The aromatic side chain of cationomycin (1) was modified by the coupling the sodium salt of deacylcationomycin (2) with several aromatic carboxylic acids. There was a linear relationship between the logarithmic reciprocal minimal inhibitory concentration of the derivatives against Bacillus subtilis IFO 3513 and the logarithmic transport efficiency for the sodium ion. The anticoccidial activity of the anisyl analog (3) was higher than that of the parent compound (1).

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From some Japanese soil, we isolated a strain of alkalophilic bacterium, Bacillus sp. OK-1, that excreted alkaline phosphatase into the culture broth. From the cell-free supernatant, the enzyme was successively purified by acetone fractionation, Sephadex column chromatography, and DEAE-cellulose column chromatography. The final preparation thus obtained showed only a single band when analyzed by SDS-polyacrylamide gel electrophoresis. The molecular weight was estimated as 108, 000, and was composed of two subunits having the same molecular weight. The purified enzyme had a pH optimum of 11 and was fairly stable between pH 5-12. This enzyme was inactivated by EDTA. Cobalt ions restored the enzyme activity. The Km was 0.037 mM.
On the basis of its properties, this metalloenzyme seems to be a novel extracellular alkaline phosphatase.

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Galactostatin obtained from the fermentation broth of Streptomyces lydicus PA-5726 strongly inhibited β-galactosidase. Its derivatives, galactostatin-lactam and 1-deoxygalactostatin, were also inhibitors. Galactostatin and 1-deoxygalactostatin were fully competitive inhibitors with high affinities for Penicillium multicolor β-galactosidase, and their Ki values were 4.0 × 10^-9 and 3.3 × 10^-8 at pH 6.0, respectively, using ONPG as substrate. In their presence, the steady-state velocities of the enzyme were reached in a matter of minutes. Galactostatin-lactam, in contrast, showed no detectable lag time on interaction with the enzyme, and the type of inhibition was also competitive with a Ki value of 1.3 × 10^-5 M. These three inhibitors bound to the enzyme in the same molar ratio (1:1).

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The effects of temperature on 6-O-α-maltosyl cyclodextrins (G₂-CDs) production from α-maltosylfluoride (α-G₂F) and cyclodextrins (CDs) by the transfer action of debranching enzymes such as pullulanase and isoamylase were studied.
The amounts of 6-O-α-maltosyl α-cyclodextrin (G₂-α-CD) production by purified pullulanase from Aerobacter aerogenes (A-pullulanase) and from Bacillus acidopullulyticus (B-pullulanase) increased with a rise in temperature, e.g., the amounts at 60°C were about 1.5 times higher than those at 30°C. Initial transfer ratios (G₂-α-CD formed/α-G₂F consumed) of A-pullulanase and B-pullulanase were about 62% and 25% (at 40°C), and about 50% and 15% (at 20°C), respectively. The transfer ratios of both A-pullulanase and B-pullulanase in the reaction using β-CD or γ-CD as acceptor also increased with a rise in temperature.
The transfer ratios were little affected by any change in temperature or any kind of acceptor CDs, in the case of isoamylase, and were about 60%.

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Auxotrophic mutants of a methanol-utilizing yeast, Candida sp. N-16, were hybridized through protoplast fusion using polyethylene glycol and Ca²⁺ ions. The complementation frequency of fusion was about 2 to 5 × 10^-4. Some of the prototrophic fusants obtained were very stable and distinguishable from the original prototroph. The fusants were uninuclear and contained 3.8 to 4.2 mg of DNA per 10¹¹ cells as compared with about 1.5 mg of DNA in the parental strains. The cell volume of the fusants was greater than that of the wild and parental strains. The growth rate of the fusants was slightly lower than that of the wild strain. There was a difference between the fusants and the wild strain with respect to the levels of alcohol-dissimilating enzymes in cells grown on methanol, and one of the fusants possessed about 2.5 times higher activity of alcohol oxidase than that of the wild strain.

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Polyphenols inactivated the plaque-forming activities of bacteriophages in the presence of cupric ion. Other metal ions did not replace cupric ion. The T-odd series of phages were more sensitive to polyphenols than the T-even series. Phage inactivation was prevented by catalase, Tiron, dithiothreitol, or 1, 4-diazabicyclo[2, 2, 2]octane, but not by superoxide dismutase or hydroxyl radical scavengers such as D-mannitol. These results indicate that free radicals of polyphenols and hydrogen peroxide were essential for polyphenol-induced phage inactivation.

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Geotrichum candidum HM-11 isolated from soil produced two types of endocellulase in a 200-1 fermentor at 30°C for 6 days. Endocellulase I (molecular weight 130, 000) was adsorbed onto insoluble cellulosic substrates such as Avicel and rapidly disintegrated them, but endocellulase II (molecular weight 63, 000) showed neither adsorbability nor disintegrating activity toward these substrates, although both enzymes rapidly decreased the viscosity of CM-cellulose. HPLC analysis revealed that both endocellulases hydrolyzed cellooligosaccharides finally to glucose, cellobiose and cellotriose.

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A limited proteolysis of Avicel-adsorbable, Avicel-disintegrating endocellulase I (molecular weight 130, 000) from Geotrichum candidum with subtilisin yielded a protein (molecular weight 80, 000) which proved fully active toward soluble substrates such as CM-cellulose, but lost both the abilities to be adsorbed onto insoluble substrates and to disintegrate the cellulose fibres. An immunological experiment showed precipitin lines between endocellulase I and subtilisin-modified endocellulase in the pattern of partial identity. N-Bromosuccinimide-oxidized endocellulase I lost cellulase activity, but retained its adsorbability onto Avicel. It is suggested that endocellulase I had both the affinity site for adsorbing onto insoluble substrates and the ordinary active site.

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An immobilized acarbose column selectively adsorbed most of glucoamylase components from a commercial glucoamylase preparation. The adsorbed enzyme was specifically eluted with maltose into a glucoamylase fraction free from α-amylase and α-glucosidase. The eluate was further fractionated into six subfractions by gel chromatography and subsequent anion-exchange chromatography. Each of the enzyme subfractions liberated β-glucose as the sole product from soluble starch and maltooligosaccharides. Thus, all the enzymes are glucoamylases, though the enzymes were apparently discriminated from one another on the basis of molecular weight and/or electrophoretic behavior. Furthermore, the enzyme subfractions were classified roughly into three groups on the structural resemblance implied by immunological cross-reactivity among them.

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A commercial preparation of glucoamylase from Aspergillus niger consisted of at least six species (A-II-IV and B-I-III), five forms of which were fully characterized. They were apparently different in molecular weight, sedimentation constant (S_{20, w}), isoelectric point (pI), chemical composition, kinetic parameters (k₀, molecular activity; k_int, intrinsic rate constant), and other enzymatic properties. When compared with enzymological characteristics as well as serological ones, B-I was found to be a peculiar enzyme among them. Furthermore, A-II was significantly discriminated from the other enzymes in respect of molecular weight and composition of amino acid, suggesting that A-II might be the product of transcription of a different DNA site.

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Glucoamylase (GA) was purified from koji, a solid culture of Aspergillus oryzae on steamed rice, by extraction with 1% NaCl solution, precipitation with ethanol, and acarbose affinity chromatography. The purified enzyme was homogeneous on gel filtration, PAGE and SDS-PAGE, ultracentrifugation, and IEF. The enzyme released β-glucose as a sole product from soluble starch and maltooligosaccharides. The other common and inherent features of GAs were also confirmed in the GA from A. oryzae. The enzyme was a glycoprotein containing about 4.8% glucosamine and 7.8% neutral saccharides.

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A gram negative bacterium isolated from soil was found to produce an endo-α-N-acetylgalactosaminidase in culture. The microorganism was identified as Alcaligenes sp. from various bacteriological characteristics. The enzyme was purified from the culture fluid by fractionation with ammonium sulfate, and column chromatographies on DEAE-Sephadex A-50 and hydroxylapatite. The molecular weight of the enzyme was estimated to be 160, 000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH was found to be 4.5 and the stable pH range was 4.5-6.5. The Km values were 3.7mM and 3.2mM with asialofetuin and asialo κ-casein glycopeptide as substrates, respectively. The enzyme released the disaccharide, Galβ1→3GalNAc, from glycoproteins possessing serine or threonine O-glycosidic linkages. The enzyme production was highly induced by porcine gastric mucin added to the medium.

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Botrytis cinerea, which was isolated from the surface of stored sunflower seed, produced a novel biologically active natural product when cultured on shredded wheat medium. The metabolite, trivially named cinereain, was a ruby red crystalline product with the molecular formula C₁₈H₂₁N₃O₃ and melting point 201-203°C. The structure was unequivocally established by single crystal X-ray diffraction; UV, IR, NMR (¹H and ¹³C) and mass spectrometry supported the crystallographic data. Cinereain significantly inhibited the growth of etiolated wheat coleoptiles (p<0.01) at 10^-3 and 10^-4M in bioassays. No effects were noted on treated intact greenhouse-grown bean and tobacco plants, but there was mild necrosis and chlorosis in corn.

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Protein disulfide-isomerase (PDI) was studied for the first time as a stimulator of refolding of recombinant proteins produced in bacteria. Experiments with reduced or scrambled ribonuclease as a substrate showed that PDI was active in low concentrations of the protein denaturants which were generally used in the refolding systems of recombinant proteins. Crude human pro-urokinase cloned and expressed in Escherichia coli was tested as an example of a recombinant protein, and PDI accelerated its refolding. The time needed to reach 50% maximum pro-urokinase refolding was shortened by half by the addition of PDI. Reduced pro-urokinase was further stimulated to refold by this enzyme. Evidence that PDI catalyzes the refolding of pro-urokinase through thiol-disulfide exchange reactions was also obtained from the repression of such stimulation by a PDI inhibitor, bacitracin. The results suggested that this refolding system can be used for the refolding of recombinant proteins produced in bacteria.

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An E. coli strain, SH209, harboring pLC9-12 exhibited 5- to 6-fold higher γ-glutamyltranspeptidase activity than the wild-type strain, at each growth temperature tested. Maximum activity was observed at 20-25°C, as was observed with the wild type. A homogeneous enzyme preparation was obtained from the periplasmic fraction of the strain by a simple three-step method. The conditions for γ-glutamyl-DOPA synthesis from L-glutamine and L-DOPA were investigated using the enzyme preparation. Under the best conditions, the maximal yield of 79%, equivalent to 158 mM (51.5 g/1) of γ-glutamyl-DOPA as to both substrates, was obtained. γ-Glutamyl-DOPA was isolated from the reaction mixture and identified using an amino acid analyzer after hydrolysis with HC1 or γ-glutamyltranspeptidase.

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The effect of various reagents on the formation and stability of heat-induced gels of sesame 13S globulins were investigated. Electrostatic interaction, the hydrophobic bond and the disulfide bond were important for forming the network structure of gels, and the hydrogen bond also had an influence on the formation of the gel. Hydrophobic bonds mainly contributed to the stability of the gel. Subunit analyses of the proteins solubilized from the gels showed the presence of a free acidic subunit (AS) and basic subunit (BS), a polymer of AS, a dimer of BS and the dimer of a fragment from AS or BS. From the results, sulfhydryl-disulfide exchange reactions during gelation are suggested.

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A thermostable aminopeptidase, called aminopeptidase T, from the extract of Thermus aquaticus YT-1 was purified and characterized. The enzyme had a dimeric structure, its relative molecular mass being 108, 000 by gel filtration, and 48, 000 by SDS-PAGE. The optimum pH of the enzyme activity was in the range of 8.5 to 9.0. The enzyme was significantly thermostable as it still retained 60% of its original activity even after heat treatment for 20 hr at 80°C. The enzyme activity was inhibited by metal-chelating agents. The enzyme had a low substrate specificity.

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Male rats of the Wistar strain (seven weeks old) were fed ad libitum with a 10%, 20% or 40% casein diet (these diets contained sufficient nitocinic acid of 6 mg per 100 g of diet) for 20 days. Urine was collected for the last 2 days, and the urinary excretion of nicotinamide, N¹-methylnicotinamide (MNA), N¹-methyl-2-pyridone-5-carboxamide (2-py) and N¹-methy-4-pyridone-3-carboxamide (4-py) was measured using the most reliable determination methods with a high-performance liquid chromatograph. The urinary excretion of nicotinamide and 2-py was about the same in the three groups. The urinary excretion of 4-py increased with increasing dietary protein levels, contrary to MNA, which decreased with increasing dietary protein levels. The total urinary excretion of nicotinamide, MNA, 2-py and 4-py was about the same amount in the three groups. The contents of total nicotinamide and MNA in the liver decreased with increasing dietary protein levels, all these results being inconsistent with the established theory.

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Three glycopeptides have been isolated from ricin D; one glycopeptide with Asp¹ Thr¹ Ile¹ Phe¹ GlcNAc² Man³ Xyl¹ and Fuc¹ was from the A-chain and two glycopeptides with the compositions of Asp² Thr¹ Gly¹ GlcNAc² Man⁶ and Asp² Thr¹ Glu¹ Pro¹ GlcNAc² Man⁶ were from the B-chain. From CBH, on the other hand, four glycopeptides were isolated; one glycopeptide with Asp¹ Thr¹ Ile¹ Phe¹ GlcNAc² Man³ Xyl¹ Fuc¹ from the A-chain, and three glycopeptides, one with Asp¹ Cm-CyS¹ GlcNAc² Man³ Xyl¹ Fuc¹, one with Asp² Arg¹ GlcNAc² Man⁶ and the third with Asp² Thr¹ Glu¹ Pro¹ GlcNAc² Man⁶, were from the B-chain.

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Ribavirin (l-β-D-ribofuranosyl-l, 2, 4-triazole-3-carboxamide; Virazole®), which is known as a potent antiviral agent, was synthesized directly from purine nucleosides and l, 2, 4-triazole-3-carboxamide (TCA) by a newly selected bacterium, Brevibacterium acetylicum ATCC 954. Among the purine nucleosides tested, guanosine and xanthosine were selected as the best substrates as to ribavirin production. The highest amount of ribavirin produced was 229 mM from 300 mM guanosine and 300 mM TCA on 96 hr reaction at 60°C. In this system involving ATCC 954, ribavirin was effectively even produced at a limited concentration of phosphate (about 10mM). ATCC 954 could also produce ribavirin directly from inosine and TCA in a high yield, but could hardly produce it directly from adenosine and TCA. In contrast, a bacterium belonging to the genus, Bacillus, could produce ribarivin directly from adenosine and TCA in a high yield.

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A 779 bp HindIII fragment of Bacillus subtilis was cloned, which in a multi-copy state renders B. subtilis cells tunicamycin resistant. Its nucleotide sequence was determined and a possible gene product of 197 or 199 amino acids was deduced.

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The bitterness of triterpenoid components, which were isolated from the fruiting body of Ganoderma lucidum, was examined. The structural similarity of the intensely bitter compounds was also elucidated, from which it was concluded that the spatial relationship of the hydrophobic methyl groups to the three functional oxygen atoms plays an important role in generating the bitterness.

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The complete assignments of four lycopodium triterpenoids, lycoclavanol, lycoclaninol, 21-epi-serratenediol and serratenediol, were accomplished by the use of a new two-dimensional technique, HMBC (heteronuclear multiple bond connectivity). It appears this technique is extremely powerful for the structural analysis of complicated compounds with many methyl groups.

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Various lengths of myosin filaments were prepared by changing the speed of lowering the ionic strength. Short filaments were formed by rapid dilution and the filament length became longer with decreasing the speed of lowering the ionic strength. The average length of myosin filaments ranged from 0.5 to 3.0 μm with varying the speed of dilution. Myosin filament suspensions were heated to form gels, and those gel strengths were evaluated as rigidity. The rigidity depended on the length of the filaments before heating. The longer the filaments, the higher the rigidities. The gels with higher rigidity had a fine strand-like network structure. In contrast, that showing lower rigidity had a coarse aggregated structure. When low protein concentrations of myosin filament suspensions were heated at 40°C, myosin filaments associated with increasing the incubation time. When heating was done at 60°C, aggregation of myosin filaments occurred in a short time. Electron microscopic observation suggests that the association and the following aggregation of myosin filaments per se cause formation of a whole network structure. Short myosin filaments began to aggregate randomly in an early stage of heating and they made clusters. But long filaments did not form a cluster; instead, some of them associated side-by-side to form bundles. These differences in the mode of aggregation among various lengths of myosin filaments seemed to be one of possible reasons for the different network structures observed in the gels.

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Advantageous mutants of Pseudomons chlororaphis B23 for the enzymatic production of acrylamide were isolated. A mucilage polysaccharide non-producing mutant, Am-3, was precipitated completely by brief centrifugation, in contrast with the parent strain. A mutant, AM-324, derived from Am-3 exhibited about 3.8-fold higher nitrile hydratase activity than that of the parent strain. These mutants are promising for acrylamide production on an industrial scale.

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D-Xylose (xylose) isomerase was extracted from xylose-grown cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. The enzyme was purified 70-fold, over the original cellfree extract, with a yield of 2.4% in a homogeneous state, as judged on sodium dodecyl sulfatepolyacrylamide gel electrophoresis and high performance liquid chromatography. The molecular weight of the enzyme was determined to be 130, 000, the enzyme being composed of two subunits of 65, 000. The optimum pH and temperature for activity were 4.5 and 37-45°C, respectively. The enzyme activity was markedly enhanced by Mn²⁺, Mg²⁺ and Co²⁺, and the enzyme isomerized aldopentoses and aldohexoses. The Km values for xylose and D-glucose were 5.6 × 10^-1M and 4.1×10^-1M, and the V_max values were 5.8 × 10² and 3.3 × 10² μmol/min/mg, respectively. NaHAsO₄•7H₂O and NaCN strongly inhibited the activity, but HgCl₂, NaN₃, dithiothreitol, monoiodoacetate and polyols such as D-sorbitol, xylitol and D-mannitol did not inhibit the activity.

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Methyl esters of gibberellins A₁₂ and A₁₄ (GA₁₂ and GA₁₄) were converted to 12- and 13-hydroxyGA methyl esters in a culture medium of prothallia of Lygodium japonicum. One of the metabolites, the methyl ester of 12α-hydroxyGA₁₄ (ent-3α, 12β-dihydroxygibberella-7, 19-dioic acid) was identified as the methyl ester of a GA occurring naturally in immature seed of Cucurbita maxima, and GA₇₄ was allocated to the free acid.

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