Two kinds of "group A saponin, " Aa and Ab, are present as the main constituent in soybean seed. The saponin composition and content in F₁ and F₂ seeds derived from the cross parents of Aa and Ab types were analyzed. The "group A saponin" was of Aa-Ab type in all the F₁ seeds, and the ratio of Aa type: Aa-Ab type: Ab type was 1:2:1 in the F₂ seeds. From these results, it appears that Aa and Ab were controlled by codominant allelic alternatives at a single locus. An investigation of the saponin composition of the seed hypocotyls of 18 wild lines revealed some lines in which "group A saponin" was absent.

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Kansho-shochu flavor (KSF) was found in the neutral fraction and was eluted by hexane-ethyl acetate (9:1) from a silica gel column. There were several monoterpene alcohols such as linalool, α-terpineol, citronellol, nerol and geraniol in the KSF. These monoterpene alcohols contributed to the sensory property of KSF. On the other hand, there were glycosidically bound geraniol and nerol in steamed sweet potatoes. In a model system of shochu mash, geraniol and nerol were transformed into linalool and α-terpineol through heat treatment and into citronellol by the shochu yeast. These results suggest that the monoterpene alcohol glycosides in steamed sweet potatoes were hydrolyzed by glycosidic enzymes in the shiro-koji of shochu mash. Subsequent transformation of the liberated nerol and geraniol into linalool and α-terpineol by the steam distillation process was also indicated.

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The changes in ¹³C-NMR and ³¹P-NMR spectra and ¹H-NMR images in soybean cotyledons during germination were investigated. Using ¹³C-NMR, fatty acid signals in the form of triglycerides were observed in dry seeds, and those were observed approximately 18 days after the start of imbibition. Sucrose signals appeared at 16 hr and disappeared at 5 days. A-⁺N-(CH₃)₃ signal was observed after 5 days, suggesting the activation of membrane metabolism in the cotyledons.
³¹P-NMR signals appeared 2hr after imbibition before any apparent change in the ¹³C-NMR spectrum. The peaks identified as sugar phosphate, inorganic phosphate in the cytoplasm and in the vacuole, and an unassigned compound, were distinguishable after 5 days. The vacuole-associated inorganic phosphate peak became prominent 18 days after imbibition in ³¹P-NMR.
Distribution maps of free water indicated that the stored macro-molecular materials which bound water were consumed heterogeneously within the cotyledon. The relaxation time (T₁) increased suddenly between 18 days and 23 days after imbibition, which indicates the consumption of stored materials.
These findings suggest that cotyledons are the source of such compounds and the energy required for plant growth for approximately 18 days from germination until tri-foliolate leaves begin developing.

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Quinoxaline derivatives derived from isomaltooligosaccharides and o-phenyIenediamine (OPD) were studied by ion-exchange chromatography. Under deoxygenated and heated alkaline conditions, the formation of quinoxaline from α-1, 6-glucan with OPD proceeded from the reducing-end glucose residue in a complicated way, and the mechanism mainly involved the following processes: 1) formation of quinoxaline derivatives of high molecular weight as a condensation with OPD and 1, 2-dicarbonyl compounds at the reducing-end residue of the glucan. 2) formation of several kinds of quinoxaline derivatives of low molecular weight from the reducing-end residue of 1, 6-linked glucan as liberated glucose; as a consequence, the one glucose unit smaller 1, 6-linked chain was liberated and again reacted with OPD. The rate of decomposition gradually decreased during the liberation of about 7 glucose residues from the reducing end. The characteristics and probable stoichiometry of this alkaline degradation of α-1, 6-glucans are discussed, related to the formation of quinoxaline derivatives.

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The effective thermal conductivity of air-impregnated gels of various air and water contents was measured at 20°C by the steady heat flow method, and the applicability of the sphere dispersion heat conduction model (the Maxwell-Eucken model) was studied. The Maxwell-Eucken model gave a fairly good approximation for the effective thermal conductivity of air-impregnated gels of the lowest water content, but that of air-impregnated gels of higher water content was underestimated by the Maxwell-Eucken model. In addition to that, the effective thermal conductivity of the air-impregnated gels of the highest water content at 40°C exceeded the maximum limit expected by the parallel layers heat conduction model. These facts suggested the existence of some heat transfer mechanism other than heat conduction and the significant role of water in it. To explain the discrepancy in applicability of known heat conduction models, a model of heat transfer in air-impregnated gels, which includes heat transfer by water vapor, is presented and discussed.

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3, 5-Dichloro-4-methoxybenzyl alcohol, a novel natural metabolite, was isolated from submerged cultures of a Stropharia species due to its inhibitory activity towards chitin synthase. In the same screening, drosophilin A and other chlorinated aromatic compounds were detected. Comparison of the activity of the natural metabolites with that of synthetic analogues showed the necessity of the chlorine substituents for the enzyme inhibition. Hexachlorophene strongly inhibited solubilized chitin synthase with an apparent Ki of 8.5μM. The inhibition could be reversed by the addition of lecithin.

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The changes of bovine casein micelles during cold storage were investigated from the standpoint of the size of micells. Large, medium, and small micelles (>50, 50-30, and <30 nm in radius) were obtained from skim-milk by differential centrifugation. After the definite-sized micelles were equilibrated at 4°C and 37°C, they were fractionated by differential centrifugation and gel chromatography. The casein contents of these fractions were measured by SDS-polyacrylamide gel electrophoresis. A high quantitative separation of caseins was achieved by this method.
The liberation of β-casein by cooling occurred easily from the larger-sized micelles. A larger amount of κ-casein was liberated from the smaller-sized micelles. The medium micelle fraction was increased by the supply from large micelles at 4°C, and the medium micelles degraded partly to the small micelle fraction and soluble casein. Small micelles degraded partly to soluble casein. The small micelle fraction was not produced from large micelles. The stability of micelles under cooling was related closely to the miceller size.

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Pyranose oxidase was purified from the crude extract of basidiomycetous fungus No. 52 about 74-fold to a single protein band on polyacrylamide gel electrophoresis with an overall yield of 23%. The molecular weight of the native enzyme was about 300, 000 on gel filtration and 69, 000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 6.3. The enzyme activity showed high stability over a wide pH range of 5.5-9.0 and below 50°C, and 60% of its initial activity remained even after heating at 70°C. The enzyme was inactivated by Ag⁺, Hg²⁺, and Cu²⁺. The enzyme contained FAD covalently bound to the protein and was a glycoprotein containing about 0.7% carbohydrate.

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In order to study the role of hydrophobicity in bitter peptides, several O-aminoacyl sugars, in which amino acids or peptides were attached to the 2- and 3-position of methyl α-D-glucopyranoside, were synthesized and sensory analyses were carried out. It was found that the bitterness increased as the hydrophobicity of compounds increased, implying that the bitterness receptor recognizes the hydrophobicity of bitter peptides. A structure for the bitterness receptor is also discussed.

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Fatty acid synthetase of Type I from Mycobacterium smegmatis was immobilized by radiationinduced polymerization of 2-hydroxyethyl methacrylate (HEMA) in the presence of trimethylolpropane trimethacrylate (TMPTMA). The stability of immobilized synthetase toward low ionic strength increased in comparison with the free form, but the stabilities of immobilized preparations assessed by pH and temperature were identical to those of the free form. The apparent Km of immobilized enzyme for acetyl-CoA and malonyl-CoA were both 6μM, essentially the same as those of the free form; acetyl-CoA, 5μM and malonyl-CoA, 6μM.

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The Maillard reaction was done with several proteins and aldoses (glucose, glyceraldehyde, and glycolaldehyde) under both neutral and alkaline conditions. Maillard reaction products (MRP) from α_s-casein and albumins (ovalbumin, bovine serum albumin, human serum albumin, and α-lactalbumin) inhibited adhesive insoluble glucan synthesis by glucosyltransferase (GTF) of Streptococcus mutans. The magnitude of the inhibitory activity in MRP from αs-casein and the three aldoses correlated with the reactivity of the aldoses with proteins. The MRP from α_s-casein and albumins inhibited GTF-I and did not affect GTF-S, while their original proteins stimulated GTF-I specifically. These results suggest that the inhibitory activity of these MRP may be related to the GTF-I stimulating property of their original proteins.

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The flocculation mechanism of a flocculent yeast, Hansenula anomala J 224, isolated from a waste water treatment tank of a Japanese sake brewing factory, was investigated by comparing some characteristics with strain J224-1 (a nonflocculent mutant of strain J224). The cell titration curves showed that strain J224 was different from strain J224-1 in charge distribution on the cell surface. The cell wall of strain J224 was more abundant in Mg²⁺, Ca²⁺, Na⁺, NH₄⁺, and total phosphorus contents but had less fatty acids than that of strain J224-1. The cell walls of both strains had almost the same amino acid composition. Acylation of strain J224 with acetic anhydride caused deflocculation. By treatment with proteinase K, an additional protein of 37 K dalton (37 K-protein) whose molecular mass was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was released in the supernatant from strain J224 cells. The 37 K-protein was purified by cation-exchange high-performance liquid chromatography. The amino acid composition of this protein was different from that of the cell wall. Flocculation of strain J224 was promoted by the addition of 37 K-protein.
We discussed the involvement of ionic interaction between 37 K-protein and phosphate and hydrophobic interaction between 37 K-proteins on the cell surface of adjacent cells in the flocculation of strain J224.

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The tripeptide Z-GlyPheLeuNH₂ was continuously synthesized in a high yield from three amino acid derivatives, Z-Gly, PheOMe, and LeuNH₂, by immobilized thermolysin (IMT) and immobilized a-chymotrypsin (IMC) in an organic solvent, ethyl acetate. The optimal conditions for the synthesis of Z-GlyPheOMe were established theoretically. The yield of Z-GIyPheOMe with IMT in ethyl acetate saturated with buffer was more than 88% after continuous synthesis for 116hr.
The optimal conditions for the synthesis of Z-GlyPheLeuNH₂ from Z-GlyPheOMe and LeuNH₂ by IMC through transesterification was established in batch reaction experiments. When the concentration of water in the reaction solution was 17-20 μl/ml, the activity of IMC was highest. The equilibrium between the water concentration in the reaction solution and that in the resin used for enzyme immobilization depended on the resin and was not affected by the presence of the enzyme immobilized. Z-GlyPheLeuNH₂ was synthesized from Z-GlyPheOMe and LeuNH₂ with a yield of 100 %, by continuous reaction for 160 hr.
The reactor for synthesis of this tripeptide was efficient and stable because of the use of transesterification and the choice of an appropriate organic solvent. The series plug-flow reactor was successfully operated for 220 hr with a yield of more than 80 %. The residual activity of IMT was 94%, and that of IMC was 100%.

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Screening was carried out of the inducers for the fruiting-body formation in a basidiomycetes Favolus avcularim from microbial metabolites. Streptomyces strain B-412 similar to Streptmyces rubiginosus was isolated from the soil as the producer of a substance which the induced the formation of incomplete fruiting bodies or stipes without pileus of F. arcularius under dark conditions. The active substance was extracted from the supernatant of the cultured medium with ethyl acetate in the acidic pH range and purified by successive chromatography as orange crystals, having its degradation point of >245-255°C. The absorption spectrum in a neutral methanol solution showed peaks at 211, 242, 270, 341 and 430 nm, which changed depending on the pH value. The active substance, named basidifferquinone, induced stipe formation in F. arcularius at 3ng/ml, which corresponds to 6.9 × 10^-9 M, and the resulting stipes completed differentiation to form pileus upon successive photoirradiation.

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Basidifferquinone is a potent inducer produced by Streptomyces strain B-412 for differentiation of a basidiomycetes Favolus arcularius to form incomplete fruiting bodies. X-Ray crystallography to-gether with MS, IR, ¹H-NMR and ¹³C-NMR spectral analyses revealed the structure of basidiffer-quinone, C₂₄H₁₆O₈, to be (1R)-4, 7, 9-trihydroxy-10-methoxy-8-methyl-l-phenyl-(1H)-anthra[1, 2-C]-furan-3, 6, 00-trione (Fig. 1).

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The gene coding for creatinase (creatine amidinohydrolase, EC 3.5.3.3) was isolated from Flavobacterium sp. U-188. The primary structure of creatinase deduced from the nucleotide sequence showed a protein (molecular weight, 42, 651) composed of 378 amino acids. The creatinase gene was over-expressed in Escherichia coli under the control of the lac promoter and the amount of this enzyme was over 20 % of the soluble protein in the cell.

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Six lipases were examined for concentrating the n-3 polyunsaturated fatty acid (n-3 PUFA) of two kinds of fish oil (cod liver oil and refined sardine oil). Although all lipases could increase the n-3 PUFA content of the remaining glycerides, Candida cylindracea and spergillus niger lipases gave glycerides with a more than two-fold increase in n-3 PUFA content over the original fish oils.
Candida cylindracea lipase seems the most promising with respect to recovery of triacylglycerol. Aspergillus niger lipase increased not only the docosahexaenoic acid (DHA) content but also eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA), although the absolute value of the latter was quite low. The effects of temperature (15-40°C) on the concentration of n-3 PUFA were investigated. Lower temperatures did not improve the concentration of n-3 PUFA, but prevented the development of an unpleasant odor in the product.

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A pectinesterase (EC 3.1.1.11) isolated from red tepals of Ficus awkeotsang was purified to a single band of protein on SDS-PAGE by column chromatography on QAE-Sephadex A-25, Q-Sepharose, hydroxylapatite, and Sephacryl S-200. The molecular weight of the enzyme was 42, 000 by SDS-PAGE. The pI was 4.4. The purified enzyme activity was stimulated in the presence of low concentrations of metal ions, in the order of Ca²⁺>Mg²⁺>K⁺>Na⁺. The Km and V_max for awkeotsang polygalacturonide were 2.94mg/ml, 66.67μmol/min/mg protein at pH 5.4 in the absence of metal ion, and 2.78mg/ml, 86.96μmol/min/mg protein at pH 7.5 in the presence of 50 mM KCI, respectively.

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A highly methyl esterified linear α-(1→4)-linked polygalacturonide, isolated from a water extract of seeds of Ficus awkeotsang Makino, was used for the study of elicitor-active oligosaccharides on host-parasite interactions in higher plants. Oligogalacturonides (OLGAs), obtained from awkeotsang polygalacturonide or low methyl esterified apple pectin by treatment with the purified endo-pectate lyase (EC 4.2.2.2) of Ermnia carotovora, were found to induce glyceollin accumulation in soybean cotyledons. The de-esterified awkeotsang-OLGAs was precisely fractionated by anion-exchange chromatography using a QAE-Sephadex A-25 column, and was assayed for the elicitor activity. Among the purified Oligogalacturonides with DP 3 to 12, it was found that 4, 5-unsaturated hexa-a-1, 4-galacturonide of the low molecular elicitor-active Oligogalacturonides (DP 5 to 7) also was a potent elicitor as well as the 4, 5-unsaturated deca-a-l, 4-galacturonide of the high molecular elicitor-active Oligogalacturonides (DP 9 to 11) previously reported by Davis et al.¹⁾

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Isoamyl acetate, one of the major characteristic aroma compounds of sake, was synthesized by the condensation of acetyl-CoA and isoamyl alcohol under the action of alcohol acetyltransferase (EC 2.3.1.84) of yeast. The enzyme, which was associated with the cell membrane, was purified about 800-fold. The purified enzyme was very labile at temperatures higher than 10°C and pHs lower than 5.5 but was stable at pH 6.0-7.5. The enzyme was most active at 30°C and pH 7.0. The isoelectric point of this enzyme was 5.5. The molecular weight was estimated to be about 50, 000 by gel filtration and SDS polyacrylamide gel electrophoresis.

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Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34, 000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain I -amino acids, L-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for L-leucine, and the k_cat/Km value for L-leucine was higher than that for L-valine. The enzyme required NAD⁺ as a natural coenzyme. The NAD⁺ analogs 3-acetylpyridine-NAD⁺ and deamino-NAD⁺ were much better coenzymes than NAD⁺. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5'-phosphate. D-Enantiomers of the substrate amino acids competitively inhibited the oxidation of L-valine.

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We isolated and purified yeast chromosome DNA molecules using pulse field gel electrophoresis (PFG). The isolated DNA had nearly the same size as the native chromosomal DNA on PFG. We could directly transform Saccharomyces cerevisiae yeasts with it, and obtain transformants that were selected by complementation of several markers. They had new chromosome DNA bands observed on PFG. The new chromosome was very stable during mitosis and mating processes, and each of the three homologous chromosomes in the derivative zygotes of transformants was separated equally in daughter cells.

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Threonine-producing mutants of Brevibacterium flavum with HD^R or DPS defect were selected by resistance to less than 8g/l of AHV, a threonine analogue, in our previous studies. Growth of a mutant with HD^R, BK 29, was still inhibited by 12g/1 of AHV. Eighteen AHV-resistant mutants derived from strain BK29 under these conditions produced more than 12g/l of threonine. All the best five strains tested showed much lower levels of or no DPS activity in addition to HD^R. The best threonine producer, strain DB185, produced 16.5g/l of L-threonine, about twice that by the parent, but the accumulation of lysine decreased to 3.8 from 10g/l as the HCl salt by the parent. The threonine production and growth were stimulated by DAP. On the other hand, growth of a mutant with DPS defect, DK330, was not inhibited by 12g/l of AHV. However, addition of lysine enhanced the growth inhibition by AHV to be almost as complete as that of strain BK29. The best strain BD122 among mutants resistant to AHV plus lysine derived from strain DK330 produced 16.6g/l of L-threonine, while the parent produced 11.2g/l. Both HD and AK of the mutant showed normal sensitivities to feedback inhibition, but its HK activity increased 2.1-fold over that of the parent.

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L-Phenylalanine produced by Brevibacterium flavum mutant M-128 from fructose as the carbon source, was 1/3 to 1/4 of that from glucose. The production from sucrose gave a similar yield as that from glucose, but that from an equimolar mixture of glucose and fructose was half of that from glucose, L-Glutamic acid production was not so strongly affected by these carbon source sugars. When mutant M-128 was producing L-phenylalanine at the maximum rate, the intracellular level of F6P was much lower with fructose as the carbon source than with glucose. The level with sucrose was similar as that with glucose, but that with the mixture of glucose and fructose was 1/2 to 1/3 of that with glucose, rather similar to that with half the amount of glucose. The intracellular AMP concentration was found to inhibit FBPase almost completely in the cells grown on any of these carbon source sugars. Toluenized cells of B. flavum strains able to grow on sucrose showed PEP-dependent PTS activity with sucrose as the sugar substrate, but those of the strains unable to grow on sucrose did not. Major products of the sucrose-PTS reaction were pyruvate and G6P in PGI-lacking mutant, while they were pyruvate, fructose, G6P, FBP, and triose phosphates in a glucose- and fructose-PTS-deficient mutant, suggesting that the reaction products are pyurvate and sucrose 6^G-phosphate, which is hydrolyzed to G6P and fructose by invertase. The fructose formed seems to be solely converted to F1P by fructose-PTS, as no fructokinase activity was detected in this bacterium.

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Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains, the A chain of 44 amino acid residues and the B chain of 50 residues. Two different primary structures have been reported for each of the A and B chains. The A and B chains corresponding to one of the reported monellin structures were synthesized by the stepwise solid-phase method using the Fmoc strategy in overall yields of 14.1% and 5.6%, respectively. The characterization of the synthetic peptides by HPLC, FAB-MS, amino acid analysis and sequencing fully supported the expected structures. The individual synthetic A and B chains were not sweet. Combination of the two chains, and subsequent HPLC purification gave monellin in a yield of 53.9 %. The synthetic monellin had a distinct, lingering sweet taste (4000 times sweeter than sucrose) and was crystallized by a vapor diffusion method. The synthetic product was identical to natural monellin by HPLC, but not by tryptic mapping. These results indicate that the reported structure for monellin differs slightly from that of natural monellin.

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(±)-Streptovitacin A (1) and its stereoisomers were synthesized by an aldol reaction of (±)-2, 4-dimethyl-4-trimethyIsiloxy-1-cyclohexanones (4b and 9) with 4-(2-oxoethyl)-2, 6-piperidinedione (5). E-73 (2) was derived from synthetic 1. (±)-1 showed moderate growth inhibition against fungi and lettuce seeds.

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Lactobacillus helveticus subsp. jugurti was transformed with plasmid, pLHR (8.5 kilobases), by electroporation. The plasmid, pLHR, consists of a cryptic plasmid, pLJl, from L. helveticus subsp. jugurti, the Escherichia coli vector pBR329, and the erythromycin resistance gene of pAMβl from Enterococcusfaecalis. Maximum transformation efficiency of 1.3×10⁴ transformants per μg of DNA was obtained by exposure to a pulse with an exponential decay waveform at 4kV/cm with 25 μF capacitance. The presence of glycine in the growth medium was essential for transformation. Plasmid DNA isolated from transformants had not undergone detectable rearrangements or deletions. In addition, it was found that L. helveticus subsp. jugurti has a restriction and modification system.

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The proglycinin synthesized in E. coli JM105 comprised approximately 20 % of the total bacterial proteins, with a yield of 39 mg per liter of culture under the optimum cultivation conditions. The proglycinin was purified to homogeneity by salt precipitation, ion-exchange chromatography, and cryoprecipitation. The purified proglycinin self-assembled to a trimer with a secondary structure similar to that of the glycinin half-molecule from soybean seeds, and had properties of gel formation by heating and precipitation with calcium salt as the native glycinin and glycinin half-molecule do. This indicated that the E. coli expression system of glycinin cDNA may be used for the evaluation of the self-assembly and the food qualities of protein-engineered soybean proteins.

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A new cDNA clone for rat IGF-I mRNA was obtained. The cDNA contained a new base sequence as cDNA in the 3' region. The sequence coincided with a part of the sequence reported earlier by Shimatsu and Rotwein⁵⁾ in exon 5 of the rat IGF-I gene. The presence of this cDNA proved the previous suggestion to be true that the size heterogeneity of IGF-I mRNA is primarily due to the heterogeneity of the 3'-untranslated region.

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