Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1, 2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydrogenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.
The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1, 2-propanediol dehydrogenated 1, 2-propanediol, 1, 2-butanediol and 2, 3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and 11 on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1, 2-propanediol and 2, 3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.

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A new chemotype mannan isolated from Candida utilis mutant was studied by fragment-ation analysis with acid. The preparation was homogeneous on ultracentrifugation and electrophoresis, and was composed of D-mannose and n-glucose in an approximate ratio of 23:2. Controlled acid hydrolysis gave 2-O-α-D-mannopyranosyl-D-mannose (1), 3-O-α-D-mannopyranosyl-D-mannose (2), 6-O-α-D-mannopyranosyl-D-mannose (3), 6-O-α-D-gluco-pyranosyl-n-mannose (4), O-α-D-mannopyranosyl-(1→2)-O-α-n-mannopyranosyl-(1→2)-α-mannose (5), O-α-D-mannopyranosyl-(1→)-O-α-D-mannopyranosyl-(1→6)-α-mannose (6), O-α-α-mannopyranosyl-(1→6)-O-α-α-mannopyranosyl-(1→6)-α-mannose (7), O-a-D-manno-pyranosyl-(l→2)-O-a-n-mannopyranosyl-(1→2)-O-a-D-mannopyranosyl-(1→6)-D-mannose (8), O-α -α-mannopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→6)-O-α-D-mannopyranosyl-(1→6)-D-mannose (9) and O-α-D-mannopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→2)-O-α-D-man-nopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→6)-D-mannose (10). Characterization of (4) has afforded firm evidence that C. utilis mannan is a glucomannan. Preferential cleavage of yeast mannan by acid hydrolysis is also discussed.

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Oxidation of norepinephrine catalyzed by Cu²⁺ was variously regulated with nucleic acid components. The reaction proceeded by a mechanism of sequential random ordered reaction via formation of the mixed complex of nucleic acid component, Cu²⁺ and aromatic reductone. Using norepinephrine as an aromatic reductone, the promoting activities of nucleic acid components on the oxidation of norepinephrine were compared and the effect of these com-ponents to the specific stage of the oxidation process was kinetically investigated. The results indicated that velocity of the oxidation was most remarkably stimulated in the presence of adenine. The velocity was followed by guanine, guanosine monophosphate, cytosine, cytidine, NAD⁺, adenosine, cytidine monophosphate, uridine monophosphate, then adenosine mono-phosphate in that order. It was also discussed that adenine was the most plausible nucleic acid component which could participate in the in vivo oxidation of norepinephrine, taking into account the concentration of Cu2+ and nucleic acid components in living tissues.

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A trypsin inhibitor was extracted from rice bran with 1% sodium chloride and partially purified by 40_??_80% ammonium sulfate fractionation. The crude inhibitor obtained showed the inhibitory activity on trypsin [EC 3. 4. 21. 4] but not on a-chymotrypsin [EC 3. 4. 21. 1] or pepsin [EC 3. 4. 23. 1]. This inhibitor was stable at acidic and neutral pHs but was gradually inactivated by the long-time incubation with pepsin. The molecular weight of the inhibitor was estimated to be 13, 500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Furthermore, gel filtration on Sephadex G-75 suggested the possible self-association of the inhibitor. Isoelectric focusing demonstrated that the inhibitor is a basic protein having a pI value of 8.2. The inhibitor was adsorbed on to CM-Sephadex C-25 at pH 5.7, indicating that it can be further purified by this chromatography.

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Two alcohol dehydrogenases (alcohol: NAD oxidoreductase, EC 1. 1. 1. 1 and alcohol: NADP oxidoreductase, EC 1. 1. 1. 2) were partially purified from extracts of strawberry seeds by conventional methods. Some of physical, chemical and kinetic properties of the enzymes are described. On the basis of gel filtration, the molecular weights were estimated to be approximately 78, 000 for NAD-dependent enzyme and 82, 000 for NADP-dependent enzyme. Thiol-reacting compounds inhibited both enzymes. NAD-dependent alcohol dehydrogenase reacted only with aliphatic alcohols and aldehydes, while aromatic and terpene alcohols and aldehydes were the better substrates for NADP-dependent alcohol dehydrogenase than ali-phatic alcohols and aldehydes.

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In the course of our studies on the polyphenol components of the cultured cells of amacha, seven polyphenol compounds were isolated as pure crystals. The chemical structures of these compounds were determined by analytical methods (IR, UV, NMR and MS spectra) and colour reactions, and the compounds were identified as phyllodulcin, hydrangenol, daphne-tin-8-monomethylether, umbelliferone, phyllodulcin-8-β-D-glucoside, hydrangenol-8-β-D-gluco-side and skiminin (umbelliferone-7-β-n-glucoside). This is the first report of the isolation of phyllodulcin-8-β-D-glucoside and daphnetin-8-monomethylether from a natural source and of the other compounds from the cells of higher plants in suspension culture.

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Effects of Aspergilhis oryzae-proteolipid (PL) on the formation of high concentrations of alcohol, more than 20% as in sake brewing, have been studied. Electron microscopy revealed that there were no vacuoles but many lipid deposits in cytoplasm of the alcohol-tolerant cells of Saccharomyces sake Kyokai No. 7, which were obtained by the anaerobic culture supplemented with PL. Sphaeroplasts from the alcohol-tolerant cells were stable in 20% alcohol, whereas those from the untolerant cells grown anaerobically in the PL-un-supplemented media were ruptured. The cell membrane became alcohol-endurable in the anaerobic cultures with PL. Synthetic phospholipid promoted yeast growth and the fer-mentative activity, whereas a small amount of sterol ester enhanced the alcohol-endurability. The supplementation of both lipids anaerobically induced physiological properties charac-teristic of the alcohol-tolerant cells.

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The reaction between formaldehyde and acetamide which affords a model compound for an amino acid having an amide group was analyzed to investigate the role of formaldehyde as a cross-linking reagent. One of the products was isolated by Sephadex G-10 column chromatography and was identified as N-hydroxymethyl acetamide (FA) by NMR spectro-metry and mass spectrometry. Another product, which could not be isolated, was estimated to be N, N-dihydroxymethyl acetamide (F₂A) by kinetic analysis and mass spectrometry. The formation of N, N'-methylene diacetamide was not observed. The mechanism of the reaction between formaldehyde and acetamide was estimated by the kinetic analyses of NMR data, and the rate constants were calculated from the data by the optimization method with a digital computer, On the other hand, formaldehyde cross-linked product was obtained in the reaction of formaldehyde with acetamide and alanine, Its decomposing reaction was analyzed with an NMR spectrometer to study the stability of the formaldehyde cross-linked product. The degradation was dominantly initiated with the release of acetamide.
Consequently, it was estimated that the C-N bond between formaldehyde and amide is so labile that amide-bound formaldehyde does not react further with amides or amines, and that the amide-formaldehyde-amine condensation product is unstable and easily decomposes by releasing the amide.

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Twelve protein-leaky mutants accumulating in culture medium at 10% to 60% of total protein produced by the bacteria were isolated from E. coii W4626Phe-. Polyacrylamide gel electrophoresis of the culture fluids of the mutants revealed many protein components, but the patterns of the stained bands in gels of exoproteins were somewhat different among mutants.
Three mutants (PE 1, 4, 10) were selected and further examined. Time course of protein accumulation showed that protein was released even after the cell mass reached maximum. The content of DNA, RNA, and protein in the culture supernatant revealed the characteristics of each mutant. The ratios of these macromolecules in PE 1 and 10 were similar to those of the parental strain. PE 4 was different from other mutants in that the content of RNA and protein was very higher than that of DNA.
Periplasmic enzymes were released by all three mutants. A large amount of cytoplasmic enzymes, such as β-galactosidase, was found in the supernatant of PE I and 10 but not in PE 4. Only PE 10 was more sensitive to lysozyme than the parent. Each mutant showed a charac-teristic cell shape.

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The antimicrobial action of trimethyl-, triethyl-, tripropyl- and tributyltin chloride on bacteria, yeasts and fungi was investigated. Trialkyltin chlorides were found to have high antimicrobial action on most microorganisms, especially on fungi. The activity of these compounds on all microorganisms, except on gram-negative bacteria, increased logarithmically with an increase in the carbon number of the alkyl chains of trialkyltin chlorides. Most microorganisms treated with trialkyltin chloride lost the ability to form colonies. The activity was influenced remarkably by pH, but not by the growth phase of the microorganism. Ethyl-enediamine tetraacetic acid had a synergistic effect on the antimicrobial action of trialkyltin chlorides on E. coli.

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The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H₂S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Ab-sorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105, 000g for 60 min.

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The effect of amino acids was examined on the production of L-lysine by AEC resistant mutant of B. lactofermentum. Among amino acids tested, only leucine showed strong specific inhibition. In order to release the production of L-lysine from this negative effect of leucine, leucine auxotrophs were derived from AEC resistant strain of B. lactofermentum. Most of these leucine auxotrophs produced larger amount of L-lysine (maximally 41mg/ml) than the parental strain which produced about 18mg/ml of L-lysine. It was confirmed that leucine auxotrophs derived from AEC resistant mutant of other glutamate producing bacteria, B. saccharolyticum and Corynebacterium glutamicum. These results suggested that leucine might directly or indirectly affect the biosynthesis of lysine.
However, this increase in lysine productivity of leucine auxotrophs could not be ex-plained by the alteration of aspartokinase (EC 2. 7. 2. 4) and homoserine dehydrogenase (EC 1. 1. 1. 3). These enzymes are key enzymes in lysine and threonine biosynthesis, respectively

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Some characteristics of polyvinyl alcohol (PVA) degrading enzyme produced by Pseudomonas O-3 which utilizes PVA as the sole carbon source were examined. The enzyme contains 1 atom iron per molecule and its solution has absorption maxima at 280, 365 and 470nm. It produced hydrogen peroxide with the degradation of PVA. Equivalence was established between the amounts of oxygen consumed and hydrogen peroxide produced, indicating that this enzyme belongs to an oxidase but not to an oxygenase. In its substrate specificity, it oxidizes secondary alcohols such as 2-pentanol, 2-hexanol, 3-hexanol, 2-heptanol, 3-heptanol and 4-heptanol. Among PVA analogues, it does not oxidize low molecular weight analogues such as dimer and trimer but oxidizes analogues with molecular weight larger than 300.

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The effects of Cu (I)- and Cu (II)-amino acid complexes on the autoxidation of linoleic acid (LA) were examined in an aqueous ethanol solution at pH 7.0 and the following results were obtained.
1) Cu (I)-amino acid complexes were rapidly oxidized by linoleic acid hydroperoxide (LAHPO) or oxygen.
2) Cu (I)-amino acid complexes were autoxidized and LA was not in the reaction mixture containing the complexes and LA.
3) The prooxidative effects of Cu (II)-amino acid complexes were proportional to the effects of the complexes on the decomposition of LAHPO.
4) Autoxidation of LA promoted by Cu (II)-amino acid complexes was inhibited by radical scavenger and hydrogen donor, e. g., n-mannitol and 2, 6-di-tert-butylphenol (DTBP).
5) Cu (II)-amino acid complexes had no effect on the autoxidation of LA promoted by radical initiator, e. g., 2-azo-bis-isobutyronitrile (AIBN).
These results suggest that Cu (II)-amino acid complexes are more stable than Cu (I)-amino acid complexes, and that Cu (II)-amino acid complexes promote the radical formation by decom-posing LAHPO in the autoxidation of LA.

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Kinetic data are given on aconitase (EC 4. 2. 1.3.) of a strain of Saccharomycopsis lipolytica excreting citric acid. Various inhibitors were tested in vitro. Sigmoid inhibition curves (Hill coefficient=4_??_6) were observed with malefic and adipic acids. The effect of the in-hibitors on excretion is discussed.

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Invertase of Candida utilis purified in an electrophoretically and ultracentrifugally pure state (molecular weight, 3×10⁵; isoelectric point, 3.8) was analyzed.
Nitrogen content of the enzyme preparation was 8.45%. The preparation contained a great deal amount of sugar moiety which consisted of mannose, glucose, and glucosamine in amounts of about 40%, 4% and 3%, respectively.
Results of amino acid analysis and the molecular weight determined by some physico-chemical methods reported previously led to the conclusion that the enzyme consisted of about 1380 moles of amino acid residues per mole. The invertase was rich in Asp, Glu, Set and Thr in the decreasing order. Amino terminal amino acid of the enzyme was determined as serine.

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A tannic substance named neocretanin was isolated from chestnut galls by chromato-graphic separation. The chemical structure of neocretanin was established as 1-O-(2', 6'-dihydroxy-4'-formyl)-phenyl-6-O-galloyl-β-D-glucopyranose (I) on the basis of spectral analyses and enzymatic hydrolyses.

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Acinetobacter sp. 351 isolated from soil grows well on butadiene oligomers and degrades 30 percent of a commercial sample of liquid polybutadiene (cis-1, 4 type, Mn=650) in 3 days. From the gel permeation chromatography of the substrate after the degradation, the strain was shown to degrade butadiene oligomers with the degree of polymerization up to about 10, but not higher oligomers. The strain also grows on n-alkanes with carbon numbers from 9 to 40, and on some alkenes and fatty acid methyl esters. The assimilation and oxidation of butadiene oligomers were compared with those of n-alkanes and alkenes.

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Inhibitory study of RNase L with ferric ion was performed to clarify the function of the enzyme. The maximal inhibitory pH of ferric ion for the enzyme is in 3.5_??_5.0 and the type of inhibition is found to be competitive with the substrate. The Km for RNA and Ki of ferric ion are 1.54×10^-1mg/ml and 22.5μm, respectively.
As the recovery of activity from iron-inactivated RNase L is almost completely realized by dialysis against EDTA solution, the inhibition of RNase L with ferric ion is considered to be a reversible reaction. Moreover, the enzyme is rapidly inactivated by methylene blue- or rose bengal-sensitized photooxidation, but the photoinactivation of RNase L is protected in the presence of the competitive inhibitor, ferric ion. It may reasonably be taken as an evidence that ferric ion attacks the active site of the enzyme.

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Volatile flavor components of cooked rice were studied. Steam distillate of cooked rice was extracted with methylene chloride and the extract was separated into four fractions: acidic, weak acidic, basic and neutral fractions. The neutral fraction was further separated into hydrocarbons and oxygenated compounds by column chromatography. All fractions were analyzed by a combination of glass capillary gas chromatography and mass spectrometry.
One hundred constituents, including 13 hydrocarbons, 13 alcohols, 16 aldehydes, 14 ketones, 14 acids, eight esters, five phenols, three pyridines, six pyrazines, and eight other compounds, were identified. Of these, 92 were newly identified volatile flavor components of cooked rice.

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Juice prepared from cabbage, broccoli, green pepper, egg plant, apple, burdock, shallot, ginger, pineapple and mint leaf were found to possess strong capacities of inactivating the mutagenicity of tryptophane pyrolysis products. In addition, radish, sweet potato, grape, Japanese ginger, cauliflower, beefsteak plant, enokidake mushroom and simeji mushroom were moderatly effective. The other forty one samples were inactive. Among the above eleven samples that inactivated the tryptophan pyrolysate efficiently, egg plant, burdock and broccoli showed the widest spectra of inactivating other mutagenic amino acids pyrolysates. The desmutagenic factor of cabbage is sensitive to heating (100°C) and pronase treatment, sug-gesting a nrotein character.

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After twelve steps of purification, a peptidyl factor named rhodotorucine A^*1 was isolated in pure form from a culture filtrate of A type cells of Rhodosporidium toruloides. Rhodo-torucine A induced the mating tube formation in a cells, opposite mating type cells of the yeast, at a concentration of 8ng/ml.

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A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3% and 840mgN/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2×10^-4M concentration. Cultures at 25°C produced the highest yield for nicotine. Con-siderable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W. basis at 4 weeks' culture. This value is near to that of the intact tobacco plants.

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Two constituent polypeptide chains of ricin D, a highly toxic protein from castor bean seeds, were cleaved with cyanogen bromide (CNBr). Four CNBr fragments, α-CB I, α-CB II, α-CB III, and α-CB IV, were isolated from the Ile (α) chain by gel filtration through Bio-Gel P-30 and characterized. N-Terminal sequence analyses revealed that the alignment of these fragments was (α-CB I)-(α-CB IV)-(α-CB II)-(α-CB III).
CNBr cleavage of Ala (β) chain, followed by reduction and S-carboxymethylation, yielded four fragments, β-CB I, β-CB II, β-CB III, and β-CB IV. Methionine residue located at 5th position from N-terminus of Ala chain was found to be resistant to CNBr cleavage. Sequence analyses of these fragments revealed that fragment β-CB IV was originated from fragment 3-CB III by acid cleavage, not by CNBr cleavage of aspartyl-prolyl bond during CNBr treat-ment, and the alignment of CNBr fragments was established to be (β-CB II)-β-CB I)-(β-CB III).

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Amino acid sequences of nine tryptic peptides derived from performic acid oxidized Ile-chain or cyanogen bromide fragments of the protein were determined mainly by the modified Edman procedure. Thus, 177 residues (67%) out of a total of 265 amino acid residues of the polypeptide chain were sequenced in the present study.

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Sixteen peptic peptides, which contain arginine(s) or lysine, were isolated from cyanogen bromide fragments CB I and CB II of Ile-chain. Sequence determination has been performed on most of these peptides to provide overlaps for the tryptic peptides. Thus, complete amino acid sequence of Ile-chain consisting of 265 amino acid residues was determined. Some structural characteristics of the protein are also discussed.

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A new procedure for the preparation of optically active 5-substituted hydantoins (5-hydantoins) has been developed. This procedure consists of (I) alkaline hydrolysis of a 5-hydantoin to its corresponding a-amino carboxylic acid, (II) oxidation of either one of the stereoisomers of amino acid with commercially available amino acid oxidase enzymes, (III) iso-lation of the remaining stereoisomer by ion exchange chromatography, and (IV) resynthesis of the 5-hydantoin by the procedure of Dakin. Because the enzymes utilized in step (II) have very broad substrate specificity, this procedure should be generally applicable to the prepa-ration of optically active 5-hydantoins. As an example, the synthesis of L-5-(4-hydroxybutyl) hydantoin is described, and the optical properties of this previously unresolved compound are reported.

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