Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 64 , Issue 7
Showing 1-36 articles out of 36 articles from the selected issue
Reviews
  • Katsuichi SAITO, Fusao TOMITA
    2000 Volume 64 Issue 7 Pages 1321-1327
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      Difructose anhydrides (DFAs) are the smallest cyclic disaccharides consisting of two fructose residues, and are expected to have novel physiological functions from their unique structures and properties.
      For mass-production of α-D-fructofuranose-β-D- fructofuranose-2′,1:2,3′-dianhydride (DFA III) and β-D-fructofuranose-β-D-fructofuranose-2′,6:2,6′-dianhydride (DFA IV), Arthrobacter sp. H65-7 and A. nicotinovorans GS-9 were selected as the best producers of inulase II, which produced DFA III from inulin and LFTase, which produced DFA IV from levan. The enzymes were purified and their genes were subsequently cloned and expressed in E. coli at higher levels than in the original bacteria. Thus, it became possible to provide a large amount of DFA III and DFA IV for evaluating their physiological properties.
      DFA III and DFA IV have half the sweetness of sucrose, but cannot be digested by the digestive system of rats. Their use by the intestinal microorganisms was observed in vivo even though their assimilation could not be detected in vitro. This implied that they were degraded by an unknown system in the intestine. It was also found that they affected calcium absorption mainly in the small intestine through mechanisms different from the known stimulants such as fructooligosaccharides and raffinose.
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  • Masaaki HIROSE
    2000 Volume 64 Issue 7 Pages 1328-1336
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      Transferrins are a group of iron-binding proteins that control the levels of iron in the body fluids of vertebrates by their ability to bind two Fe3+ and two CO32-. The transferrin molecule, with a molecular mass of about 80 kDa, is folded into two similarly sized homologous N- and C-lobes that are stabilized by many intrachain disulfides. As observed by X-ray crystallography, each lobe is further divided into two similarly sized domains, domain 1 and domain 2, and an Fe3+-binding site is within the interdomain cleft. Four of the six Fe3+ coordination sites are occupied by protein ligands (2 Tyr residues, 1 Asp, and 1 His) and the other two by a bidentate CO32-. Upon uptake and release of Fe3+, transferrins undergo a large-scale conformational change dependng on a common structural mechanism: domains 1 and 2 rotate as rigid bodies around a rotation axis that passes through the two antiparallel β-strands linking the domains. The extent of the rotaion is, however, variable for different transferrin species and lobes. As a Fe3+ release mechanisms at low pH from the N-lobes of serum transferrin and ovotransferrin, the structral evidence for ‘dilysine trigger mechanism’ is shown. A structural mechanism for the Fe3+ release in presence of a non-synergistic anion is proposed on the basis of the sulfate-bound apo crystal structure of the ovotransferrin N-lobe. Domain-opened structures with the coordinated Fe3+ by the two tyrosine residues are demonstrated in fragment and intact forms, and their functional implications as a possible intermediate for iron uptake and release are discussed.
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Analytical Chemistry Note
Organic Chemistry Regular Papers
Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Koji KISHI, Yoshifumi WATAZU, Yoshiaki KATAYAMA, Hiroaki OKABE
    2000 Volume 64 Issue 7 Pages 1352-1358
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      Mass production of an r-CDH derived from Nocardia species was made possible by gene technology. (Horinouchi et al., Applied and Environmental Microbiology, 57, 1386-1393 (1991)). However, the characteristics of the r-CDH have not been studied in detail and have not been improved enough for industrial use. We accordingly characterized both the native-CDH and the r-CDH prepared from Streptomyces lividans. Both CDHs were monomers with molecular masses of 37 kDa. The Km of r-CDH was 2.50×10-3 M for cholesterol and 2.33×10-4 M for NAD. The activators of CDHs were TritonX-100 and cholate. TritonX-405, Ag+, and Zn2+ inhibited both enzymes. The residual activity of native CDH after heat treatment was 32% (37°C, 60 min), while the r-CDH showed a residual activity of 87% (37°C, 60 min). The r-CDH is an enzyme with high substrate specificity for cholesterol as well as native CDH and higher thermal stability than native CDH.
      We have developed a novel serum cholesterol assay using the r-CDH, which permits the direct measurement of cholesterol by measuring NADH reaction products. We conclude that this r-CDH enzyme is useful and can be used to measure cholesterol in a clinical chemistry setting.
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  • Shin-ichi KASHIWABARA, Sayuri AZUMA, Masao TSUDUKI, Yuzuru SUZUKI
    2000 Volume 64 Issue 7 Pages 1379-1393
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      The gene that coded for the subunit of an molecular weight (Mr) 540,000 homohexameric α-glucosidase II (α-D-glucoside glucohydrolase, EC 3.2.1.20) produced by Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477) growing at 30 to 66°C was expressed in Escherichia coli HB101. The resulting homohexameric enzyme had a half-life of 10 min at 80°C. Its purification and characterization showed that the enzyme was identical with the native one except for the latter deleting 7 N-terminal residues found in the former. The primary sequence of the subunit with 787 residues and an Mr of 91,070 deduced from the gene was 24-34% identical to the corresponding sequences of 15 α-glucosidases in the glycosyl hydrolase family 31 from 14 eukaryotic origins and the archaeon Sulfolobus solfataricus 98/2.
      From the sequence analysis by the neural network method of Rost and Sander [Rost, B. and Sander, C., Proteins: Struct. Funct. Genet., 19, 55-72 (1994)], we inferred that α-glucosidase II might make each subunit of 3 secondary structural regions, i.e., one N-terminal β region, one central α/β region with two catalytic residues Asp407 and Asp484, and one C-terminal β region.
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  • Takeshi YAMAGAMI, Kazuki TSUTSUMI, Masatsune ISHIGURO
    2000 Volume 64 Issue 7 Pages 1394-1401
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      A cDNA encoding tulip bulb chitinase-1 (TBC-1) was cloned using a combination of immunoscreening from a λ ZAP cDNA library with anti-TBC-1 antiserum and the 5′ rapid amplification of cDNA end (RACE) method, and sequenced. The cDNA consists of 1,106 nucleotides and included an open reading frame encoding a polypeptide of 314 amino acids. Comparison of the deduced amino acid sequence and the determined protein sequence indicated the presence of a signal peptide and an extra peptide composed of 26 and 13 amino acids at the N- and C-termini, respectively. The deduced sequence of TBC-1 had 10-20% and 63% sequence similarities to plant class III chitinases and gladiolus bulb class IIIb chitinase (GBC-a), respectively. The cDNA encoding mature TBC-1 was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant TBC-1 (rTBC-1) expressed in E. coli was purified by gel filtration followed by ion-exchange chromatography. Specific activity of the rTBC-1 was almost same as the authentic TBC-1 toward glycolchitin. This is the first report on the cDNA cloning of a class III chitinase having C-terminal extra peptide.
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  • Akiko NISHII, Miho TAKEMURA, Hidetomo FUJITA, Masahito SHIKATA, Akiho ...
    2000 Volume 64 Issue 7 Pages 1402-1409
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      By differential screening of an arrayed normalized cDNA library from the inflorescence apex in Arabidopsis, a cDNA clone having a deduced amino acid sequence with a motif for a zinc finger was isolated as one of the genes expressed specifically in the reproductive phase. The deduced protein has a modular structure with a putative single C2-C2 zinc-finger motif distantly related to a GATA-1-type finger, a basic region with a sequence resembling a nuclear localization signal, and an acidic region. The gene seemed to have been formed by the exon-shuffling during its molecular evolution, since individual domains are encoded by discrete exons. RNA gel blot analysis showed its expression in shoot apex and flowers in the reproductive phase. The gene was named ZIM for Zinc-finger protein expressed in Inflorescence Meristem. The nuclear localization of ZIM was detected using GFP as a reporter. These results suggest that ZIM is a putative transcription factor involved in inflorescence and flower development.
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  • Takafumi KUBODERA, Nobuo YAMASHITA, Akira NISHIMURA
    2000 Volume 64 Issue 7 Pages 1416-1421
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      A pyrithiamine (PT) resistance gene (ptrA) was cloned from a genomic DNA library prepared from a PT resistant mutant of Aspergillus oryzae. It conferred high resistance to PT on an A. oryzae industrial strain as well as A. nidulans. Nucleotide sequence analysis showed that the ptrA gene contained one intron (58-bp) and encodes 327 amino acid (aa) residues. Additionally, the deduced aa sequence has 72% and 63% identity to Fusarium solani sti35 encoding a stress-inducible protein and Saccharomyces cerevisiae THI4 encoding an enzyme involved in thiamine biosynthesis, respectively, indicating that ptrA is a mutated allele of a gene belonging to the THI4 family. The mutation point was identified in the conserved motif in 5′-flanking region of these three THI4 homologous genes (ptrA, sti35, and THI4). The introduction of the ptrA gene allowed an A. oryzae industrial strain to grow on the minimum medium containing PT (0.1 mg/l) on which an untransformed strain did not grow. This result indicates that the ptrA is applicable as a dominant selectable marker for transformation of A. oryzae.
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  • Naoko HASEGAWA, Yoshinobu KIMURA, Tatsuya ODA, Nobukazu KOMATSU, Tsuyo ...
    2000 Volume 64 Issue 7 Pages 1422-1429
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      We have previously reported that ricin, a toxic lectin that inhibits protein synthesis induced apoptotic cell death. In this study, we have found that isolated ricin CM-B-chain, which has no effect on cellular protein synthesis, induced DNA fragmentation in U937 cells in a dose- and time-dependent manner, albeit it required a longer incubation time and higher concentration than those of holotoxin ricin. Z-Asp-CH2-DCB, a caspase family inhibitor and serine protease inhibitor, 3,4-dichloroisocoumarine (DCI) effectively inhibited the CM-B-chain-mediated DNA fragmentation as well as in ricin. Thus, like ricin, multiple proteases with different substrate specificity may also be involved in the CM-B-chain-mediated apoptotic pathway. Furthermore, BFA inhibited both ricin- and CM-B-chain-mediated DNA fragmentation, suggesting an intracellular vesicle transport system through the Golgi complex may be involved in the apoptotic induction by these proteins as a common feature. On the other hand, cycloheximide (CHA) strongly increased the CM-B-chain-mediated DNA fragmentation, but inhibited ricin-mediated DNA fragmentation. The opposite effects of CHA may reflect the difference in the apoptotic mechanism between ricin and CM-B-chain. In conclusion, our results suggest that ricin-B-chain can induce apoptosis through its lectin activity, but the underlying mechanism may be distinct from that of ricin in which the A-chain contributes profoundly to the apoptotic induction.
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  • Hirofumi NAKANO, Hiromi MURAKAMI, Motohiro SHIZUMA, Taro KISO, Teomar ...
    2000 Volume 64 Issue 7 Pages 1472-1476
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      β-Fructofuranosidase fructosylated not only the hydroxyl group but also the thiol group of 2-mercaptoethanol in a transfer reaction using sucrose as a donor substrate. The enzymes from Candida utilis and Saccharomyces cerevisiae (bakers’ yeast) were effective catalysts for the thio-fructofuranosylation. The thio-fructosylation product was isolated by activated carbon chromatography and its structure was confirmed by Fab-mass spectrometry and NMR spectroscopy. The thio-fructofuranoside was synthesized effectively at around 3.0 M for the acceptor concentration. The product increased with the sucrose concentration at least up to 1.9 M. O-Fructofuranoside was simultaneously synthesized at an early stage of the reaction, although it was hydrolyzed on further incubation. On the contrary, the thio-furctofuranoside accumulated efficiently after synthesis, indicating it was very stable against the hydrolytic action of the β-fructofranosidase.
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  • Kazuhisa HATAKEYAMA, Makoto GOTO, Miki KOBAYASHI, Masato TERASAWA, Hid ...
    2000 Volume 64 Issue 7 Pages 1477-1485
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.
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  • Yuji FURUYA, Hidemi SAWADA, Toshikatsu HIRAHARA, Kazue ITO, Takashi OH ...
    2000 Volume 64 Issue 7 Pages 1486-1493
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      A basidiomycete, Coprinus sp. SF-1, was found to produce an L-Trp-oxidizing enzyme by screening from the culture collection of our laboratory. After solubilization by 1 M NaSCN from the particulate fraction of disrupted cells of the strain, the enzyme was purified about 76-fold to essential homogeneity. The enzyme had a molecular mass of about 420 kDa and the subunit molecular mass was 68 kDa. The enzyme contained 1 mol of non-covalently bound FAD per mol of the subunit. It catalyzed the simultaneous reactions of oxidative deamination and oxygenative decarboxylation of L-Trp to form indolepyruvic acid and indole-3-acetamide, the former of which was further oxidized to indole-3-acetic acid. The molar ratio of the respective reaction products was about 9:1. The enzyme specifically oxidized L-Trp, and slightly acted on L-Phe and L-Tyr. The Km for L-Trp was about 0.5 mM in both oxidase and oxygenase reactions. Thus, the enzyme is a novel one and was tentatively designated “L-Trp oxidase (deaminating and decarboxylating)”. The optimum pHs of oxidase and oxygenase activities were 7.0 and 9.0, respectively. The optimum temperatures of both activities were 50°C. The enzyme was stable at pH 6.0-10.5 and below 50°C, and at 4°C for 1 year.
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  • Hideki SATO, Kazuhiro NAKAJIMA, Yukio MAENO, Takashi KAMAISHI, Tatsuo ...
    2000 Volume 64 Issue 7 Pages 1494-1499
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      Yellowtail ascites virus (YAV) is a member of the family Birnaviridae and causes viral ascites among juvenile yellowtail (Seriola quinqueradiata). We have reported the cloning and expression of two viral cDNAs, the first being segment A encoding a polyprotein of viral capsid proteins (VP2 and VP3) and a protease (NS), and the second being VP2-epitope encoding serotype-specific epitope region on VP2, using a baculovirus expression system. Another viral cDNA encoding a polyprotein of NS and VP3 was cloned and expressed in this study. For the expression of NS/VP3 (YAV nt 1626 to 3066) in insect cells a 31-kDa protein, corresponding to VP3 was detected, indicating an appropriate posttranslational processing of NS/VP3 polypeptide by NS protease itself. The analysis of the N-terminal amino acid sequence of this protein showed that NS protease may cleave an Ala-Ser bond. A study of the potential for vaccination of yellowtail fry by injection of insect cell lysates infected with baculovirus, containing either cDNA of segment A, VP2-epitope, or NS/VP3 was undertaken. Only a vaccination with cell lysates infected with a recombinant virus carrying the full length of YAV segment A gene demonstrated approximately the same effect as that of inactivated YAV. This result suggested that all proteins VP2, VP3, and NS are required for an effective vaccination.
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  • Keisuke NAKASHIMA, Jun-ichi AZUMA
    2000 Volume 64 Issue 7 Pages 1500-1506
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      A multi-enzyme distribution of endo-β-1,4-glucanase activity was found in the digestive system of a worker caste of the lower termite Coptotermes formosanus (Shiraki) by zymogram analysis. Its distribution analysis demonstrated that about 80% of this activity was localized in salivary glands from where only one component (EG-E) was secreted into the digestive tract.
      EG-E was isolated by a combination of chromatographic and electrophoretic techniques. Its molecular mass, optimal pH and temperature, isoelectric point, and Km were 48 kDa, 6.0, 50°C, 4.2, and 3.8 (mg/ml on carboxymethylcellulose), respectively. EG-E hydrolyzed cellooligosaccharides with a degree of polymerization of 4 and larger, and had low activity on crystalline cellulose. Main reaction products from low molecular weight cellulose were cellobiose and cellotriose. The N-terminal amino acid sequence of EG-E has similarity with fungal endo-β-1,4-glucanases and cellobiohydrolases of the glycosyl hydrolase family 7 rather than the other insect endo-β-1,4-glucanases of family 9.
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Biochemistry & Molecular Biology Notes
Food & Nutrition Science Regular Papers
  • Koji TAKAHASHI, Hiroshi KONDO, Hiroshi KUROIWA, Yoshiko YOKOTE, Makoto ...
    2000 Volume 64 Issue 7 Pages 1365-1372
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      The reversible thermal transition of soluble branched starch chains prepared from slightly acid-treated potato starch granules (ATS) was investigated. Potato starch was immersed in 15% sulfuric acid to obtain ATS with a 1% hydrolysis rate. About half of the molecules of ATS, which spontaneously formed large aggregates with a mass of a few million daltons in aqueous solution, was fractionated and soluble branched starch chains with a relative molecular weight (Mr) of 8.91×104 were obtained. Structural analysis indicated that the soluble branched starch chains consisted of three unit chains with Mr 7,900 and 21 unit chains with Mr 2,700. DSC and FT-IR measurements showed that the soluble branched starch chains underwent a reversible thermal transition, which is considered to be a helix-coil transition, during heating and cooling, but a debranched sample and β-limit dextrins showed substantially different thermal behavior, indicating the contribution of the ordered structure of the branched chains.
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  • Mingzhou SUN, Hiroyuki SAKAKIBARA, Hitoshi ASHIDA, Gen-ichi DANNO, Kaz ...
    2000 Volume 64 Issue 7 Pages 1373-1378
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      We have earlier found that flavones and flavonols in vegetables specifically inhibited one of the carcinogenesis-related enzymes, cytochrome P450 (CYP) 1A1, and subsequently suppressed the mutagenicity of food-derived carcinogens. In this study, we explored other candidates for the enzyme inhibitor in Chinese medicinal plants. Some of them were antimutagenic toward 3-amino-1-methyl-5H-pyrido[4, 3-b]indole (Trp-P-2). For example, Rheum officinale contained large amounts of anthraquinones as the active compounds, 3.4 mg of emodin, 2.1 mg of chrysophanol and 1.8 mg of rhein in 10 g of dry matter. Anthraquinones showed similar IC50 values for antimutagenicity against Trp-P-2 to those for inhibition of the N-hydroxylation activity of CYP1A1 toward Trp-P-2, indicating that the antimutagenicity was attributable to CYP inhibition. The structure-activity relationships were then examined with 14 commercial chemicals, and it was found that the interaction with an enzyme required three rings and an oxygen group in the side ring. This characteristic is similar to that of flavones and flavonols.
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  • Noritaka MATSUO, Koji YAMADA, Mitsuo MORI, Kentaro SHOJI, Takashi UEYA ...
    2000 Volume 64 Issue 7 Pages 1437-1443
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      Male Wistar rats were given purified diets containing safflower (SAF), perilla (PER), or palm (PAL) oils with or without 1% tea polyphenols (TP) for 3 weeks, and chemical mediator releasing activity from rat peritoneal exudate cells (PEC) was measured. Histamine releasing activity was not influenced by TP, while histamine release and intracellular histamine content were significantly increased in the PAL-fed group. On the contrary, leukotriene B4 (LTB4) release was significantly lower in rats fed PER than in those fed SAF and PAL, and TP significantly decreased the release in all fat groups. TP also significantly inhibited the release of LTB5, which was generated only in rats fed PER. TP significantly decreased the proportion of arachidonic acid (AA) in PEC in the SAF-fed group and that of eicosapentaenoic acid (EPA), the precursor of LTB5 in the PER-fed group, but did not influence that of AA in the PAL- and PER-fed group. These results suggest that ingestion of TP improves type I allergic symptom through the inhibition of LT release though the inhibition by TP could not be totally explained by the reduction of substrate fatty acid.
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  • Shinya NAGAFUCHI, Mamoru TOTSUKA, Satoshi HACHIMURA, Masao GOTO, Takes ...
    2000 Volume 64 Issue 7 Pages 1459-1465
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      We have investigated the effects of dietary nucleotides on intraepithelial lymphocytes (IEL) and intestinal epithelial cells (IEC) in weanling mice. The proportion of T-cell receptor (TCR) γδ+ IEL in BALB/c mice fed a diet supplemented with nucleotides (NT(+) diet) was significantly higher than that in mice fed the nucleotide-free diet, while the proportion of TCRαβ+ IEL in NT(+) diet-fed mice was significantly decreased. The change of the TCRαβ+/TCRγδ+ ratio was mainly observed in a CD8αα+ subset of IEL. IEC from NT(+) diet-fed mice produced a higher level of IL-7, which is important in the development of TCRγδ+ IEL, than those from control diet-fed mice. The expression levels of IL-7 and IL-2 receptors on IEL were not different between the two dietary groups. Our findings suggest that the increased population of a TCRγδ+ IEL subset by feeding nucleotides may be caused by the increased production of IL-7 by IEC.
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  • Miho KOIE, Miyuki TANAKA, Kazutoshi HAYASE, Hidehiko YOKOGOSHI
    2000 Volume 64 Issue 7 Pages 1466-1471
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      The purpose of this study was to find whether the addition of dietary lysine affected the rate of brain protein synthesis in aged rats fed on a gluten diet. Experiments were done on two groups of aged rats (30 wk) given the diets containing 5% gluten or 5% gluten+0.3% lysine for 10 d. The fractional rates of protein synthesis in brain, liver, and kidney increased with an addition of dietary lysine. In brain, liver, and kidney, the RNA activity [g protein synthesized/(g RNA•d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the addition of the limiting amino acid for the low quality protein elevates the rate of protein synthesis in the brain of aged rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.
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Food & Nutrition Science Note
  • Masaki TERAHARA, Sachiko NISHIDE, Tsutomu KANEKO
    2000 Volume 64 Issue 7 Pages 1531-1533
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      We examined the 3-amino-1,4-dimethyl-5H-Pyrido-[4,3-b]indole (Trp-P-1) concentration in the blood after administering Trp-P-1 (0.25 mg/ml) with or without Streptococcus thermophilus 1131 cells (10 mg/ml) to rats. The Trp-P-1 concentration in the blood from the portal vein was significantly lower in the rats that had been administered with Trp-P-1 with the strain 1131 cells than without them. However, there was no difference in the Trp-P-1 concentration in the blood taken from the abdominal aorta of these rats.
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Microbiology & Fermentation Technology Regular Papers
  • Masaru NAGAI, Akira OZAWA, Tohoru KATSURAGI, Takuo SAKAI
    2000 Volume 64 Issue 7 Pages 1337-1344
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      Aspergillus awamori IFO 4033 produced an acid-stable protopectinase in solid-state fermentation using wheat bran as the medium. The enzyme was purified to a homogeneous preparation with anion-exchange, hydrophobic, and size-exclusion chromatography. The enzyme was a monomeric protein of 52 kDa, by SDS-PAGE analysis, with an isoelectric point of pH 3.7. The optimum pH for enzyme activity was 2.0, and it was most active at 50°C (at pH 2.0) and was stable up to 50°C (at pH 2.0). The enzyme showed pectin-releasing activity toward protopectins from various origins, especially on lemon protopectin. An outstanding characteristic of the enzyme was its extreme stability in acidic conditions: the enzyme activity was not lost after incubating at pH 2.0 and 37°C for 24 h.
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  • Yoshihiko YASOHARA, Noriyuki KIZAKI, Junzo HASEGAWA, Masaru WADA, Mich ...
    2000 Volume 64 Issue 7 Pages 1430-1436
    Published: 2000
    Released: February 10, 2005
    JOURNALS FREE ACCESS
      An NADPH-dependent carbonyl reductase (S1) isolated from Candida magnoliae catalyzed the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE), with a 100% enantiomeric excess, which is a useful chiral building block for the synthesis of pharmaceuticals. The gene encoding the enzyme was cloned and sequenced. The S1 gene comprises 849 bp and encodes a polypeptide of 30,420 Da. The deduced amino acid sequence showed a high degree of similarity to those of the other members of the short-chain alcohol dehydrogenase superfamily. The S1 gene was overexpressed in Escherichia coli under the control of the lac promoter. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as the enzyme from C. magnoliae did. An E. coli transformant reduced COBE to 125 g/l of (S)-CHBE, with an optical purity of 100% enantiomeric excess, in an organic solvent two-phase system.
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