Mutation experiments were performed to decrease the protease productivity of Aspergillus awamori var. kawachi using ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine. The selected mutant HF-15 showed reductions in protease productivity of 93%, 84% and 50% in solid wheat bran culture, shaking Medium B and wheat bran cultures, respectively, as compared with the parent. Protease-less mutant HF-15 failed to produce α-mannosidase, and N-acety1-β-Dglucosaminidase productivity decreased by 35%. Mutant HF-15 specifically produced a high amount of raw starch-adsorbable and raw starch-digestive glucoamylase similar to GAI under all tested cultural conditions. On the contrary, high protease-producing mutant HF-10 produced a glucoamylase with very limited adsorption and digestion capacity on raw corn starch, and lower hydrolysis toward gelatinized potato starch and glycogen that was similar to GA I'.

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A debranching enzymewas extracted from the endospermof germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.

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Endo β-1, 3-ghicanase IV (E.C. 3.2.1.6, endo-1, 3(4)-β-D-glucanase) from Flav. dormitator var. glucanolyticae FA-5 was shown to be a glycoprotein by gel filtration and sodium dodecyl sulfate gel electrophoresis. The carbohydrate moiety was composed of 17 hexose units. The enzyme had an apparent molecular weight of 3.3×10⁴, determined by gel nitration, sodium dodecyl sulfate gel electrophoresis and ultracentrifugation. The enzyme showed maximum reactivity at pH 6.0 and 6.5 for living yeast cells and laminaran, respectively. The enzyme predominantly released laminaripentaose from a variety of linear β-1, 3-glucans and showed transglucanosylation activity. The amino acid composition of the enzyme and some of its physicochemical and enzymatic properties are described.

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The action pattern and amino acid residues involved in the active site of endo β-1, 3-glucanase IV from Flav. dormitator var. glucanolyticae FA-5 were investigated. The glucanase was specific for straight chains of β-1, 3-glucosidic linkages. The enzyme endowisely hydrolyzed β-1, 3-ghicans to produce laminaripentaose and showed transglucanosylation activity. The glucanase was inactivated by modification of histidine residues with diethylpyrocarbonate and by photooxidation, and by modification of carboxyl residues with Woodward's reagent K and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. The presence of β-1, 3-ghicans resulted in protection of the enzyme from inactivation by modifications, suggesting that these residues are at the β-1, 3- glucan binding and/or active site of the glucanase molecule.

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The distribution of oligomeric proanthocyanidins in legume seeds was investigated in connection with organoleptic astringency. The seeds contained various kinds of oligomers (dimers-hexamers). The total oligomer contents were at least 0.040% in azuki beans, 0.017% in black soybeans, 0.005% in mung beans and a trace amount in regular soybeans. Each aliquot of aqueous solution of partially purified proanthocyanidin oligomers tasted astringent even at a concentration of 0.001 %(w/v). The possible contribution of oligomeric proanthocyanidins and polymeric proanthocyanidins to the astringent off-taste of legume seeds is discussed.

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Six kinds of dimeric procyanidins were isolated from azuki beans by combination column chromatographies on Polyamide C-200 and Sephadex LH-20. Judging from the chemical properties, UV absorbance, MS pattern of the isolated procyanidins and the analysis of the products through acid-hydrolysis, they were identified as: a procyanidin with a C₄-C₈ linkage (C₃₀H₂₆O₁₁) and five conformational isomers of Type-B procyanidins with C₄-C₈ linkages (C₃₀H₂₆O₁₂). Each aliquot of aqueous solution (0.001 %) of dimeric procyanidins tasted astringent. The total content of dimeric procyanidins was assumed to be at least 0.011% in azuki beans. These compounds may contribute to the astringency of the bean and its products.

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A thermo-labile antigen (TLA) on the yeast cell surface was isolated from a yeast cell autolyzate and purified to a homogeneous state by chromatography on an immunoadsorbent affinity column. The molecular weight of TLA was about 1.45 x 10⁵ on SDS-polyacrylamide gel electrophoresis and about 1.5x 10⁵ on gel chromatography on Sephadex G-200. The TLA contained 74.5% protein and 25.5% sugar. It was characterized by high contents of glycine, glutamic acid, serine and aspartic acid. Half-cystine, methionine, histidine and arginine were not found. The sugar moiety was composed of galactose, mannose, 7V-acetylglucosamine and fucose. The antigenic determinant of TLAwas distinct from that of cell wall mannan in the Ouchterlony immunodiffusion test. No precipitin line against anti-TLA serum was observed, when TLAwas heated at 90°C for 10 min. Oxidation with periodate had little effect on antigenicity, but digestion with Pronase or treatment with protein denaturants resulted in loss of the antigenicity. These results suggest that the protein moiety plays an important role as the antigenic determinant of TLA. Moreover, the antiserum specific to TLAagglutinated fresh yeast cells, and the distribution ofTLA was apparent on the yeast cell surface by immunofluorescence staining. These findings suggest that TLAmolecules were exposed on the outer surface of the yeast cell wall.

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Benzyloxycarbonyl-L-cysteine and glycine benzhydrylamide were condensed by papain in a yield of 39.5%. After elimination of the N-protecting group, L-cysteinylglycine benzhydrylamide was condensed with benzyloxycarbonyl-L-glutamic acid by acid protease from Irpex lacteus Fr. in a yield of 21.0%. From the isoglutathione derivative thus obtained, αγ-linkage between glutamic acid and cysteine was formed by α→γ transpeptidation in alkaline conditions after esterification of γ-carboxylic acid.

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An enzyme in Pseudomonas diminuta showed hydrolyzing activity of a benzhydrylamide (=diphenylmethylamide) bond in S-benzylcysteinylglycine benzhydrylamide. The enzyme was purified 225-fold by precipitation with ammoniumsulfate, and column chromatography with ECTEOLA-cellulose, DEAE-cellulose and hydroxyapatite. It showed an optimum pH of 6 to 8 and it was markedly inhibited by Hg²⁺ or p-chloromercuribenzoate. The preparation was more specific against S-benzylcysteinylglycine benzhydrylamide than other substrates tested.

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A β-D-glucan was isolated on fractionation of a 4% potassium hydroxide extract (hemicelluloses) of immature barley plants (Hordeum distichum L.). Most of the glucose residues in the extract were found to be derived from the glucan. Methylation analysis and enzyme degradation studies showed that the glucan had (1→3)- and (1 →4)-linked D-glucopyranosyl residues in an approximate molar ratio of 1.0:2.3. The molecular weight of the glucan was estimated to be 1.8 x 10⁵ by gel filtration on Sepharose CL-6B.

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A xyloglucan occurring in the hemicellulose II fraction of cell walls of immature barley plants was characterized by methylation and fragmentation analyses. The results indicated that the xyloglucan was mainly composed of the following repeating units:

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The effects of partial denaturation were investigated on the surface properties of ovalbumin and lysozyme. The surface tension of proteins decreased greatly as denaturation proceeded. The emulsifying and foaming properties of the proteins were remarkably improved by heat denaturation without coagulation. The emulsifying properties of the proteins increased with denaturation, and correlated linearly with surface hydrophobicity. On the other hand, the protein foaming properties incresed with denaturation, correlating curvilinearly with surface hydrophobicity. The foaming power and foam stability of SDS-bound ovalbumin did not improve as much with those of heatdenatured ovalbumin, although surface hydrophobicity increased to the same extent as by heat denaturation. The relationship between the conformation and functionality of proteins is discussed.

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The aqueous extract of dried bonito (Katsuobushi) was distilled by using a thin film evaporator. The resulting distillate was extracted with diethyl ether, and the extract was separated into basic, acidic, weak acidic, and neutral fractions.
The basic, acidic, and weak acidic fractions were analyzed by gas chromatography and gas chromatography-mass spectrometry.
Seventy-four compounds, including 24 acids, 24 phenols, 8 pyridines, 12 pyrazines, 3 thiazoles, and 3 other compounds were identified. Thirty-six of these compounds were newly identified as volatile flavor compounds of Katsuobushi.

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Various alkyl 3-phenylamino-2-cyano- and 2-ethoxycarbonylacrylates and 3-phenylamino-2-cyanoacrylonitriles were synthesized and assayed as inhibitors of the Hill reaction in isolated pea chloroplast suspensions and as pre- and post-emergent herbicides on mustard and barnyard grass. Many of the compounds were potent Hill reaction inhibitors and several showed significant herbicidal activity.

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Intrinsic viscosity, Stokes radius and the hydrophobic coefficient of Keshavarz and Nakai [Biochim. Biophys. Acta, 576, 269 (1979)] were measured to compare the shape and surface hydrophobicity of ovalbumin and s-ovalbumin. Both the intrinsic viscosity and Stokes radius of s-ovalbumin were smaller than those of ovalbumin, which suggests that the configuration of s-ovalbumin became more compact during the ovalbumin-s-ovalbumin transformation. The hydrophobic coefficient of s-ovalbumin was larger than that of ovalbumin, which suggests that the surface hydrophobicity of s-ovalbumin was larger than that of ovalbumin. Further, these properties were measured for ovalbumin samples obtained at various stages of ovalbumin-s-ovalbumin transformation. Changes in the shape and surface hydrophobicity of ovalbumin were not found in the first stage of ovalbumin-s-ovalbumin transformation. They changed rapidly in the last stage of the ovalbumin-5-ovalbumin transformation.

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Phosphoglycerate mutases (EC 2.7.5.3) of several lactic acid bacteria were adsorbed to blue dextran-Sepharose 4B and eluted quantitatively with 1 m KC1. Phosphoglycerate mutase of Leuconostoc dextranicum AHU 1078 was eluted with 3-phosphoglycerate, 2-phosphoglycerate or 2, 3-bisphosphoglycerate at 1 mM. Furthermore, ATP, ADP, GTP and GDP were also effective for eluting the mutase from the blue dextran-Sepharose column, but AMP and GMP were not. Although NADP(H) eluted the mutase, NAD(H) did not. By single step purification using linear gradient elution of 2, 3-bisphosphoglycerate between 0 and 0.5 mM, the Leuconostoc mutase was purified to electrophoretic homogeneity with a 62.5% yield. The difference in spectrum of Cibacron Blue in the presence of the mutase was quite similar to that of the dye in the presence of KC1, but not in the presence of glycerol. The Leuconostoc mutase was inactivated by pyridoxal phosphate. This inactivation was prevented in the presence of blue dextran, as well as 3-phosphoglycerate.

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Successive feeding of phenol at concentrations of less than 5.5 mM into a thick suspension of Trichosporon cutaneum WY 2-2 precultured in MPY-medium resulted in a high yield (approximately 28.7 g wet cells/liter) of intact cells capable of decomposing phenol actively (3.7 μmol/min/g of wet cells).
The effects of pH and additions of ethanol and 2-mercaptoethanol were tested on the stability of crude extracts from the strain grown on phenol. The crude extracts were stable at a pH range of 7.6 and 8.3, and were stable for 35 days when 10% ethanol and 5 mM 2-mercaptoethanol were added.
A highly purified preparation of catechol 1, 2-oxygenase was obtained from strain WY 2-2 grown on phenol. The purified enzyme was homogeneous on polyacrylamide disc-gel electrophoresis. The enzyme had a molecular weight of about 105, 000 and gave rise to subunits of molecular weight of 35, 000 by SDS gel electrophoresis. Therefore, the enzyme appears to be a trimer of subunits with identical molecular weight. The Michaelis constants were 9.0 μM for catechol and 6.8 μm for 4-methylcatechol. The enzyme exhibited higher activities towards 4-methylcatechol and hydroxyquinol than towards catechol, and had essentially the same substrate specificity as the crude extracts. 4-Methylcatechol completely inhibited the enzyme activity towards catechol.

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Volatile components of wines from Koshu and Zenkoji grapes (Vitis vinifera orientalis) were identified by the combined use of a gas chromatograph-mass spectrometer. The odor of dichloromethane extracts was very similar to that of the original wines. The neutral fraction of the extracts was especially close to the original wine flavor. The phenolic volatile compound 2-methoxy-5-vinylphenol was newly found as a wine components in both wines. A large amount of terpinen-4-ol and a trace of linalool were found in Koshu wine, while a small amount of terpinen-4-ol was detected in Zenkoji wine. Only slight differences in constituents of esters, alcohols and hydrocarbons were recognized in the flavor of both wines.

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β-Hydroxyisobutyrate dehydrogenase was purified and crystallized from Candida rugosa IFO 0750. The overall purification was about 3700-fold with a yield of 34.5%. The molecular weight was estimated to be 80, 000 by the gel filtration method and 75, 000 by the sedimentation velocity method. The enzyme consisted of two identical subunits with molecular weights of 40, 000. The enzyme dehydrogenated both _L- and _D-β-Hiydroxyisobutyric acid (β-HIBA) with NAD⁺ as a coenzyme. The specific activities of the enzyme for _L- and _D-β-HIBA were 36.1 and 8.94 units per mg protein, respectively. The Km values for _L-, _D-β-HIBA and NAD⁺ were 3.70x 10^-4_M, 1.25x 10^-3_M and 5.00x 10^-5_M, respectively. It was suggested from kinetic analysis that the dehydrogenations of _L- and _D-β-HIBA were catalyzed at a single active center of the enzyme.

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Benzaldehyde (BA) was found to have an inhibitory effect on the proliferation of chemicaltransformed rat liver epithelial cells, Culb TC/R/TC. Such cells treated with BA (2×10^-10_M) ceased proliferation by the 4th or 6th day of cultivation, while control cells continued to proliferate throughout the period (14 days). BA also inhibited the piling-up of cells with no marked morphological change. These effects of BA on cells were seen as temporary and reversible. These data suggested the restoration of the cell-cell contact inhibition to Culb TC/R/TC cells by BAtreatment.

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2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase), a new enzyme functioning at the methylcitric acid cycle of propionyl-CoA oxidation, was present in the cell-free extract of Yarrowia (Saccharomycopsis) lipolytica. The enzyme was separated from the usual aconitate hydratase (EC 4.2.1.3) of the yeast with DEAE-Sephadex A-50 column chromatography. The enzyme was able to catalyze a reversible reaction between 2-methylcitrate and 2-methyl-cis-aconitate, but showed no activity on threo-D_s-2-methylisocitrate, citrate, cis- or trans-aconitate, threo-D_s-, threo-D_L- or erythro-L_s-isocitrate, _DL-homocitrate or other hydroxy-acids tested.
In contrast, the other enzyme fraction separated as aconitate hydratase by chromatography showed no activity on synthetic 2-methylcitrate, but was able to catalyze strongly a reversible reaction between 2-methyl-cis-aconitate and threo-D_s-2-methylisocitrate.
From these findings, the previously proposed cycle sequence was revised at the following broken arrows: propionyl-CoA+oxaloacetate → (CoASH+) 2-methylcitrate → 2-methyl-cis-aconitate → threo-D_s-2-methylisocitrate → pyruvate + succinate (→→ oxaloacetate).
2-Methylcitrate dehydratase showed maximum activity at pH 6.5 to 7.0 and at 25 to 40°C. The enzyme was stable at temperatures up to 40°C and at pH 6.5 to 7.5, but labile in Tris-HCl buffer. The synthesis of this enzyme was constitutive in this yeast, although it was slightly repressed by glucose.

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2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase) functioning at the methylcitric acid cycle of propionyl-CoA oxidation was purified from a cell-free extract of Yarrowia (Saccharomycopsis) lipolytica. Disc gel electrophoresis proved that the enzyme preparation was homogeneous. The molecular weight was about 79, 000 in determinations by gel filtration and SDS-disc electrophoresis. The enzyme was composed of 685 residues of amino acid per molecule. The enzyme showed an isoelectric point of 3.9. No cofactor was required for full enzyme activity. The enzyme was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate, but not by any chelating reagents. The enzyme competitively inhibited by citrate (Ki=4.5 x 10^-4_M), threo-D_s-isocitrate (Ki=l.2x 10^-2_M), threo-D_s-2-methylisocitrate (Ki=1.1 x 10^-3_M), tricarballylate (Ki=3.1 x 10^-2_M), and _DL-fluorocitrate (Ki=9.5 x 10^-4_M).

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Syntheses of various γ-glutamylpeptides were examined taking use of the highly purified γ-glutamylcysteine synthetase from Proteus mirabilis. The accumulation of each peptide was measured after long time incubation, and good formation was observed in the synthesis of peptides of following amino acids, L-cysteine, L-α-aminobutyrate, L-serine, L-homoserine, glycine, L-alanine, L-norvaline, L-lysine, L-threonine, taurine and L-valine. Peptide syntheses were confirmed by analyses of the component amino acids, after hydrolysis of the peptides.
The structure of the glutamylpeptides, especially the peptide-linkage at the γ-carbonyl residue of L-glutamate, was determined by mass spectrometry of the N-trifluoroacetyl methylester derivatives of the glutamylpeptides. Enzymatic synthesis of γ-glutamyl-L-α-aminobutyrate was also confirmed by PMR spectrometry in the comparison with chemically synthesized compound.

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The relationship between soy sauce gas chromatographic (GC) patterns precisely analyzed on a glass capillary column and ranked order in sensory analysis was investigated by stepwise multiple regression analysis. The most negative and positive correlation between the GC peaks and sensory data were shown by trans-2-hexen-l-ol contributing to preferable aroma and iso-butyric acid concerning to unpleasant smell, respectively. Highly predictable multiple regression models were calculated in the analysis. The variety of volatile components selected for the equations indicated that these constituents represented not only different groups of chemical structures in aroma compounds but also growth of various microorganisms during the soy sauce making process.

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8a-Hydroperoxy tocopherones, prepared from the photooxidation of α-, γ- and δ-tocopherols, were allowed to react with ascorbic acid in ethanol. The hydroperoxy tocopherones were reduced to 8a-hydroxy and 8a-ethoxy tocopherones which in turn were reduced to tocopherols by ascorbic acid. The tendency of the hydroperoxy tocopherones to form tocopherols during the reaction correlated with vitamin E activities of tocopherols. The results indicate that α-tocopherol and ascorbic acid can act synergistically on the quenching of ¹O₂. The inhibitory effects of α-tocopherol and ascorbic acid were examined on the chlorophyll-sensitized photooxidation of methyl linoleate and soybean oil. At the initial stage of the oxidation, the inhibitory effect of α-tocopherol was lengthened in the presence of ascorbic acid.

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A new type of β-conglycinin was found in precipitates of a crude glycinin fraction at pH 6.4 and low ionic strength. The protein was purified by ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography and ion-exchange chromatography on DEAE-Sepharose CL-6B. The purity check and identification of the protein were performed by gel electrophoresis, gel filtration, N-terminal amino acid analysis, immunochemical analysis and ultra-centrifugal analysis. This protein (designated as B₀-conglycinin) was composed of three identical subunits which were identical with the β-subunit in β-conglycinins at 0.5 ionic strength, but differed from the other β-conglycinins in that B₀-conglycinin formed an insoluble aggregate at ionic strengths less than 0.2.

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A component responsible for flocculation was extracted from Pseudomonas strain C-120 by treating the cells with 3 M guanidine hydrochloride. The guanidine hydrochloride-extracted cells were reflocculated, not only with the guanidine hydrochloride extract but with DNA prepared from various bacteria. The reconstituted flocs were deflocculated by deoxyribonuclease or guanidine hydrochloride which indicated that the reconstituted flocs closely resembled natural flocs. In reconstitution experiments using Escherichia coli DNA at different molecular weights, it was found that DNA with a molecular weight higher than about 6 x 10⁶ was required to flocculate the guanidine hydrochloride-extracted cells. Heat-denatured DNA did not flocculate the guanidine hydrochloride-extracted cells. DNA with a high molecular weight was detected in the guanidine hydrochloride extract. It was concluded that the component involved in flocculation of this organism was highly polymerized double stranded DNA.

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The effects of adding oils to soybean protein were examined with respect to hardness of the formed gel. The gel hardness increased with a decrease in the chain length of the added fatty acid methyl esters or triglycerides. When the gels were observed under a scanning electron microscope, the addition of fatty acid methyl ester with a shorter chain length produced a good, hard gel, while the gel formed with fatty acid methyl ester of a longer chain length was easily broken. There were few differences in the hardness of gels formed after adding various unsaturated fatty acid methyl esters, triolein or edible oils, but these gels were firmer than those without oils. The behavior of the protein solution containing oils on thermo-denaturation was the same as that without oils. The amount of protein adsorbed on oil droplets increased with a decrease in the chain length of the fatty acid methyl ester. The hardness of the gel formed with oils may be strengthened by the protein-oil interaction after thermo-denaturation of protein.

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By controlled reduction of 1, 3, 4, 5, 6-pentachlorocyclohexene isomers with lithium aluminum hydride, three isomers of 1, 3, 4, 5-tetrachlorocyclohexene were prepared. Their structures were (34/5)-, (3/45)- and (35/4)- by PMR analyses.

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Bacillus subtilis protoplasts regenerate on media containing horse serum, bovine serum or gelatin. These compounds could be replaced by polyvinyl pyrrolidone or dextran, and a medium which contained 30 g polyvinyl pyrrolidone and 20 mg casamino acids per liter with chemically defined ingredients was especially useful for selection of prototrophs, e.g., by protoplast fusion. Polyvinyl pyrrolidone and other plasma expanders stimulated protoplast division in liquid media and improved protoplast survival on agar media.

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The level of C2-casein mRNA in the mammary gland was estimated after weaning litters from 7 day-lactating rats. After a steep deline (1/34th) within 2 days of weaning, it gradually declined until 4 day-weaning. After different periods of weaning, it was tested whether the mRNA level is restored by resuckling. The mRNA level was increased greatly in a system in which the rats in 1 or 2 day-weaning litters were resuckled for 2 days, suggesting that this system serves as an useful model for investigating regulation mechanisms of casein mRNA levels.

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