Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 58 , Issue 1
Showing 1-50 articles out of 57 articles from the selected issue
  • Masaharu Ishii, Tetsuya Ono, Ken-ichiro Suzuki, Yasuo Igarashi, Tohru ...
    1994 Volume 58 Issue 1 Pages 1-4
    Published: January 23, 1994
    Released: February 08, 2008
    JOURNALS FREE ACCESS
    The electron transport system in Pseudomonas hydrogenothermophila strain TH-1, a thermophilic, aerobic hydrogen-oxidizing bacterium, was studied using a membrane fraction, an ubiquinone-depleted membrane fraction (Q- MF), and an ubiqunone-reconstituted membrane fraction (Q+ MF). From spectrophotometric and TLC analyses, cytochromes b and c, ubiquinone-8 (Q8), and cytochrome 0 were detected as respiratory components in the membrane fraction of P. hydrogenothermophila. Spectrophotometric analyses also showed that reduction of all the components in the membrane fraction depends on a membrane-bound hydrogenase reaction. In Q- MF, reduction of cytochromes b and c depending on membrane-bound hydrogenase reaction could not be observed. However, the reduction was restored in Q+ MF. From these results, a possible electron transport system in P. hydrogenothermophilia strain TH-1 is proposed
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  • Hirofumi Higaki, Haruki Yamaguchi
    1994 Volume 58 Issue 1 Pages 5-8
    Published: January 23, 1994
    Released: February 08, 2008
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    The reconstitution of Phaseolus vulgaris α-amylase inhibitor from its two kinds of subunits was studied. The inhibitor was reconstituted by quick dilution from its subunits denatured in 6M guanidine hydrochloride with a concomitant restoration of the inhibitory activity. Various environmental factors had a profound influence on the rate and extent of the reconstitution. Especially low protein concentration and high ionic strength were essential for a high yield of regained activity, and Zn2+ was also effective in promoting reconstitution. The maximum specific activity of reconstituted inhibitor was obtained at a 1 : 1 molar ratio of α-subunit : β-subunit. Under the optimal conditions obtained, the activity regain was about 60% of the activity of the native inhibitor. Further, this report advances the possibility that the glycan chains of each subunit may be important in proper folding and assembly of the subunit polypeptides.
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  • Nobuaki Ishida, Hiromi Kano, Hidejiro Ogawa
    1994 Volume 58 Issue 1 Pages 9-13
    Published: January 23, 1994
    Released: February 08, 2008
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    A micro-imaging probe, the best planar resolution of which was 0.0125mm × 0.0125mm, was devised using an NMR spectrometer with a superconducting magnet (JEOL GSX-270 WB). A strong Z-axis magnmtic field (slice) gradient was required to lessen slice thickness. Application of a strong slice gradient around the 180°refocus pulse of the spin-echo 2D-FT method induced extra-reduction of signal intensity beyond that expected. The problem which was considered to be due to a diffusive effect, was overcome by saving the slice gradient on application of the 180°refocus pulse. Using the probe, measuring conditions (combinations of pamameters) for a young tomato fruit of several mm in diameter were examined. Reduction of spatial resolution (voxel) was limited by a decline of image quality and the minimum voxel suitable for observation of the sample was 0.05mm × 0.05mm planar resolution and 0.25mm slice thickness corresponding to two or three plant cells vertically aligned. NMR micro-imaging can be used for studying cell conditions in plant materials of small sizes
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  • Ikuo Suda, Shu Furuta, Yoichi Nishiba
    1994 Volume 58 Issue 1 Pages 14-17
    Published: January 23, 1994
    Released: February 08, 2008
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    A fluorometric method has been developed to measure a 1, 3-diethyl-2-thiobarbituric acid (DETBA)-malondialdehyde (MDA) adduct as an index of lipid peroxidation in plant materials. Plant tissue samples were prepared under nitrogen gas and then added to an assay system containing butylated hydroxytoluene. Following the reaction between DETBA and the plant tissue samples, the DETBA-MDA adduct was extracted with ethyl acetate and measured by spectrofluorometry or high-performance liquid chromatgraphy (HPLC) with a fluorescence detector. The species of influencing substances with spectrofluorometry were fewer and their interfering concentration was higher than that by traditional colorimetry. When this method was applied to plant materials, the detection limits for the DETBA-MDA adduct were 2.5nmol/g of fresh weight and 0.0625nmol/g of fresh weight by spectrofluorometry and HPLC with a fluorescence detector, respectively. Using this sensitive, specific and simple fluorometric method, DETBA-MDA adducts ranging from 0.8 to 18.0pmol/g of fresh weight could easily be detected from vegetables, fruits, and potatoes.
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  • In-Eui Lee, Nguyen Van Chuyen, Fumitaka Hayase, Hiromichi Kato
    1994 Volume 58 Issue 1 Pages 18-23
    Published: January 23, 1994
    Released: February 08, 2008
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    The desmutagenic activities of low-molecular weight melanoidins (LM-MEL, MW 500-1000) and nondialyzable melanoidins (ND-MEL, MW ahove l000) prepared from a glucose-glycine reaction system were examined by Ames' test on such mutagens/carcinogens as nitro or amino compounds of aromatics or heterocycles, azo compounds, nitroso compounds, and epoxides. ND-MEL and LM-MEL exhibited a desmutagenic activity of 25-75% against mutagenic aromatic or heterocyclic compounds such as aflatoxin B1, B[α]P, 2-aminofluorene, 4-aminobiphenyl, and 2-aminonaphthalene, as well as heterocyclic amines, using S. typhimurium TA98 in the presence of S9 mix. In the case of using S. typhimurium TA100 in the absence of S9 mix, ND-MEL and LM-MEL showed a desmutagenic activity of 20-50% against 2-nitrofluorene, 4NQO and 2-nitronaphthalene. The strong desmutagenic activity of MEL from various kinds of amino acids, peptides, or egg albumin hydrolyzates with glucose was also observed against Trp-P-1. Furthermore, MEL had marked desmutagenic activity against the Trp-P-1 activated by S9 mix and synthetic N-OH-Trp-P-2.
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  • Shinji Tokuyama, Kazunori Hatano, Takeshi Takahashi
    1994 Volume 58 Issue 1 Pages 24-27
    Published: January 23, 1994
    Released: February 08, 2008
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    A novel enzyme, N-acylamino acid racemase (acylamino acid racemase) which catalyzes the interconversion of the enantiomers of N-acylamino acid, but does not act on amino acids, was found in an actinomycete strain Y-53 isolated from soil. A taxonomic study on the strain identified Y-53 as a strain of Streptomyces atratus. This strain also produced L-and D-aminoacylases simultaneously. Furthermore, another 13 strains of actinomycetes with the enzyme activity from the type culture collection of the Institute for Fermentation, Osaka (IFO) were observed.
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  • Jei-Fu Shaw, Wen-Chieh Chen
    1994 Volume 58 Issue 1 Pages 28-31
    Published: January 23, 1994
    Released: February 08, 2008
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    Three alkaline phosphatase isozymes, named APase-I, II, and III, were isolated from the cephalothoraxes of bighead shrimp (Solenocera melantho). The molecular weights of APase-I, II, and III estimated by SDS-PAGE were 88.6, 53, and 20kDa, respectively. They were all glycosylated monomeric enzymes. The optimum pH of APase-I and II were 10 and 8, and the pH ranges for maximum stability were 8.0-12.5 and 5.0-8.0, respectively. Both enzymes showed optimum temperature around 37°C. Arrehnius plots for enzyme thermal inactivation showed that APase II was more stable than APase I above 18°C but less stable below this temperature. Both enzymes showed the highest activities toward aryl phosphate esters among several phosphoesters tested. The Km for p-nitrophenyl phosphate were 0.14 and 4.44mM for APase I and II, respectively. Na+ stimulated both enzyme activities in physiological concentration range. APase I was less susceptible to denaturating agents and inhibitors than Apase II.
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  • Yusuke Matsuda, Tohru Okuda, Shonosuke Sagisaka
    1994 Volume 58 Issue 1 Pages 32-37
    Published: January 23, 1994
    Released: February 08, 2008
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    Using two sets of cultivars, two hardy and two less hardy ones, we studied differences in the regulation of the synthesis of cold-inducible proteins at an early stage of cold acclimation. During 11 days of cold treatment, all four cultivars had changes in rates of protein synthesis, which were divided into four phases. The difference in the regulation of synthesis of the protein was evident at the third and fourth phases. In the less hardy cultivars, rates of synthesis of cold-inducible proteins started to decline and the profiles of proteins being synthesized resembled those of control samples (without cold treatment). In hardy cultivars, rates of synthesis of typical cold-inducible proteins remained unchanged, suggesting that some regulatory mechanisms regulate the continued cold-inducible synthesis of proteins in the latter cultivars by acting, presumably, at particular target sites within the crown cells. Analysis of freezing tolerance showed that in stems and crowns of winter wheat, the cold-inducible proteins do not contribute directly to the freezing tolerance, while some may have roles in bringing about increased longevity in cold environments
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  • Satoshi Kitao, Hiroshi Sekine
    1994 Volume 58 Issue 1 Pages 38-42
    Published: January 23, 1994
    Released: February 08, 2008
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    Transglycosylation from sucrose to phenolic and related compounds by sucrose phosphorylase (EC 2.4.1.7) from Leuconostoc mesenteroides was studied. The enzyme had a rather broad acceptor specificity and transferred glucosyl residue of sucrose to various acceptors such as hydroxybenzenes, hydroxybenzoic acids, benzyl alcohol, and hydroxybenzyl alcohols. The enzyme could transfer the glucosyl moiety of sucrose to phenolic OH groups and alcoholic (hydroxymethyl) OH groups, though not to carboxyl groups. The phenolic OH group was more effective than a hydroxymethyl group as the acceptor. Phenolic OH groups adjacent to hydroxyl, hydroxymethyl, or carboxyl groups had an increased effectiveness as acceptors. About 2.3g of the purified transfer product was obtained from 2.0g of hydroquinone. Its structure was identified as hydroquinone-O-α-D-glucopyranoside (α-arbutin) on the bases of the secondary ion mass spectrometry analysis, the component analysis of its enzymatic hydrolysates, and the carbon-13 nuclear magnetic resonance analysis. The browning resistance of α-arbutin to light irradiation was extremely increased compared to that of hydroquinone. The inhibitory activity on tyrosinase of α-arbutin was almost equal to that of arbutin.
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  • Elizabeth Waters J, Patrice Pellerin, Jean-Marc Brillouet
    1994 Volume 58 Issue 1 Pages 43-48
    Published: January 23, 1994
    Released: February 08, 2008
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    An arabinogalactan-protein which reduced the heat-induced turbidity of protein in wine was purified from a red wine. The arabinogalactan-protein (AGP) was composed of a central 3-linked galactan core carrying, at position 6, 6-linked galactan chains which were in turn heavily substituted at positions 3 and 4 by terminal arabinofuranose residues and short 5-linked arabinan chains. The protein (13% by weight) was dominated by serine, alanine, hydroxyproline, and threonine, and also contained a relatively high amount of the basic amino acids lysine and arginine. The molecular weight, estimated by universal calibration, was 210, 000. Enzymatic removal of the terminal arabinofuranosyl units of the active AGP and subsequent partial shortening of the outer 6-linked galactan chains did not affect the haze protective activity. Periodate oxidation and then Smith degradation greatly reduced the size of the polysaccharide, its amount of protein, and eliminated its absorption at 280nm and its haze protective activity.
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  • Masatoshi Goto, Kei Tanigawa, Werasit Kanlayakrit, Shinsaku Hayashida
    1994 Volume 58 Issue 1 Pages 49-54
    Published: January 23, 1994
    Released: February 08, 2008
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    Glucoamylase I (GAI) bound to β-cyclodextrin (β-CD) with the binding constants of Kd = 17.7μM and n = 1.69. Binding resulted in formation of an inclusion complex, as indicated by the fact that organic guest compounds for β-CD inhibited the binding of GAI to β-CD. Titration of GAI (A1-R615) with β-CD or Amylose A showed critical spectral perturbations at 286 nm that reflected the aromatic side chains of Tyr and Trp, but no spectral changes were observed in GAI' (A1-V469) and Gp-I (A470-V514). Titration of GAI with derivatives of β-CD, namely, 6-hydroxypropyl-β-CD (H-CD), 2, 6-O-dimethyl-β-CD, and 2, 3, 6-O-trimethyl-β-CD, yielded spectral changes only in the case of H-CD. Therefore, the Cp region (A515-R615) of GAI seems stereospecifically able to recognize the structure of the secondary OH-side but not the primary OH-side of β-CD. Chemical modifications specific for aromatic amino acids of the GAI-CD inclusion complexes were done with N-bromosuccinimide and tetranitromethane. In the presence of β-CD, one Trp residue was protected from oxidation but Tyr residues were not. The analysis of a Cp region/β-galactosidase fusion protein indicated that Trp562 contributed to formation of inclusion complexes.
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  • Hitoshi Ashida, Kazuki Kanazawa, Gen-Ichi Danno
    1994 Volume 58 Issue 1 Pages 55-59
    Published: January 23, 1994
    Released: February 08, 2008
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    A decrease in hepatic phosphoglucomutase activity was found after intraperitoneal doses to rats with pro-oxidative drugs ; carbon tetrachloride, ethanol, paraquat, phenobarbital, thiopental, 3-methylcholan-threne, benzo[a]pyrene, Trp-P-1, benzene, and polychlorinated biphenyl. When the drugs were given at half of their LD50 values, except ethanol, hepatic phosphoglucomutase activities were decreased, and aldehydes which were detected by the thiobarbituric acid test were simultaneously accumulated 24 h after administration, in the liver. A significant correlation between phosphoglucomutase activity and the accumulations of aldehydes was obtained (r=-0.536). No other changes in hepatic enzymes and metabolites were commonly observed. On the other hand, the low-molecular-weight aldehyde fraction in secondary products of linoleic acid decreased phosphoglucomutase activity in hepatocytes cultured with dexamethasone. It was concluded that aldehydes originating from endogenous lipid peroxidation decreased phosphoglucomutase activity in the liver, and the assay of hepatic phosphoglucomutase activity was useful as a marker of oxidative stress induced by pro-oxidative drugs.
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  • Koji Hara, Koki Fujita, Hirofumi Nakano, Nobuhiro Kuwahara, Toshiko Ta ...
    1994 Volume 58 Issue 1 Pages 60-63
    Published: January 23, 1994
    Released: February 08, 2008
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    Jack bean α-mannosidase had a wide acceptor specificity and could transfer mannosyl residues to various acceptors such as D-fructose, L-arabinose, maltose, lactose, and sucrose. The structures of the transferred products of branchd cyclodextrins (CDs) (glucosyl-βCD, maltosyl-αCD, and maltosyl-βCD) were found to be α-D-mannosyl-(1→6)-α-D-glucosyl-(1→6)-βCD, α-D-mannosyl-(1→6)-α-D-glucosyl-(1→4)-α-D-glucosyl-(1→6)-αCD and α-D-mannosyl-(1→6)-α-D-glucosyl-(1→4)-α-D-glucosyl-(1→6)-βCD, respectively. Almond α-mannosidase also produced the same transmannosylated products of branched CDs.
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  • Michiko Watanabe, Jun Watanabe
    1994 Volume 58 Issue 1 Pages 64-66
    Published: January 23, 1994
    Released: February 08, 2008
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    Some foods from plant sources, particularly of Moringeceae origin, contained compounds that enhanced bacterial ice-nucleation activity. A hot water extract from mustard seeds was so potent in its enhancing activity that the compounds responsible could be isolated from the seeds as yellow crystals. By instrumental analyses, this enhancer was identified as 4-hydroxy-3-nitrophenylacetic acid. The addition of a purified preparation of this compound at 0.1-1ppm to the cultivation medium for Xanthomonas campestris was effective for enhancing its ice-nucleation activity.
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  • Mutsumi Matsue, Shinichi Tomita, Shoko Nyui, Jun Matuyama, Isao Kiyosa ...
    1994 Volume 58 Issue 1 Pages 67-71
    Published: January 23, 1994
    Released: February 08, 2008
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    The effects of the iron ion and reductones on bleomycin (BLM)-induced DNA degradation were observed by measuring the amount of malondialdehyde (MDA) formed. The formation of MDA was detected after adding Fe2+ or Fe3+ + Asc combined to a solution consisting of DNA and BLM, but not after adding Fe3+ alone, catecholamines or phenolic compounds. Marked suppressive effects of lactoferrin (Lf) and ovotransferrin on MDA formation were observed in the solution with Fe2+ or Asc. However, serum albumin, β-lactoglobulin, α-lactalbumin, and ovoalbumin had no effect on suppressing MDA formation. The suppression by bovine Lf (bLf) of MDA formation decreased with increasing iron saturation at 1.0mM Fe2+, but at 100% iron saturation, 51.2% of apo-bLf remained suppression. In the solution with 0.25mM Asc, 100% iron saturated bLf markedly enhanced MDA formation.
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  • Kenji Yamamoto, Setsu Kadowaki, Masatoki Fujisaki, Hidehiko Kumagai, T ...
    1994 Volume 58 Issue 1 Pages 72-77
    Published: January 23, 1994
    Released: February 08, 2008
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    Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M) was proved to act on complex type biantennary oligosaccharides of glycoproteins by using dansylated asparagine-linked and pyridylaminated oligosaccharides, as the substrate. The enzyme could act on both asialo- and sialo-biantennary oligosaccharides. This is the only endo-β-N-acetylglucosaminidase known to act on sialo glycans, though their activity for them was weak. The enzyme could liberate complex type biantennary oligosaccharides from native human asialotransferrin, which was ascertained by a combination of the pyridylaminated method and HPLC. The enzyme had substrate specificity for high-mannose type oligosaccharides different from those of the endo-β-N-acetylglucosaminidases of other microorganisms : ovalbumin glycopeptide-IV was a better substrate for Endo-M than glycopeptide-V. The enzyme could act on complex type triantennary oligosaccharides of dansylated glycopeptide prepared from calf fetuin. The enzyme had various novel specificities in regard to activities on complex type and high-mannose type oligosaccharides in glycoproteins.
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  • Satoshi Nakamura, Ryuichiro Nakai, Kenji Wakabayashi, Yutaka Ishiguro, ...
    1994 Volume 58 Issue 1 Pages 78-81
    Published: January 23, 1994
    Released: February 08, 2008
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    Alkaliphilic and thermophilic Bacillus sp. strain TAR-1, isolated from soil, produced a xylanase extracellularly. The xylanase was most active over a pH range of 5.0 to 9.5 at 50°C. Optimum temperatures of the crude xylanase preparation were 75°C at pH 7.0 and 70°C at pH 9.0. Zymogram analyses of the culture supernatant showed that the molecular mass of the xylanase was 40kDa and the isoelectric point was pH 4.1. The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase. Production of the thermophilic alkaline xylanase was induced by xylan and xylose, but was repressed in the presence of glucose.
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  • Kinya Washizu, Keiichi Ando, Satoshi Koikeda, Susumu Hirose, Akira Mat ...
    1994 Volume 58 Issue 1 Pages 82-87
    Published: January 23, 1994
    Released: February 08, 2008
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    The microbial transglutaminase (TGase)-producing strain S-8112 [Agric. Biol. Chem., 53, 2613-2617(1989)] was identified as a variant of Streptoverticillium mobaraense. We amplified a partial gene fragment by polymerase chain reaction (PCR) using oligonucleotides synthesized from the amino acid sequence of TGase, and cloned the gene for TGase using the PCR amplified fragment as a probe. The gene encoded a precursor of TGase consisting of 406 amino acid residues, which comprised the prepro region of 75 amino acid residues and the mature region of 331 amino acid residues. We expressed the TGase gene in Streptomyces lividans under a tyrosinase promoter, and found an active and mature recombinant enzyme, indicating the processing of the gene product.
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  • Shino Takehana, Kinya Washizu, Keiichi Ando, Satoshi Koikeda, Kazuyuki ...
    1994 Volume 58 Issue 1 Pages 88-92
    Published: January 23, 1994
    Released: February 08, 2008
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    The gene coding for microbial transglutaminase (TGase) from Streptoverticillium, which consists of 331 amino acids, was chemically synthesized. The codons have been substituted for those mainly favored in yeast. Our strategy involved the construction of the TGase gene in five sections (54 oligomers) that contained unique restriction enzyme sites at both ends, which could readily be ligated to form the full-length product. The chemically synthesized gene was inserted downstream from the ompA signal peptide of the E. coli expression vector, pIN-III-ompA, which carries lpp and lac promotors. The resultant plasmid directed the expression of TGase, with the activity being secreted mainly into the periplasmic space of E. coli. The induced gene product was identical with native TGase in size and in immunological properties, though the enzyme activity was low.
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  • Tatsuya Moriyama, Kazuteru Wada, Manabu Oki, Takao Matsuura, Makoto Ki ...
    1994 Volume 58 Issue 1 Pages 93-98
    Published: January 23, 1994
    Released: February 08, 2008
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    The mechanism of arachidonic acid (AA) release in collagen-activated human platelets was studied. An arachidonic acid metabolite, thromboxane B2 (TXB2), was formed in parallel with the formation of phosphatidic acid (PA) without formation of lysophosphatidic acid (lysoPA) or lysophosphatidylinositol (lysoPI) in the absence of extracellular Ca2+, suggesting that AA was released from PI via a PI-specific phospholipase C (PI-PLC)/diacylglycerol (DG) lipase/monoacylglycerol (MG) lipase pathway under the cytosolic low Ca2+ concentrations. Moreover, solubilized DG lipase and MG lipase could hydrolyze the substrates at basal cytosolic free Ca2+ concentrations. Subsequently, the relationship of cytosolic free Ca2+ concentrations and formation of AA metabolites was analyzed using Ca2+ ionophre, A23187. Collagen was able to induce a release of small amounts of AA under basal cytosolic Ca2+ conditions. However, a release of large amounts of AA was induced by phospholipase A2 activated by both collagen-receptor occupancy and elevated Ca2+ levels. A TXA2 mimetic agonist, STA2 induced all the responses except for AA release. From these results, the mechanism of AA release and signal transduction in collagen-activated human platelets is discussed.
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  • Yuzo Yamada, Kojiro Maeda, Kozaburo Mikata
    1994 Volume 58 Issue 1 Pages 99-103
    Published: January 23, 1994
    Released: February 08, 2008
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    The partial base sequences of 18S and 26S rRNAs of Rhodosporidium fluviale, R. lusitaniae, and Erythrobasidium hasegawianum were analyzed. In the 26S rRNA partial base sequencings, R. fluviale CBS 6568 and R. lusitaniae IGC 4599 and IGC 4641 had 81-82 and 77 percent similarities compared with R. toruloides (type species of genus Rhodosporidium) IFO 0559 and IFO 0880. Erythrobasidium hasegawianum IFO 1058 showed 69-71, 59, 63, and 61 percent similarities with R. toruloides IFO 0559 and IFO 0880, L. scottii (type species of genus Leucosporidium) IFO 1923, R. dacryoidum IFO 1930 and IFO 1931, and Kondoa malvinella IFO 1936, respectively. In the 18S rRNA partial base sequencings, R. fluviale CBS 6568 and R. lusitaniae IGC 4599 and IGC 4641 had zero and two base differences with R. toruloides. Erythrobasidium hasegawianum IFO 1058 showed ten, sixteen, three, and twenty base differences with R. toruloides IFO 0559 and IFO 0880, L. scottii IFO 1923, R. dacryoidum IFO 1930 and IFO 1931, and K. malvinella IFO 1936, respectively. Based on the sequence data obtained, a new genus, Sakaguchia was proposed for R. dacryoidum with a new combination, Sakaguchia dacryoides.
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  • Junji Magae, Toshimi Nagi, Kazuaki Takaku, Takao Kataoka, Hiroyuki Kos ...
    1994 Volume 58 Issue 1 Pages 104-107
    Published: January 23, 1994
    Released: February 08, 2008
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    Phagocytosis is one of the basic and characteristic properties of macrophages. We screened metabolites of Actinomyces for low molecular weight substances that selectively inhibited phagocytosis of dried yeast but not pinocytosis of neutral red by thioglycollate-elicited peritoneal macrophages. Inhibitors of actin filament organization, protein kinases, respiration, and lipid synthesis selectively inhibited phagocytosis, and blockers of proton gradients selectively inhibited pinocytosis. This suggests that these functions are differently regulated. We applied this system to screening of metabolites of Actinomyces, and identified mycotrienin, piericidin, and genistein as selective inhibitors of phagocytosis.
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  • Yoichi Ueda, Takako Tsubuku, Ryuichi Miyajima
    1994 Volume 58 Issue 1 Pages 108-110
    Published: January 23, 1994
    Released: February 08, 2008
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    Sulfur-containing components in an ethanol extract and boiled water extract of onion (Allium cepa L. )were analyzed by HPLC. Trans-(+)-S-propenyl-L-cysteine sulfoxide (PeCSO) and its γ-glutamyl peptide (γ-Glu-PeCSO) were the major constituents in the ethanol extract, whereas cycloalliin was the most abundant one in the boiled water extract. The large amount of cycloalliin found in the boiled water extract was mostly derived from PeCSO by heating. PeCSO and γ-Glu-PeCSO showed a characteristic kokumi flavor (continuity, thickness, and mouthfulness) by a sensory test in an umami solution containing 0.05% (w/v) each of monosodium glutamate and disodium inosinate.
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  • Youn-Sung Kim, Hwanghee-Blaise Lee, Kang-Duk Choi, Seonyang Park, Ook ...
    1994 Volume 58 Issue 1 Pages 111-116
    Published: January 23, 1994
    Released: February 08, 2008
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    A gene (estC) coding for an esterase (esterase III) of Pseudomonas fluorescens was cloned into Escherichia coli JM83. DNA sequencing showed a single open reading frame with GTG as a translation initiation codon for esterase III. This was confirmed by N-terminal amino acid sequence analysis of the purified esterase III protein from an E. coli clone. The promoter sequence and a potential Shine-Dalgarno sequence were followed by the coding sequence of the estC gene. The amino acid sequence deduced from the nucleotide sequence contains the consensus active site sequence, G-X-S-X-G, of serine esterase. The esterase III expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. The native form of the enzyme was a monomer with a molecular weight of 41, 000. The results of substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC 3.1.1.1) and a serine residue is present at the active site of the enzyme.
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  • Ali-Azam Talukder, Satoshi Yanai, Mamoru Yamada
    1994 Volume 58 Issue 1 Pages 117-120
    Published: January 23, 1994
    Released: February 08, 2008
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    A partial library of the Escherichia coli genomic genes has been constructed, in which each clone has part of a genomic gene fused in frame with the lacZ gene in addition to its promoter and operator. DNA of randomly selected clones was sequenced, and the resultant deduced N-terminal amino acid sequences showed that 17 out of 26 genes analyzed encode unknown proteins. Genomic locations of the cloned genes and their expressional regulations under the aerobic and anaerobic conditions were also analyzed. These results suggest that this library is useful for the global analysis of the E. coli genomic genes and that this strategy may be applicable to the genomic analysis of other microorganisms.
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  • Kenji Yoshikawa, Kazuya Yamamoto, Shigetaka Okada
    1994 Volume 58 Issue 1 Pages 121-125
    Published: January 23, 1994
    Released: February 08, 2008
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    The transfer action of α-D-xylosidase I and II from Aspergillus flavus MO-5 on p-nitrophenyl-α-D-xylopyranoside (α-p-NPX) was investigated, as compared with that of commercial α-D-xylosidase (from Bacillus sp.). In reaction mixtures with various concentrations of α-p-NPX, both enzymes (I and II) liberated p-nitrophenol and xylose at low concentrations, and showed transfer products in addition to hydrolyzates at high concentrations. IP-1 and IP-3, IIP-1 and IIP-2, and CP-1 and CP-3 were main transfer products by α-D-xylosidase I, α-D-xylosidase II, and commercial α-D-xylosidase, respectively. They were isolated by active charcoal column chromatography and silica gel column chromatography. Their structures were analyzed by enzymatic digestion, methylation analysis, and 13C-NMR analysis. As the results, IP-1, IIP-2, and CP-1 were found to be the same structure, p-nitrophenyl-4-O-(α-D-xylopyranosyl)-α-D-xylopyranoside. Then, it was proved that the structure of IIP-1 was p-nitrophenyl-3-O-(α-D-xylopyranosyl)-α-xylopyranoside. On the other hand, IP-3 and CP-3 were found to be the same structure, D-xylopyranosyl-α-1, 4-D-xylopyranose without a p-nitrophenyl group. We found that α-D-xylosidase I showed α-1, 4 xylosyl transfer action as well as commercial α-D-xylosidase, and α-D-xylosidase II showed α-1, 3 xylosyl transfer action in addition to α-1, 4 xylosyl transfer action on α-p-NPX.
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  • Bunzo Mikami, Keiichi Nomura, Yuhei Morita
    1994 Volume 58 Issue 1 Pages 126-132
    Published: January 23, 1994
    Released: February 08, 2008
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    The less reactive SH groups of soybean β-amylase, SH4, SH5, and SH6, were modified with p-chloromercuribenzoic acid or N-ethylmaleimide, after the reactive SH groups, SH1, SH2, and SH3, were blocked with 5, 5'-dithiobis-(2-nitrobenzoic acid) and cyanide. The enzyme activity decreased, accompanied by the modification of SH4. α-Cyclodextrin protected SH4 from the modification more effectively than maltose. The SH4-modified enzyme still bound to glucose, maltose, and α-cyclodextrin. SH4 was concerned with neither the catalysis nor substrate binding but its large substituent affected the substrate binding site. The sequencing of the 5-(iodoacetoamidoethyl)-aminonaphthalene-1-sulfonate-labeled peptides showed that SH4, SH5, and SH6 are Cys343, Cys82, and Cys208, respectively. Comparison of the primary structure of β-amylases also showed that the sequence around SH4 (Cys343), as well as SH2 (Cys95), is strongly conserved between higher plant and bacterial β-amylases. These results agree with the structure model deduced from X-ray crystallography of soybean β-amylase.
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  • Akio Kobayashi, Yutaka Koguchi, Hiroshi Kanzaki, Shin-ichiro Kajiyama, ...
    1994 Volume 58 Issue 1 Pages 133-134
    Published: January 23, 1994
    Released: February 08, 2008
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    Peroxidase (POD) catalyzed the polymerization of naturally occurring phenolics to give a variety of bioactive products. The guaiacol readily reacted in the POD system and afforded potential anti-microbial compounds. Three active phenols, 1, 2, and 3, were isolated from the reaction mixture. Spectroscopic analyses elucidated that 1 and 2 were a dimer and a trimer respectively originating from guaiacol. Compound 3 was readily oxidized to 4 by exposure to air. The structure of 4 was determined by a combination of chemical modification and spectroscopic analyses, and 3 was elucidated to be the hydroxyquinone form of 4.
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  • Yotaro Konishi, Aki Okamoto, Jun Takahashi, Michio Aitani, Nobuji Naka ...
    1994 Volume 58 Issue 1 Pages 135-139
    Published: January 23, 1994
    Released: February 08, 2008
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    To examine the significance of α-glucosidase in starch metabolism in vivo, wheat seedlings and seeds were treated with Bay m 1099 (Miglitol, N-hydroxyethyl-1-deoxynojirimycin), an α-glucosidase inhibitor. Bay m 1099 did not affect germination, but it inhibited growth of seedlings at the high dosage (100μg/ml medium). Treatment of normally grown seedlings with Bay m 1099 (10 and 100 μg/ml) decreased α-glucosidase activity with a dose response and caused accumulation of maltose in tissues with decreases in glucose. The decrease in glucose formation would inhibit plant growth, which was observed particularly at higher dosages of the inhibitor. When wheat seeds were treated with Bay m 1099 at 10μg/ml for 4 days, under which the growth of the plants after germination was minimized, the induction of α-glucosidase, but not amylase, in kernels was considerably suppressed during germination and maltose metabolism to glucose was disturbed. In addition, the Bay m 1099 treatment decreased the initial rate of starch degradation by 48%, compared with the control. These results suggest that wheat α-glucosidase participates in maltose hydrolysis as well as in the onset of starch degradation with collaboration of amylase.
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  • Kenji Matsui, Tadahiko Kajiwara, Akikazu Hatanaka, Doris Waldmann, Pet ...
    1994 Volume 58 Issue 1 Pages 140-145
    Published: January 23, 1994
    Released: February 08, 2008
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    Cooxidation activity of highly purified soybean lipoxygenase-2 and -3 was investigated with all-trans-retinoic acid as a co-substrate. Both the isoenzymes rapidly degraded retinoic acid in the presence of linoleic acid, but lipoxygenase-2 had more cooxidation activity than lipoxygenase-3. During cooxidation, 5, 6-epoxyretinoic acid was formed as a major product. HPLC on a chiral phase (Ceramospher Chiral RU-1) found a racemic mixture of the epoxide, indicating its formation by oxidative attack by peroxyl radicals.
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  • Puming He, Sakiyo Yamaoka-Koseki, Kyoden Yasumoto
    1994 Volume 58 Issue 1 Pages 146-150
    Published: January 23, 1994
    Released: February 08, 2008
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    The effect was assessed of paraquat administration on the antioxidative defense mechanism in blood and liver. Ten-week-old senescence-accelerated mice were fed with either a 20% casein diet or the same diet supplemented with 200ppm paraquat for 12 weeks. About 0.2ml of blood withdrawn periodically from the supraorbital vein was assayed for the activities of antioxidative enzymes and oxidative damage. Blood and liver collected after decapitating the mice were equivalently treated at the end of the experimental period. Paraquat ingestion depressed the antioxidative enzyme activities, and increased the level of oxidized protein in erythrocytes and the thiobarbituric acid-reactive substance (TBARS) value in plasma. Similar changes were observed for the plasma and liver in the antioxidative enzyme activites, and in the level of oxidized protein and the TBARS value. These changes produced by paraquat administration were closely associated with accelerated aging leading to cell death. We discuss the effect of paraquat on senescence in mice, in relation to the oxidative damage caused.
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  • Takeshi Matsumoto, Yasuo Iwasawa, Hideaki Shima, Hiroki Shirai, Tetsuy ...
    1994 Volume 58 Issue 1 Pages 151-155
    Published: January 23, 1994
    Released: February 08, 2008
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    Plasma transferrin (Tf), transthyretin (TTR), and retinol-binding protein (RBP) have been proposed to be sensitive nutritional parameters. We investigated the effect of laparotomy and/or starvation on these plasma protein levels in rats. Starvation decreased the Tf, TTR, and RBP levels to 62.2%, 41.2%, and 52.0% of the initial values, respectively, until day 3, but then sustained the day-3 levels on day 4. In the starved and laparotomized rats, the TTR level on day 1 and the RBP level on day 2 were more decreased than those in the only-laparotomized rats. Recovery of the TTR and RBP levels in the laparotomized rats fed on a stock diet ad libitum was very slow. In conclusion, the plasma Tf, TTR, and RBP levels showed different responses according to the metabolic phase of starvation. Laparotomy affected differently each of these levels, but the decrease in these protein levels was mainly dependent on the nutritional condition of the laparotomized rats.
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  • Kazuhiro Iwasaki, Hiroo Uchiyama, Osami Yagi
    1994 Volume 58 Issue 1 Pages 156-159
    Published: January 23, 1994
    Released: February 08, 2008
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    The survival of genetically engineered and wild-type Pseudomonas putida PpY101, that contained a recombinant plasmid pSR134 conferring mercury resistance, were monitored in andosol and sand microcosms. The survival of genetically engineered and wild-type P. putida was not significantly different in andosol. The population change of the two strains was dissimilar in andosol and sand. The survival of genetically engineered and wild-type P. putida strains was affected by the water content of andosol, and increased with the increment of the water content. The impact of the addition of genetically engineered and wild-type P. putida strains on indigenous bacteria and fungi was examined. Inoculation of both strains had no apparent effect on the density of indigenous microorganisms.
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  • Haruhiko Kawasaki, Tohru Toyama, Takuya Maeda, Humiko Nishino, Kenzo T ...
    1994 Volume 58 Issue 1 Pages 160-163
    Published: January 23, 1994
    Released: February 08, 2008
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    The 2-haloacid dehalogenase of Pseudomonas putida No. 109 was mediated by a 74-kb conjugative plasmid, which was transferred by mating into Pseudomonas and Escherichia coli and there expressed the dehalogenase. A 2.8-kb EcoRI-fragment generated from the plasmid was cloned and sequenced. The dehalogenase gene (dehH109) was identified by comparison with the N-terminal amino acid sequence and the molecular weight of the enzyme protein. The gene dehH109 coded for a 224-amino acid protein of Mr 25, 231, which showed significant homology to the other four L-specific 2-haloacid dehalogenases from Pseudomonas sp. CBS3, P. putida AJ1, and Xanthobacter autotrophicus GJ10 and also to the haloacetate dehalogenase H-2 from Moraxella sp. strain B, but no homology with another haloacetate dehalogenase H-1 and the D-specific 2-haloacid dehalogenase from P. putida AJ1.
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  • Yoshio Koide, Toyohiko Noso
    1994 Volume 58 Issue 1 Pages 164-169
    Published: January 23, 1994
    Released: February 08, 2008
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    Cystatin, a cysteine proteinase inhibitor, was isolated from chum salmon (Oncorhynchus keta) pituitary glands by ion-exchange chromatography on Mono-Q, gel filtration on Superdex 75, and reverse-phase HPLC on an ODS following ethanol-ammonium acetate extraction. Salmon pituitary cystatin was equipotent to chicken egg-white cystatin in the papain inhibitory assay. The cystatin consists of 111 amino acid residues with two disulfide linkages formed between 66-75 and 89-109, and has 43% identical sequences with chicken egg-white cystatin with consensus sequences of reactive sites, Gly(4), Gln-X-Val-X-Gly (48-52), and Ile(Val)-Pro-Trp (96-98).
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  • Kimiyasu Isobe, Hiroshi Nishise
    1994 Volume 58 Issue 1 Pages 170-173
    Published: January 23, 1994
    Released: February 08, 2008
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    A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent Kms were 2.96 M and 28.6 mM, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent Km for glycolaldehyde was found to be 83.3mM. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 M ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.
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  • Makoto Hattori, Shigeo Imamura, Koichi Nagasawa, Koji Takahashi
    1994 Volume 58 Issue 1 Pages 174-177
    Published: January 23, 1994
    Released: February 08, 2008
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    Hen egg lysozyme-carboxymethyl dextran (HEL-CMD) conjugate was prepared by using water-soluble carbodiimide as a model protein-acidic polysaccharide conjugate for improving the protein function. An acid-amide bond between HEL and CMD was confirmed by SDS-PAGE, isoelectric focusing and IR spectra. The molar ratio of CMD to HEL in the conjugate was 1:1. The isoelectric point of the conjugate was 5.5-6.0, which is much lower than that of HEL. Spectroscopic studies suggested that the conformation around the Trp residue had not changed but the α-helix content had decreased to about 1/3 that for native HEL. The conjugate maintained about 60% of the enzymatic activity of native HEL at 40-60°C, while it was about 1.4 times as active as native HEL at 4°C and 80°C. The conjugate was more stable to proteolysis than native HEL. The denaturation temperature of the conjugate was about 73°C, which is almost the same as that of native HEL. However, the enthalpy for denaturation of the conjugate was about 1/3 that of native HEL, which corresponds to the decrease in α-helix content.
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  • Koji Takahashi, Makoto Hattori
    1994 Volume 58 Issue 1 Pages 178-182
    Published: January 23, 1994
    Released: February 08, 2008
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    Phosphated polysaccharides were used to regulate the rate of matrix reconstruction from a neutral-salt soluble collagen (NSC) solution and the stability of the matrix in comparison with various chemically modified polysaccharides. Non-charged polysaccharides such as soluble starch, dextran, acetyl starch, hydroxypropyl starch and starch propylsulfonate had no substantial effect on matrix reconstruction. The positively charged polysaccharides, 3-trimethylamino-2-hydroxypropyl starch, slightly accelerated the molecular rearrangement, but was little incorporated in the matrix and had no effect on the thermal denaturation behavior of the matrix. Sulfated polysaccharides, polyanions, caused reconstruction without a lag phase because of too-rapid aggregation, and resulted in the reconstruction of the matrix with a less stable and heterogenous macrostructure. However, starch phosphate and dextran phosphate markedly accelerated the reconstruction of the matrix with uniform intermolecular cohesion similar to that of the control. This regulatory function of phosphated polysaccharides was enhanced with decreasing the pH from 9 to 5.
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  • Kyohei Mizokami, Hiroaki Katsura, Yoshinori Okita, Shinya Sekitou, Hid ...
    1994 Volume 58 Issue 1 Pages 183-184
    Published: January 23, 1994
    Released: February 08, 2008
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  • Cun Zhuang, Takashi Mizuno, Hitoshi Ito, Keishiro Shimura, Toshimitsu ...
    1994 Volume 58 Issue 1 Pages 185-188
    Published: January 23, 1994
    Released: February 08, 2008
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    We developed a method for the fractionation and purification of antitumor polysaccharides, considered to be a type of immunopotentiator or BRM (biological response modifier), from the mycelium of liquid cultured Grifola frondosa. The active polysaccharide fractions that showed higher antitumor activity were considered to be heteroglycans or their protein complexes as follows, in water-soluble fractions : FI0-a-α : fucogalactomannan-protein complex ; FI0-a-β : mannogalactofucan ; FA-1 : galactoglucomannofucan-protein complex ; FA-2-b-α : glucogalactomannan-protein complex ; in water-insoluble fractions : FIII-1-a : mannofucoglucoxylan ; FIII-1-b : mannoglucofucoxylan-protein complex ; FIII-2-a : mannofucoglucoxylan-protein complex ; FIII-2-b : glucomannofucoxylan-protein complex.
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  • Masao Arimoto, Sachiko Matsuura, Chikaaki Muro, Hiroshi Tsujibo, Eiko ...
    1994 Volume 58 Issue 1 Pages 189-190
    Published: January 23, 1994
    Released: February 08, 2008
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    All the lignans tested in a bioassay with Brassica campestris L. subsp. rapa Hook. fil. et ANDERS inhibited the root growth of this plant, except for deoxypicropodophyllin. The effects of functional groups in the molecule on the inhibitory activity of these compounds were studied. It is suggested that the methylenedioxyl group and the stereochemical configuration of the lactone junction of podophyllotoxin derivatives were closely related to the inhibitory activity. The O-methyl derivative of two hydroxyl groups of matairesinol greatly enhanced the inhibition of root growth in this plant.
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  • Kimikazu Hashino, Ikunoshin Kato, Yoshikazu Kurosawa, Kiyotoshi Sekigu ...
    1994 Volume 58 Issue 1 Pages 191-192
    Published: January 23, 1994
    Released: February 08, 2008
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    A new artificial cell adhesive protein was engineered by grafting the Arg-Gly-Asp (RGD) sequence, the minimal recognition signal of fibronectin for interaction with integrin, to immunoglobulin G (IgG) by in vitro mutagenesis. The mutagenized protein showed cell adhesive activity on baby hamster kidney (BHK) cells.
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  • Naoko Yamano, Shizu Fujishima, Rika Miwatani, Fumiko Yaku, Ryutaro Tan ...
    1994 Volume 58 Issue 1 Pages 193-195
    Published: January 23, 1994
    Released: February 08, 2008
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    Vibrio cholerae non-O1 (1148A) produced β-N-acetylglucosaminidase and N-acetylglucosamine (GlcNAc) deacetylase intracellularly when grown in chitin or GlcNAc containing medium. It also secreted chitinase only in the chitin-containing medium. The partially purified GlcNAc deacetylase deacetylated GlcNAc but not chitin oligosaccharides, the dimer to hexamer of GlcNAc. We also detected the reaction product by capillary electrophoresis.
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  • Yoichi Noda, Koji Yoda, Makari Yamasaki
    1994 Volume 58 Issue 1 Pages 196-197
    Published: January 23, 1994
    Released: February 08, 2008
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    TmrB protein, which endows Bacillus subtilis with tunicamycin resistance, was found to bind more tightly to the membrane in B. subtilis than in E. coli. Although only the C-terminal amphiphilic α-helix was responsible for membrane-TmrB interaction in E. coli, there should be other binding factor(s) in the original strain, B. subtilis.
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  • Hiroko Chiue, Kimikazu Iwami, Takanori Kusano, Fumio Ibuki
    1994 Volume 58 Issue 1 Pages 198-199
    Published: January 23, 1994
    Released: February 08, 2008
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    Ten samples with different deamidation rates were prepared from commercially available maize zein by heat treating with 0.05 N HCl in 70% ethanol, and were examined for their antioxidative activity in simply mixed protein-fatty acid (10:1) model systems at moderate humidity. A regression line with a correlation coefficient of r = -0.946 was obtained, when the linoleate/palmitate ratio on day 3 was plotted against the percentage of deamidation.
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  • Takashi Fukada, Kenji Iida, Noriyuki Kioka, Hiroshi Sasaki, Tohru Koma ...
    1994 Volume 58 Issue 1 Pages 200-201
    Published: January 23, 1994
    Released: February 08, 2008
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    We isolated a cDNA of N-acetylglucosaminyltransferase I (GnT-I) from rat liver and analyzed GnT-I mRNA expression in several rat tissues. We found that GnT-I mRNA was expressed in a tissue-specific manner and the pattern of its expression was suggested to be species-specific by comparison with the reported result in mice.
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  • Yoshihisa Tomoda, Kenji Umemura, Takashi Adachi
    1994 Volume 58 Issue 1 Pages 202-203
    Published: January 23, 1994
    Released: February 08, 2008
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    Alginate lyase-lysate (A.L.L., Algin-Oligo(R)), which had been prepared by degrading sodium alginate by alginate lyase, was found to have a growth-promoting effect on the elongation of barley roots, and especially that of the radicle, the effective concentration of A.L.L. for elongation of the roots being 100-3000μg/ml, with no inhibition at the highest concentration. When a radicle was brought into contact with A.L.L., it responded by initiating elongation within 2 to 4h. The elongation rate increased from 2.9mm/h to 5.3mm/h. Treatment with A.L.L. resulted in about a 2-fold increase in the alcohol dehydrogenase activity of the control under hypoxic conditions.
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  • Yunn-Bor Yang, Hsiang-Ling Hu, Ming-Chung Chang, Hung Li, Ying-Chieh T ...
    1994 Volume 58 Issue 1 Pages 204-205
    Published: January 23, 1994
    Released: February 08, 2008
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    The L-aminoacylase produced intracellularly by Alcaligenes denitrificans DA181 was purified to homogeneity. This enzyme had an apparent molecular weight of 80, 000, and was composed of two subunits of identical molecular weight. Its isoelectric point was pH 5.1. The optimal reaction temperature and pH were 65°C and 8.0, respectively. This enzyme showed specificity toward N-acetyl-derivative of hydrophobic L-amino acids with N-acetyl-L-valine as the favored substrate, followed by N-acetyl-L-alanine.
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  • Yoko Murakami, Young-Sook Hahn, Kumio Yokoigawa, Kinji Endo, Hiroyasu ...
    1994 Volume 58 Issue 1 Pages 206-207
    Published: January 23, 1994
    Released: February 08, 2008
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    Freeze-sensitive strains of yeast were induced from a freeze-tolerant yeast Torulaspora delbrueckii by incubation with ethyl-methane sulfonate as a mutagen. A maximum ratio of mutation was attained by the incubation at 30°C for 75min. One-hundred and fifty strains of freeze-sensitive yeast were selected by plating-culture for the first screening. The freeze-tolerance ratio of each strain was examined based on the fermentative activity before and after freezing in liquid medium and dough. Strain 60B3 showed the highest freeze-sensitivity in a pre-fermented frozen dough (pre-fermented at 30°C for 2h, and frozen at -20°C for 7days)among eight strains finally selected.
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  • Yoshiaki Nishiya
    1994 Volume 58 Issue 1 Pages 208-210
    Published: January 23, 1994
    Released: February 08, 2008
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    The ADE1 gene from Candida utilis CA(u)-37, a strain used for commercially producing enzymes, was cloned by complementation of the adel mutation of Saccharomyces cerevisiae. It was composed of 903bp, and the deduced amino acid sequence was 70% homologous to those of the ADE1 genes of S. cerevisiae and Candida maltosa. The highly preserved region of SAICAR synthetase, the ADE1 gene product, was also found by a homology search.
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