direct antioxidative and superoxide anion generation-suppressing activities of flavonoids and/or their metabolites absorbed from the gastrointestinal tract.

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The inhibitors of 2, 3-oxidosqualene : lanosterol cyclase were investigated by comparative studies between pig's liver and Baker's yeast. The fundamental skeleton of the inhibitors was 8-azadecalin. To the nitrogen atom, an isoprenoid-like chain [nerylacetone (Z-form), geranylacetone (E-form) or its hydrogenated form] was attached by the reaction of reductive amination with NaCNBH₃. Among the three forms, the Z-isomer was the most potent inhibitors toward both the pig's liver and yeast cyclases. To examine the effect of carbon chain length (lipophilicity), various fatty acids (C6-C18) were appended to the 8-azadecalin derivatives. Strong inhibitory activity was observed for those compounds having carbon chains around C12. Interestingly, the amide compounds (not the carbocationic intermediate) exhibited remarkably strong inhibition toward the liver cyclase, whereas they had an insignificant effect on the yeast cyclase (about 10²-fold less active). The yeast cyclase needed the amine functionality (carbocationic intermediate), which was prepared by using LiAlH₄ from the corresponding amides, to exhibit potent inhibition. We found that N-dodecyl-8-aza-4, 4, 10β-trimethyl-trans-decal-3β-ol (7i) was the most potent inhibitor (IC₅₀ = 1μM) toward the yeast cyclase amongst any known material. Kinetic studies showed that the inhibition pattern was dependent only on whether the side chains on the 8-azadecalin were linear or branched ; the compounds having isoprenoid-like chains were non-competitive inhibitors, while those having linear hydrocarbon chains (amides or amines) were competitive inhibitors.

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A glutamate racemase gene of Lactobacillus brevis ATCC 8287 was cloned into Escherichia coli TM93 by the phenotypic complementation of a phosphoenolpyruvate carboxylase deficiency on minimum agar medium containing D-glutamate. The gene was localized to a 1.4-kb HindIII-EcoRI DNA fragment and the total nucleotide sequence of the fragment was analyzed. The gene has typical promoter and SD sequences which appeared to function in E. coli. The deduced amino acid sequence of the enzyme had 276 amino acids and the molecular weight was calculated as 29, 426. Two cysteine residues and their surrounding regions of the enzyme are homologous to those of other cofactor-independent racemases. The glutamate racemase was puritied from recombinant E. coli to homogeneity and characterized. The enzyme required no cofactors for the activity, and retained its activity even in 2M (300g/l) L-glutamate.

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The effect of theanine, one of the components of green tea, on the blood pressure and brain 5-hy-droxyindoles in spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) was investigated by intraperitoneally administering theanine. The effect of glutamine, which is structurally similar to theanine, was also examined. When SHR were injected with various amounts of theanine (0, 500, 1000, 1500, and 2000mg/kg), the change was dose-dependent, and a significant decrease in blood pressure was observed with the high doses (1500 and 2000 mg/kg). A dose of 2000 mg/kg of theanine did not alter the blood pressure of WKY, while the same dose to SHR decreased it significantly. On the other hand, glutamine administration to SHR did not change either the blood pressure or the heart rate. The brain 5-hydroxyindole level was significantly decreased by theanine administration to both WKY and SHR, the decrease being dose-dependent.

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The thermostable α-galactosidase from Candida guilliermondii H-404 synthesized self transfer products in the absence of a suitable acceptor. The main self transfer product, using melibiose as a donor substrate, was O-α-D-galactosyl-(1, 6)-O-α-D-galactosyl-(1, 6)-D-glucose. This enzyme had a wide acceptor specificity. D-Glucose, D-galactose, maltose, maltitol, and 1, 4-butandiol were the most effective acceptors in the transgalactosylation catalyzed by this enzyme. The enzyme could also transfer α-galactosyl residues to pentoses (L-arabinose, D-xylose, and D-ribose) and methyl pentoses (D-fucose and L-rhamnose). The main transfer products to lactose, maltose, and sucrose as acceptors were identified as O-α-D-galactosyl-(1, 6)-O-β-D-galactosyl-(1, 4)-D-glucose, O-α-D-galactosyl-(1, 6)-O-α-D-glucosyl-(1, 4)-D-glucose, and O-α-D-ga-lactosyl-(1, 6)-O-α-D-glucosyl-(1, 2)-β-D-fructoside (raffinose), respectively.

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The interaction of IL-4, IL-5, and free bile acids with the immunoglobulin production by mouse spleen lymphocytes was studied to examine their immunoregulatory activity. In the absence of lipopolysaccharide (LPS), IL-4 enhanced the IgE and IgG production significantly and the IgA production weakly, but not the IgM production. On the other hand, IL-5 had an inhibitory tendency on the IgE and IgA production, though not significantly. In the presence of LPS, both IL-4 and IL-5 significantly enhanced the IgE production by mouse splenic lymphocytes. When the lymphocytes were cultured with the physiological concentration of free bile acids (10μM) and LPS for 3 days, chenodeoxycholic acid inhibited the IgE production, but cholic and deoxycholic acids did not. In the presence of lL-4 or IL-5, these bile acids cancelled the stimulatory effects of interleukins and rather significantly inhibited the IgE production. These results suggest that these free bile acids act as an anti-allergic agent.

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Enzyme solutions of glucoamylase, acid protease, alkaline protease and lipase were treated with micro-bubbles of supercritical carbon dioxide (SC CO₂) fed from a cylindrical filter nozzle. The microbubbles of SC CO₂ could increase the CO₂ concentration in the sample solution from 0. 4 to 0. 92mol/l at 25 MPa and 35°C, and hence could improve the efficiency of inactivation by about 3 times compared to treating without the filter nozzle. Alkaline protease and lipase in the solution could be completely inactivated by the treatment at 35°C and 15 MPa for 30 min. With the inactivation of glucoamylase and acid protease, their residual activity-CO₂ density profiles consisted of two straight lines with intersections at density values of 0. 82 and 0. 60g/cm³, respectively. These enzymes showed an abrupt decrease in activation in regions above the CO₂ density of their intersections.

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An alginate lyase from the culture supernatant of Enterobacter cloacae M-1 was purified by ammonium sulfate precipitation, cation-exchange chromatography (SP-Toyopearl), and gel filtration (Ultrogel AcA44). The final preparation thus obtained showed a single band on SDS-PAGE. The purified enzyme had the molecular weight of 38, 000 and 32, 000 by SDS-PAGE and gel filtration, respectively. The pI of the enzyme was 8. 9. The optimum pH and temperature for the enzyme reaction were around 7. 8 and 30°C, respectively. The enzyme was unstable on heating. EDTA completely inhibited the enzyme activity, but the activity was completely restored by the treatment with CaCl₂. The enzyme was specific for poly-guluronate and produced several kinds of unsaturated oligomers from the gluluronate. This suggested that the enzyme could be classified as an endo poly-guluronate lyase.

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A partially purified sialidase that preferentially hydrolyzes colominic acid, which is a homopolymer of N-Acetylneuraminic acid linked by α-2, 8 linkages, was prepared from Bacteroidesfragilis SBT3182. This enzyme had a K_m of 0. 63mM for α-2, 3-sialyllactose, 0. 97mM for α-2, 6-sialyllactose, and 0.01mM for colominic acid. Colominic acid was a good substrate of this enzyme. By using this kinetic property of the enzyme, we transferred sialic acid from colominic acid to lactose by the transferase activity of the sialidase. Both α-2, 3-sialyllactose and α-2, 6-sialyllactose were synthesized with a total yield of 0.14%. We also observed that the ratio of synthesized sialyllactose isomers changed with the reaction time while the yield was constant.

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The selective incorporation of n-3 and n-6 unsaturated fatty acids into phospholipids was investigated by changing the ratio of n-3 unsaturated fatty acids against n-6 unsaturated fatty acids in the medium where Chinese hamster V79-R cells were grown. Unsaturated fatty acids with lower degrees of unsaturation were abundantly incorporated into phosphatidylcholine independently of n-3 and n-6. Unsaturated fatty acids with higher degrees of unsaturation were more predominantly incorporated into phosphatidylethanolamine. When the difference in the numbers of double bond between unsaturated fatty acids was more than two, the unsaturated fatty acids with a higher degree of unsaturation was more selectively incorporated into phosphatidylethanolamine than the unsaturated fatty acids with a lower degree of unsaturation. By the analysis of molecular species, n-3 and n-6 unsaturated fatty acids with higher degrees of unsaturation were incorporated competitively into phosphatidylethanolamine. Finally, docosahexaenoic acid was incorporated into diacyl phosphatidylethanolamine more selectively than the other unsaturated fatty acids.

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A γ-glutamylmethylamide (γ-GMA)-dissimilating enzyme system, H protein and L protein, was purified to homogeneity from a cell-free extract of Methylophaga sp. AA-30. H protein contained flavin and had a molecular mass of 360kDa. It consisted of two dissimilar subunits with molecular masses of 55 and 29kDa. L protein had a molecular mass of 38kDa and contained 1 mol of thiol group per mol of protein. Since L protein in the native form was very labile, the protein was treated with 5, 5'-dithiobis(2-nitrobenzoate) to produce a stable but inactive derivative of the protein, which was purified then reactivated with dithiothreitol. The γ-GMA-dissimilating enzyme system catalyzed the formation of glutamate, formaldehyde, 2-ketoglutarate, and ammonia from γ-GMA, 2-ketoglutarate, and ammonia via the two-step reaction shown below.

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Two chitinases, designated PLC-A and PLC-B, were purified from the leaves of pokeweed (Phytolacca americana) using DEAE-cellulose column chromatography followed by gel filtration on Sephadex G-75, hydrophobic column chromatography, and ion-exchange FPLC. PLC-A and PLC-B are acidic and basic proteins having molecular masses of 25 and 29kDa, and isoelectric points of 3.7 and 9.5, respectively. On the basis of their partial amino acid sequences, it was seen that PLC-A and PLC-B belong to class II and class III chitinases, respectively. The optimal pH of PLC-A toward glycolchitin is pH 4.5 and hydrolyzed (GlcNAc)₄ into 2(GlcNAc)₂, and (GlcNAc)₅ - ₆ into (GlcNAc)₂ and (GlcNAc)₃. On the other hand, PLC-B has two optimal pHs at 3 and 7 toward glycolchitin and hydrolyzed (GlcNAc)₅ into GlcNAc and (GlcNAc)₄, and (GlcNAc)₆ into GlcNAc, (GlcNAc)₂, and (GlcNAc)₄.

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Alkalophilic Bacillus sp. KSM-1378 produces an alkaline amylopullulanase that hydrolyzes both α-1, 4 linkages in amylose, amylopectin, and glycogen and α-1, 6 linkages in pullulan. The hydrolytic activities against amylose and pullulan were specifically inhibited by maltotriose (K_i=0. 5mM), isomaltitol (K_i=5. 2mM), and methyl α-D-galactoside (K_i=40mM) and by β-cyclodextrin (K_i=0. 9mM), α-cyclodextrin (K_i=11mM), and raffinose (K_i=31mM), respectively, in a competitive manner in each case. Inhibition by N-bromosuccinimide of the α-amylase activity was prevented by amylose but not by pullulan, while inhibition by N-bromosuccinimide of the pullulanase activity was prevented by pullulan but not by amylose. Kinetics of reactions in the simultaneous presence of amylose and pullulan indicated that the observed rates of formation of products closely matched those predicted by a kientic model in which the α-1, 4 and α-1, 6 hydrolytic reactions were catalyzed at two independent active sites. Incubation of the enzyme at 40°C and pH 9. 0 caused complete inactivation of the amylase activity within 4 days, but the pullulanase activity remained at the original level under the same conditions. This alkaline amylopullulanase can, therefore, be considered to be a "two-headed" enzyme molecule.

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An emulsification method using a gel-like phase of a saccharide and protein mixture has been developed. In the method, which is called a gel emulsification method, an oil is added to the highly concentrated saccharide solution containing protein to form a clear gel-like phase, which followed by dilution with water to form a fine oil-in-water emulsion. This emulsion was investigated as to its emulsifying activity and emulsion stability as compared with that obtained by high-shear equipment, which was called a homomixer method. The emulsifying activity of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method. The emulsions prepared by both methods were highly stable in terms of the stability against coalescence. On the other hand, the stability against creaming of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method. The surface hydrophobicity of the protein and the unfreezable water content in the highly concentrated saccharide solution containing protein were not correlated to the emulsifying properties of the emulsions prepared by the gel emulsification method, which appeared to be dependent on the viscosity of the highly concentrated saccharide solution containing protein.

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Four molecular forms of α-glucosidase were isolated from spinach seeds by several kinds of chromatography. The molecular masses of α-glucosidases I, II, III, and IV were 78, 78, 82, and 82kDa by SDS-PAGE, and 62, 62, 190, and 70kDa by gel filtration, respectively. α-Glucosidases I and II showed similar enzymatic properties, in which the K_m for soluble starch was about 10 times lower than that for maltose, and they had higher activity not only toward malto-oliosaccharides but also toward α-glucans. The optimum pH was 4.5-5.5 and about 50% of the activity remained after incubation at 70°C for 20min. On the other hand, α-glucosidases III and IV showed similar enzymatic properties, in which the K_m for maltose was 3-4 times lower than that for soluble starch, and they had high activity toward malto-oligosaccharides but faint activity toward α-glucans. The optimum pH was 4.5-5.0 and no activity was found after incubation at 70°C for 20min. However, anti-α-glucosidase III serum formed precipitation specifically with α-glucosidase III.

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A DNA fragment coding for a part of a putative phosphatidylinositol 3 kinase was cloned from Schizosaccharomyces pombe by cross-hybridization with Saccharomyces cerevisiae VPS34 gene, a yeast homologue of mammalian PI-3 kinase. The clone contained an open reading frame of 797 amino acids but lacked the initiation codon, ATG. The predicted amino acid sequence was homologous to those of S. cerevisiae VPS34 and mammalian PI-3 kinase genes. Disruption of the gene resulted in extremely low levels of PI-3-P and higher levels of PI-4-P, supporting the idea that the gene codes for the PI-3 kinase of S. pombe. The disruptants harbored large vacuoles and were sensitive to stresses such as high temperature or high concentration of monovalent and divalent cations.

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Three mitogenic lectins, designated PL-A, PL-B, and PL-C, were purified from the roots of pokeweed (Phytolacca americana) using Q-Sepharose column chromatography followed by gel filtration on Sephadex G-75, hydrophobic chromatography on Butyl-Toyopearl, and FPLC on a Mono-Q column. PL-A, PL-B, and PL-C are acidic proteins having isoelectric points of 4. 35 and their apparent molecular masses were 22, 48, and 21kDa by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, respectively. The three lectins have similar amino acid compositions rich in half-cystine and similar N-terminal sequences, indicating that they are homologous proteins. Identical sequences of N-terminal regions and six corresponding tryptic peptides in PL-A and PL-B suggested that PL-A may be an N-terminal half fragment of PL-B. Although all of three lectins have mitogenic activities, PL-B is a mitogenic lectin with the most potent hemagglutinating and mitogenic activities, and PL-C has almost no hemagglutinating activity.

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The role of the acidic fragment (Asp-Glu-Glu) in delicious peptides was investigated in detail by using the Na⁺ or K⁺ salts of acidic oligopeptides so that amount of Na⁺ or K⁺ intake of peptides composed of acidic amino acids could be varied by changing their sequences. The taste of these peptides was confirmed to vary with Na⁺ or K⁺ intake. Additionally, in order to study the role of basic (Lys-Gly) and acidic (Asp-Glu-Glu) fragments in delicious peptides for producing the taste, five delicious peptide analogs, Ser-Leu-Ala-Lys-Gly-Asp-Glu-Glu, Ser-Leu-Ala-Asp-Glu-Glu-Lys-Gly, Lys-Gly-Ser-Leu-Ala-Asp-Glu-Glu, Lys-Gly-Asp-Glu-Glu, and Glu-Glu-Asp-Gly-Lys, were synthesized. The intensity of the umami and/or salty taste of these peptides and their Na salts was almost the same, despite their chemical structures being different. These results indicate that the acidic fragment as well as the basic fragment plays an important role in the taste production and intensity of delicious peptides, and that an umami or salty taste can be produced by the localization of the cation of the basic fragment and the anion of the acidic fragment.

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The nucleotides of a bifunctional enzyme FAD synthetase gene, which showed both flavokinase and ATP : FMN adenylyltransferase activities, from Corynebacterium ammoniagenes were sequenced. The FAD synthetase gene product consisted of 338 amino acids and had a calculated molecular weight of 37, 712. The deduced protein sequence of the FAD synthetase shared a homology with those of the protein X of Escherichia coli, which has been reported to have both flavokinase and ATP : FMN adenylyltransferase activities like the FAD synthetase of C. ammoniagenes, and the protein X of Pseudomonas fluorescens. From the analysis of the flanking sequences of the FAD synthetase gene, the gene organization and the operon structure around the FAD synthetase gene of C. ammoniagenes were thought to be different from those of Gram-negative bacteria. An over-expression system of the FAD synthetase of C. ammoniagenes was constructed in E. coli to study the structure and function of the protein. Under the tandem tryptophan promoter, the FAD synthetase activity increased 2231 times compared to that of non-transformed C. ammoniagenes.

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A bacterial sex pheromone, cOB1, which induces conjugal transfer of the Enterococcus faecalis hemolysin-bacteriocin (Hly/Bac) plasmid, pOB1, was isolated from the culture broth of pOB1-free E. faecalis. Its structure was found to be a hydrophobic octapeptide, H-Val-Ala-Val-Leu-Val-Leu-Gly-Ala-OH. The cOB1 peptide induced the mating response of not only pOB1 but also another incompatibility group Hly/Bac plasmid, pYI1.

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The purpose of this study is to prepare Worcestershire sauce with higher levels of ethanol and aromatic components by a trickle bed bioreactor. Ethanol productivity in a trickle bed bioreactor was measured with changing circulation rates from 40 to 280ml/min with a fixed aeration rate (200ml/min) at 28°C. Compared with the lower circulation rate (40ml/min), at 210ml/min of circulation rate or higher, ethanol productivity increased 10. 3-fold at 41h of fermentation.

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Tastes of Leu-Lys-Tyr (LKY) analogues, a series of potent angiotensin I converting enzyme (ACE) inhibitory peptides were evaluated. Some of these analogues were found to be sweet, such as Val-Lys-Tyr and Ala-Orn-Tyr. Furthermore, the structural requirements for sweetness or decreasing the bitterness were investigated by considerations of the structure-taste relationship with LKY analogues.

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The pesticidal activities of trehazolin (TRZ), a specific and potent trehalase inhibitor, are described. TRZ inhibited trehalase of Rhizoctonia solani, a plant pathogenic fungus, with an IC₅₀ value of 66nM. Spraying rice seedlings with TRZ at a concentration of l00μg/ml suppressed infection by Rhizoctonia solani. TRZ showed insecticidal activity toward silkworms by injecting 50 to 100μg.

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The concentration of ceruloplasmin (CP) in human milk was obtained by measuring the increased absorbance of a solution containing apotransferrin and Fe²⁺ at 460nm after adding the milk. The CP concentrations in colostrum and mature milk at less than 1 month after parturition were 4.45±1.23mg and 4.09mg±1.47mg/1OOml, respectively. In mature milk after more than one month, the CP concentration had decreased to 1.72±0.91mg/100ml.

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The effects of amino acid composition on the conversion ratio of Trp to niacin in rats were investigated. The ratio in the group fed with a nicotinic acid-free, 9% casein-sucrose diet (basal diet) was 3.5%. This ratio was statistically decreased to 0.2% by the addition of 2% glycine, 0.078% L-threonine, and O.20% L-cystine, and restored to 3.2% by the further addition of 0.1% tryptophan. From these results and our previous findings, it was considered that Trp imbalance might be a manifestation of niacin deficiency.

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Swelling of a polyelectrolyte complex gel prepared from xanthan and chitosan was sensitive to the ambient pH. In NaOH solutions, the complex gel swelled at pH l0-12, and the maximum swelling was observed at pH l0. The gel also swelled in HCl solution at pH 0, though it dissolved at pH 0.2-1.0 in the course of swelling. In NaCl solutions and Carmody buffer solutions, the pH at which the maximum swelling occurred shifted to pH 8 though the equilibrium swelling ratio decreased.

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Pseudomonas sp. B21C9, a new nitrile-degrading microorganism, was isolated from a soil sample. By the hydrolysis of (RS)-iso-propyl-4'-chlorophenylacetonitrile using cells of B21C9, (S)-2-isopropyl-4'-chlorophenylacetic acid having excellent optically purity could be obtained. It appeared that the microbial hydrolysis proceeded via stepwise reactions by a nitrile hydratase having poor (S)-selectivity and an amidase having strict (S)-selectivity.

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The proportion of hexanal to total carbonyl compounds in soybean extracts was about 1-2%. When the extract was incubated in the presence of exogenous linoleic acid, the content of carbonyl compounds increased considerably. The proportion of hexanal to total carbonyl compounds derived from linoleic acid by enzymatic action was about 20%.

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A series of homo and heterocyclic alkanoates were prepared and evaluated as insect attractants toward Blattella germanica (L.) and Supella longipalpa (F.). Among these compounds, the tetrahydrofurfuryl alkanoates and 4-tetrahydropyranyl hexanoate showed relatively better activity. It was also observed that, in general, the five-membered compounds were better performers than their sixmembered counterparts. The introduction of an oxygen atom in the ring enhanced the activity, irrespective of the ring size.

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Thiobacillus ferrooxidans AP19-3 has a novel NADH-dependent sulfite reductase in the periplasmic space. The gene responsible for the appearance of NADH-dependent sulfite reductase activity was cloned into a vector plasmid pBR322 to give a 5.7-kb hybrid plasmid, pTHS1, which contains a 1.3-kb DNA fragment of T. ferrooxidans AP19-3. When pTHS1 was used to transform sulfite reductase deficient E. coli mutants, strain AT2455 (cysG), JM246 (cysl), and AT2427 (cysJ), it complemented only the E. coli cysG mutation. Since cysG codes for S-adenosyl-L-methionine : uroporphyrinogen III methyltransferase, the enzyme involved in siroheme synthesis, the results indicate that the DNA region that codes for S-adenosyl-L-methionine : uroporphyrinogen III methyltransferase is present in a T.ferrooxidans 1.3kb DNA fragment on pTHS1.

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Peptide mixtures with high and low Fischer's ratios (i.e., the ratio of branched-chain amino acids to aromatic amino acids), obtained from an enzymatic hydrolysate of casein, and the corresponding amino acid mixtures were intragastrically force-fed to rats. Cerebral cortex monoamines were more influenced by the ingestion of the peptide than that of the amino acid mixture.

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The minimum size DNA fragment (3011 bp) containing the entire phosphoenolpyruvate carboxylase [EC 4.1.1.31] gene of E. coli was cloned into a modified plasmid vector of high copy-number. The gene expression was directed by its own promoter and the content of the enzyme reached about 30% of total soluble protein of the transformed cells.

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Benzyl 6-0-β-D-xylopyranosyl-β-D-glucopyranoside (β-prime-veroside) (1), 2-phenylethyl β-primeveroside (2), and 2-phenylethyl 6-O-α-L-rhamnopyranosyl-β-D-glucopyranoside (β-rutinoside) (3) were isolated as aroma precursors of benzyl alcohol and 2-phenylethanol from flower buds of Jasminum sambac Ait. The isolation was guided by an enzymatic hydrolysis and GC and GC-MS analyses.

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Hop bitter acids, humulones (1) and lupulones (2), were shown to have potent DPPH radical scavenging activity (RSA) and lipid peroxidation inhibitory activity (LIA). Furthermore, 5-acetyl lupulones (3) and 4-methyl lupulones (4) had more potent LIA than native lupulones but no RSA. This result indicates that the β, β'-triketone moiety of the lupulones has LIA.

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Many protein-free media have been deveoped, because protein-free media are usually more economical than serum-free or serum-containing media and facilitate the purification of bioactive materials. We evaluated various iron salts and chelating agents replacing transferrin to develop a protein-free medium for a human-human hybridoma, HB4C5, and found out that ferric citrate was favorable for the production and the productivity of monoclonal antibodies.

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Hordeumin stored at -40 to -80°C in 1% HCl-methanol suffered neither from color reduction nor discoloration. After heating at 80°C for 60min, hordeumin showed a pigment retention rate of 100%. This characteristic is because the pigment is a composite high-molecular weight compound consisting of anthocyanins and polyphenols. It was determined, however, that discoloration and browning occurred more rapidly than color reduction during storage and heating of the pigment.

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L-Asparaginase was extracted from Candida utilis cells using various reducing agents, 2-mercaptoethanol, dithiothreitol, or cysteine. The extraction of the enzyme depended upon the kind and concentration of reducing agents, temperature, time of incubation, and pH of buffer used. The enzyme was typically extracted by incubating the cells at 50°C for 4h in extraction solution containing 20mM 2-mercaptoethanol in 20mM potassium phosphate buffer (pH 7.0). The enzyme can be extracted from either cell precipitate or cell culture broth. The yeast cells were viable after extraction of L-asparaginase.

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A zinc-binding protein was purified to homogeneity from a scallop, Patinopecten yessoensis. The protein was synthesized under zincenriched conditions like metallothioneins (MTs). The protein, however, did not satisfy the criteria for classification as MTs in amino acid composition because the cysteine content was extremely low and asparatic and glutamic acids were the predominant residues. These results suggested that the Zn-binding protein purified in this study was a new inducible protein and involved in zinc storage in the scallop.

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Bacteria cell suspensions were sterilized by direct electric current and the cell death rate was proportional to the current. When repeated hydrolysis of casein by immobilized mycelia was done under 0.06 A of direct electric current, contaminants were inhibited while the hydrolysis was stable for more than 10 batches (250h).

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Besides corn cystatin I (CC-I) we found, by cloning, another molecular species of cystatin, named corn cystatin II (CC-II), which was found by screening with CC-I cDNA as a probe. The dissimilarity in amino acid sequence between CC-I and CC-II was distinct around the N-terminal region. Enzymologically, CC-II protein expressed in Escherichia coli was a stronger inhibitor of cathepsin L than CC-I.

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We cloned four kinds of goat insulin-like growth factor-I (IGF-I) cDNAs. Each of them encodes the same mature IGF-I and different signal peptides. In this study, we analyzed expression of the four kinds of mRNAs in tissues in various developmental stages by a reverse transcriptase-polymerase chain reaction assay. The results suggest that their expression may be controlled by different promoters, the activities of which are tissue- and development-specific.

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2, 6-Dideoxy-2, 6-imino-L-gulonic acid (1) with inhibitory activity against β-D-glucuronidase and α-L-iduronidase was stereoselectively synthesized from 2, 3-di-O-benzyl-N-benzyloxycarbonyl-6-O-t-butyldiphenylsilyl-1, 5-dideoxy-1, 5-imino-D-glucitol (4). Through a deoxygenation reaction at the C-4 position of the latter, 2, 3, 6-trideoxy-2, 6-imino-L-gulonic acid (2) was also prepared.

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Purified recombinant sheep angiotensinogen (rsAngn) lost 74% of the reactivity with human renin during storage at pH 8.0 and 4°C. The inactivated rsAngn was reactivated by incubation at acidic pHs. This indicates that pH-dependent inactivation and reactivation occur in rsAngn.

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We developed an accurate method to separate and detect proteases using isoelectric focusing electrophoresis. The simultaneous addition of 0.3% octyl-D-glucopylanoside or 8M urea to acrylamide gel containing gelatin and of cytochrome c and pl marker proteins to the sample solution prevented interaction between protease and substrate during electrophoresis. Picogram to nanogram quantities of commercial proteases, papain, chymotrypsin, and proteinase K, were detected at the estimative isoelectric point of these proteases.

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