Fucogalactomannan was obtained from the walls of Rhodotorula glutinis K-24 by alkaline extraction, pronase digestion, and alcohol precipitation. This purified polysaccharide was composed of fucose, galactose and mannose in the molar ratio of 1:2.5:2, and occupied about 20% of the cell wall. In the successive Smith degradation, mannose residues were resistant to periodate oxidation, whereas most of galactose residues were degraded, releasing threitol and arabinose. It appeared that mannose residues consisted of a backbone of the polysaccharide and all or part of the galactose residues might be of a furanose type. By partial acid hydrolysis of fucogalactomannan, three kinds of oligosaccharide containing fucose and galactose were obtained. Their structures were identified as Gal_f(1→6)Gal_f, Fuc(1→2)Gal_f(1→2)Gal_f(1→6)Gal_f, and Fuc(1→2)Gal_f(1→6)Gal_f-(1→2)Gal_f(1→6)Gal_f, respectively. The methylation analysis revealed that the polysaccharide consisted of (1→3)-linked mannose residues, all of which were branched at the C-2 position with fucose and galactose residues. Most of the fucose residues existed in the nonreducing end of the side chain. Galactose residues were mostly contained as (1→2) and (1→6) linkages in the side chain.

View full abstract

Glucose isomerase purified from cells of Streptomyces phaeochromogenus was irradiated in dilute solution, and the effect of γ-irradiation was investigated. The activity of the enzyme decreased exponentially with the dose under all conditions investigated. The inactivation yields (G₀-value) in neutral solution were 0.069 in air and 0.115 in nitrogen.
The role of the radicals produced by water radiolysis in the inactivation of glucose isomerase was estimated by using nitrous oxide tert-butanol as selective radical scavengers. Under these conditions, the hydroxyl radical and the hydrogen atom were found to be important in the enzyme inactivation, and the hydrated electron contributed very little. The inactivation efficiencies of the hydroxyl radical, the hydrogen atom and the hydrated electron were 0.032, 0.025 and 0.005, respectively.

View full abstract

Two acid phosphodiesterases (phosphoric diester hydrolase, E.C. 3.1.4) were purified to apparent homogeneity from cultured cells of Nicotiana tabacum var. Bright Yellow. These enzymes appeared to differ in some physicochemical properties but exhibited similar broad substrate specificities towards cyclic and p-nitrophenyl nucleotides, and bis-p-nitrophenyl phosphate. Of these compounds, the most active substrates were bis-p-nitrophenylphosphate and 2', 3'-cyclic nucleotides. Both p-nitrophenyl 3'- and 5'-thyrnidylates were hydrolyzed at comparable rates, whereas the hydrolysis of 3', 5'-cyclic AMP occurred preferentially at the 5'-phosphodiester bond. A unique catalytic property of these acid phosphodiesterases was the ability to liberate inorganic phosphate from ATP, ADP and p-nitrophenyl phosphate. Ribonucleic acid, its breakdown products, and inorganic phosphate were found to be effective as inhibitors.

View full abstract

Useful cryophilic killer strains of Saccharomyces cerevisiae were bred by back-crossing KL-88, which is a wild killer sake yeast, with a repetitive parent WL-7and is one of the best cryophilic wine yeasts. The hybrids maybe aid in producing good wines in pure cultures at relatively low temperature (1315°C).
A haploid of KL-88 was crossed with haploids of WL-7to yield first-generation hybrids. Haploids possessing both killing activity and SO₂ tolerance were selected from the hybrids and back-crossed with a haploid of WL-7 to yield second-generation hybrids. Several strains of killer hybrids were comparable to WL-7in fermentation ability at 15°C, SO₂ tolerance, TTC stain, and growth on β-alanine medium at 35°C. One strain (2HYL-2) aided in producing wine as good as with WL-7, in fermentation at 15°C on pilot-plant scale.
The hybrid killed only Saccharomyces yeasts in grape must. In the last-stage of fermenting
Koshu grape must with the killer hybrid (2HYL-2), no sensitive yeasts were detected, because they were killed by the killer yeast. Moreover, most of the neutral yeasts, such as Kloeckera and Torulopsis, that were abundant in grape juice before the addition of the starter yeast, disappeared in the last-stage. On the other hand, many sensitive pseudo-film-forming yeasts of the genus Saccharomyces were detected in the last-stage of fermenting must with WL-7strain. These results show the possibility of fermenting musts with only a single strain of cryophilic killer yeasts at a relatively low temperature without contaminating other yeast strains.

View full abstract

Five derivatives of partially N-acetylated chitosan [degree of substitution (d.s.) for N-acetyl group: 0.05, 0.2, 0.4, 0.6 and 0.9] were prepared from chitosan by reaction with acetic anhydride (<1 mol GlcN). Each derivative was exhaustively oxidized with NaIO₄, reduced with NaBH4and hydrolyzed with 0.5 n HC1by the conventional method. The reaction products were fractionated by gel chromatography using Bio-Gel P-2. Glycol aldehyde, meso-Qrythritol and a series of 2- acetamido-2-deoxy-β-D-glucopyranosyl(1 --[→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl(1-]_n→2)-D-erthritols were detected in the hydrolyzates of all these derivatives, regardless of low d.s. for Nacetyl groups. These results indicate that free amino groups were heterogeneously present in the partially N-acetylated chitosans because chitosan was heterogeneously N-acetylated under the present conditions.

View full abstract

S-Methyl thioacetate (MeSAc) production by brewer's yeast from methyl mercaptan (MeSH) was investigated under various conditions. At optimum, 98 mg/liter of MeSAc was produced from 500 mg/liter of MeSH contained in culture broth. The MeSAc level in yeast growth mediumwas increased with increasing MeSH at relatively low levels (10 to 500 mg/liter). However, higher MeSH levels in medium (over 1 g/liter) inhibited yeast growth, and no MeSAc was produced. MeSAc was formed readily by incubating MeSH with yeast resting cells. Furthermore, S-ethyl or S-n-propyl thioacetate accumulated in yeast cell suspension when ethyl or n-propyl mercaptan, respectively, was incubated with resting cells. MeSAc was also produced from L-methionine by brewer's yeast, but its formation was dramatically inhibited by copper ions. This finding suggests that MeSHis an intermediate product between L-methionine and MeSAc.

View full abstract

The radiosensitivity of glucose isomerase was investigated under various irradiation conditions. The inactivation of glucose isomerase irradiated in a cell-bound state was exponential, and an increase in inactivation was recognized in an oxygenated condition. The cell-free enzyme was highly radiosensitive and had a small oxygen effect compared to that in a cell-bound state. The oxygen enhancement ratio (OER) decreased with a degree in enzyme purification.
Released glucose isomerase was protected by the addition of glutathione, and the inactivation curve in nitrogen almost agreed with that in the cell.
The protective effect of glutathione in oxygen decreased at higher doses because glutathione in oxygen was easily decomposed by irradiation.

View full abstract

The aggregation pheromone produced by the male red flour beetle Tribolium castaneum, and confused flour beetles, T. confusum was identified as 4, 8-dimethyldecan-l-al by GLC, GC-MS, PMRspectra, and synthesis of the compound. The synthetic pheromone was less attractive compared with the natural pheromone, because the synthetic sample was composed of four optical isomers.

View full abstract

The thermal denaturation of 7S globulin has been examined as a function of ionic strength from 0 to 4.0 by using immunochemical methods, disc electrophoresis and turbidity. Changes in immunochemical properties were a measure of the extent of denaturation and provided information on the structural changes in 7S globulin at the molecular level. The 7S globulin did not lose its antigenicity even after heating, and both dissociates and aggregates maintained partial cross reactivity against anti-7S. The nature of thermal denaturation was divided into two main types at an ionic strength of approximately 2.0. At an ionic strength near zero, stable dissociation products were actually formed, and at ionic strengths from 0.1 to 2.0, the presence of salts increased in aggregate formation. The stabilizing effect of salt on thermal denaturation appeared at 2.0 2.2 μ. In the range of 3.54.0μ, 7S globulin was heated to 100°C for 5 min without changes in both antigenic specificity and electrophoresis pattern.

View full abstract

Galactans from albumen glands and from freshly collected egg masses of intermediary hosts of bilharzia in Brazil (Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea) were investigated. All these galactans had D-galactopyranose characterized as the sole sugar component. No evidence was found for the presence of L-galactopyranose in these polymers, using specific enzymes. Methanolysis of the methylated galactans and periodate oxidation data indicated a multibranched structure for these galactans. Yields of O-methyl sugars showed a equimolecular proportion of non-reducing end groups and disubstituted residues linked through positions 3 and 6 and a predominance of β-(l→3)- over β-(l →6)-linkages. In view of the structural identity presently observed in the galactans from all our sources, as compared to the distinctive structure proposed for the galactan after five days of egg masses laying [M. Iacomini et al., Arq. Biol. Tecnol., 23, 329 (1980)], it is inferred that the enzymic system for galactan degradation is still inactive in freshly collected egg masses.

View full abstract

New types of stable chrysanthemic acids and esters were synthesized, and their ¹³C-NMR were examined and fully analyzed. The configurations of the cyclopropanecarboxylic acid and halomethylvinyl group were reflected on the spin-lattice relaxation time of the substituted methyl carbon involved in their structure. The long-range spin-spin coupling constants (³J_Ch) correlated
well to the NOE and T₁ measurements, which can generally be used to distinguish the geometry of the substituted double bond.

View full abstract

Microorganisms that hydrolyze methyl 2-phenylpropionate (1) or reduce 4-phenyl-2-butanone (3) were screened from 250 type cultures. Several Aspergilli and two bacteria hydrolyzed ester 1, and Asp. sojae IAM 2703 preferentially hydrolyzed CR)-isomer of (±)-l, whereas Bacillus subtilis var. niger IFO 3108 and Mycobacterium smegmatis ATCC10143 preferentially hydrolyzed (5)-isomer. The hydrolysis of the related esters of 1 with these organisms was also examined.

View full abstract

Adult rats fed ad libitum and on restricted-feeding (food available between 10 : 00 and 16 : 00) regimen showed feeding-cued circadian rhythms in the activity patterns of glycyl-L-leucine and L-leucine absorption by the proximal small intestine. Absorption of the two compounds by the distal small intestine followed these similar, but less overt, rhythmic patterns. The glycyl-L-leucine hydrolase activity of cytosolic fraction of the mucosal homogenates from the proximal intestine also exhibited a similar daily rhythm in the rats on restricted-feeding regimen, but no discernible rhythm in the rats fed ad libitum. Rhythmicity of the hydrolase activity was hardly noticeable in the membrane fraction on either feeding regimen. Restricted-feeding resulted in an enhanced daily average level for those intestinal activities. In the presence of bestatin, rhythmicity of glycyl-L-leucine absorption by the proximal intestine was unimpaired while cytosolic and membrane hydrolysis of the dipeptide was severely inhibited. These results were interpreted as indicating that the glycyl-L-leucine absorption rhythm is associated to a limited extent with daily fluctuations in the activities to hydrolyze the dipeptide and to transport the resulting free amino acids, but to a large extent with change in the activity to transport the dipeptide intact. It is suggested that the peptide absorption rhythm is related to someaspect of the luminal content rather than to its cyclic ingestion alone.

View full abstract

Chemiluminescence of a Cypridina luciferin analog, 2-methyl-6-phenyl-3, 7-dihydroimidazo[ l, 2-a]pyrazin-3-one, was applied to immobilized enzyme sensors. Xanthine oxidase, peroxidase, glucose oxidase, uricase and cholesterol oxidase were immobilized by using photocrosslinkable resin prepolymer or ion-exchangeable cellulose beads. The immobilized enzyme sensor system was composed of a photoncounter and a test tube in which the immobilized enzyme membrane or particles were placed. A linear relation between the concentration of substrates and luminescence rate was obtained on a logarithmic scale. This immobilized enzyme sensor system could be used repeatedly. Hydrogen peroxide, xanthine and hypoxanthine were measured sensitively and rapidly within 100 sec. Glucose, cholesterol and uric acid were measured sensitively within 10 min but could be measured within 100 sec, although less sensitive. The detection limits for xanthine, hypoxanthine, hydrogen peroxide, glucose, cholesterol and uric acid were 0.02, 0.02, 0.2, 0.4, 2 and 2?M, respectively. Concentrations of hypoxanthine in tuna muscle, and glucose and cholesterol in serum measured using this sensor system were comparable with those measured by the standard methods.

View full abstract

Curdlan gel shrinks even in water caused syneresis. This syneresis is repressed almost completely by the addition of starch to curdlan before heating, but not repressed by the addition of different sugar compounds even of 10%concentration. The role of starch in repressing syneresis is discussed.

View full abstract

Liposomes of soybean and egg yolk phosphatidylethanolamine (PE) were oxidized in the presence of copper. Fatty acyl moieties with hydroxy and hydroperoxy groups attached were converted to methyl esters. The methyl esters were separated in TLC and then separated on silicic acid thin layer plates containing AgNO3. After GLC purification, hydrogenated and trimethylsilylated samples were analyzed for positional isomers in MS. Fatty acids, 18:2 and 18:3 in soybean PE and 18:2, 20:4, and 22:6 in egg yolk PE were examined. A singlet oxygen mechanism was presumably involved in part, but to explain the whole isomer pattern, some yet unexplained mechanism appears to be involved.

View full abstract

A methanol-utilizing bacterium, which produced red to pink pigments and assimilated methanol via icl^- serine pathway, was isolated from soil and tentatively designated as Pseudomonas MS31. This bacterium produced L-serine when glycine was added to the growth medium at the late exponential phase of growth. The cells showed high L-serine degradation activity. Chelating agents and some metal ions, which inhibited L-serine degradation, stimulated the L-serine accumulation. In the presence of 0.11 mM EDTA, o-phenanthroline, 8-hydroxyquinoline, αα'-dipyridyl, cobalt sulfate or nickel sulfate, this bacterium produced 0.72.1 mg L-serine from 4 mg glycine per ml culture.

View full abstract

Temperature-sensitive mutants producing L-serine efficiently from glycine were obtained from the facultative methylotroph Pseudomonas MS31. Forty-five mutant strains showed adequate growth on methanol at 30°C but little or no growth at 37°C. Fourteen of these mutants produced L-serine more efficiently than the wild-type strain. The typical mutant strain ts 162 showed a high conversion rate in glycine-to-L-serine when the cultivation temperature was changed from a permissive (30°C) to non-permissive state (38 42°C) together with the addition of glycine and methanol after adequate growth. The mutant strain accumulated 6.8 mg L-serine from 12 mg glycine per ml culture under optimum conditions. The reduction of L-serine degrading activity in the mutant strain seemed to contribute to the high productivity of L-serine.

View full abstract

The highest inhibition rate of conidial germination of Pyricularia oryzae was shown by extracts of rice plant leaves inoculated by a pathogen after treatment with probenazole, a rice blast controlling agent. Four anti-conidial germination substances were isolated from these extracts. Substances A, C and D inhibited the germination of the conidia at concentrations between 100 and 200 mcg/ml, and substance B caused morphological changes in the germination tubes of the conidia with a little inhibition of germination. These substances were differentiated from momilactone A, B and the degraded or metabolized products of probenazole. Besides anti-conidial germination activity, they showed antimicrobial activities against several kinds of phytopathogenic bacteria of fungi on agar plates by diffusion method.

View full abstract

Substance B, the major component, isolated from rice plant treated with probenzaole and inoculated, having anti-conidial germination activity against blast fungus, was found to be a mixture of fatty acids, including palmitic acid, linoleic acid and linolenic acid. The main compound of substance B was linolenic acid, having strong anti-conidial germination activity. It was determined as a-linolenic acid by gas chromatographic analysis. The minor components showed little or no anti-conidial germination activity.

View full abstract

Nuclease activity associated with cells and protoplasts was analyzed by agarose gel electrophoresis. Datura innoxia protoplasts were found to possess a high exonuclease activity. On the other hand, Datura innoxia cells had an endonuclease activity, but no apparent exonuclease. The exonucleases from the protoplasts were active at pH 5 and 6, but not at pH 9. Endonuclease activity from the cells was also inhibited at pH 9. Cultured cells of Daucus carota, Glycine max, Pisum sativum and Vicia hajastana had endonuclease activity, but did not exhibit exonuclease activity. Nicotiana suaveolens cells had both types of nuclease activity. On the other hand, cells from cereals such as Triticum monococcum, Oryza sativa, and Zea mays had active exonuclease activity.

View full abstract

The four sesquiterpenes shizukanolide (1), dehydro-shizukanolide (2), glechomanolid (3) and isofuranodiene (4) were isolated from Chloranthus japonicus Sieb. (Hitori-shizuka in Japanese, Chloranthaceae). Their absolute structures have been established on the basis of their physical and chemical properties. Dehydro-shizukanolide showed moderate antifungal activity.

View full abstract

meso-α, ε-Diaminopimelate D-dehydrogenase was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and HgCl₂. Two sulfhydryl groups were titrated per molecule in the presence and absence of 6 M guanidine hydrochloride: the enzyme contained one sulfhydryl group per subunit. Modification of the sulfhydryl groups with p-chloromercuribenzoate, 5, 5' -dithiobis(2-nitrobenzoic acid), 4, 4'-dithiopyridine, N-ethylmaleimide, and iodoacetic acid was accompanied by a loss of enzymeactivity. However, modification of sulfhydryl groups of the enzyme with cyanide did not affect the activity. Thus, the introduction of bulky or charged substituents to sulfhydryl groups decreased the catalytic activity of the enzyme, but modification of the groups with the small and uncharged group, a cyano group, did not. The sulfhydryl groups did not play an essential role in catalysis.

View full abstract

Biological asymmetric hydrolysis of ethyl (±)-cycloheptadienecarboxylate with Rhodotorula minuta var. texensis IFO 1102 and chemical resolution of the corresponding carboxylic acid with (-)-quinine provided (R)-(+)-ethyl 2, 5-cycloheptadienecarboxylate (78% e.e.) and (S)-(+)-2, 5-cycloheptadienecarboxylic acid (95% e.e.), respectively. The (R)-(+)-carboxylate was converted to (R)-(-)-2, 5-cycloheptadienylcarbaldehyde and the (S)-(+)-carboxylic acid to (S)-(+)-2, 5-cycloheptadienylcarbaldehyde. Ectocarpene (78% e.e.), male-gamete attractant of marine brown alga, and its antipode (95% e.e.) were synthesized by stereoselective Wittig reaction between the (R)-(-)- and (S)-(+)-aldehydes and propyltriphenylphosphonium bromide in a liquid-solid two phase system using 18-crownether-t-BuOK, respectively.

View full abstract

Zein was resolved into several components by electrophoresis on polyacrylamide gel in the presence of sodium dodecylsulfate (SDS). Treated with 2-mercaptoethanol (2-ME) in advance, zein wasresolved into only two components by the electrophoresis. These two components were tentatively named α- and β-zein. Both were isolated by gel filtration on Sephacryl S-200 in the presence of SDS and 2-ME. Amino acid analysis showed that a- and β-zein were similar to each other, except that the number of methionine residue was three in the former and one in the latter. Whenprotein bodies isolated from corn endosperms were subjected to electrophoretical analysis, the same characteristics as those found in zein were observed.

View full abstract

Four isobutyl amides were isolated from the fruits of white pepper (Piper nigrum L.) and identified to be N-isobutyl-1 3-(3, 4-methylenedioxyphenyl)-2E, 4E, 12E-tridecatrienamide (3, guineensine), N-isobutyl-2E, 4E, 8Z-eicosatrienamide (5), N-isobutyl-2E, 4E-octadecadienamide (6) and N-isobutyl-2E, 4E-decadienamide (7, pellitorine).

View full abstract

Plasmid pUO1 which specifies haloacetate dehalogenase H-l and H-2 and mercuric reductase was isolated from a fluoroacetate-assimilating Moraxella1 strain B. From the spontaneous mutant deficient in H-2 enzyme derived from strain B, plasmid pUOl 1 specifying H-l enzyme and mercuric reductase was also isolated. The molecular sizes of pUOl and pUOll were estimated to be 43.7± 1.6 Mdal and 40.1 + 1.3 Mdal, respectively, by electron microscopy. These values were in good agreement with those estimated by electrophoretic analyses of the cccDNAand its restriction endonuclease digests. The digestion patterns of both plasmid DNAswere analogous, suggesting that plasmid pUOl l was the deletion mutant derived from pUOl. It could be presumed that the deleted DNAsegment had a molecular size of about 3.6 Mdal.

View full abstract

A strongly acidic amino acid-N-carboxymethyl-L-serine-, not previously knownin nature, has been isolated from asparagus (Asparagus officinalis) shoots. Some unique properties of this amino acid, such as a muchbigger mobility to anode on high voltage paper electrophoresis (pH 3.6) than aspartic acid and characteristic changes of NMR spectra in aqueous solution with various pD, were discussed in relation to its structure.

View full abstract

The level of glutamine synthetase in Micrococcus glutamicus ATCC1 3032 varied in response to the nitrogen source in culture medium; it was 10-20 fold higher in glutamate-, peptone- or yeast extract-grown cells than in ammonia-or urea-grown cells. Ammonia(3 mM)reduced the enzyme level to 50% when added to glutamate medium. No difference between nitrogen sources was observed in extent of inhibition by Mg²⁺ of γ-glutamylhydroxamate-forming (transferring) reaction in crude extracts.
The optimum pH was 7.0-8.0 for glutamine-forming (synthesizing) reaction and 7.0 for transferring reaction. The enzyme was stable to heating at 50°C for 10 min in 0.05M potassium phosphate buffer (pH 6.0) containing 0.1mM MnCl₂. Km values for glutamate, ammonia and ATP in synthesizing reaction were 7.9, 5.0 and 1.2 mM, respectively. GTP and hydroxylamine could be substituted for ATP and ammonia with about 10 and 30% reactivity. Mg²⁺ was effective as a cofactor in synthesizing reaction and Mn²⁺ showed 34% of the reactivity of Mg²⁺ at a concentration of 30 mM. Glutamine synthetase was inhibited by adenosine, AMP and ADP but not by amino acids other than D-threonine. The regulation system of glutamine synthetase in M. glutamicus is discussed.

View full abstract