Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 67 , Issue 10
Showing 1-41 articles out of 41 articles from the selected issue
Organic Chemistry Regular Papers
Organic Chemistry Note
  • Hiroshi YAMAGUCHI, Hiroaki KODAMA, Satoshi OSADA, Fumio KATO, Masood J ...
    2003 Volume 67 Issue 10 Pages 2269-2272
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      A tryptic [EC 3.4.21.4] digestion assay of 2-aminoisobutyric acid (Aib)-containing peptides was carried out to investigate the effect of α,α-dialkyl amino acid residues on the protease resistance. The introduction of Aib residues to the P1′ positions exhibited a 19-fold higher protease resistance than the peptide with Aib residues introduced to the P2 position or the non-Aib peptide. The peptide having Aib residues introduced to the P1′ and P2 positions resulted in complete resistance.
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Biochemistry & Molecular Biology Regular Papers
  • Akira TSUKAMOTO, Yasushi KOJIMA, Yukio KITA, Jun SUGIURA
    2003 Volume 67 Issue 10 Pages 2075-2082
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      An efficient transformation system for the basidiomycete Coriolus hirsutus was developed. A double-auxotrophic mutant of C. hirsutus, deficient both in ornithine carbamoyltransferase (OCTase) and 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase), was transformed to Arg+ with each allelic type of the C. hirsutus genomic OCTase gene (arg1) newly cloned. The transformation frequency of 103-104 transformants per μg DNA per 106-107 oidial protoplasts was reached. Southern blots showed that the transforming DNA was integrated into chromosomal DNA with multi-copies. The Arg+ phenotype of the transformants was stably inherited through mitosis.
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  • Mitsuo MIYAZAWA, Masayoshi HISAMA
    2003 Volume 67 Issue 10 Pages 2091-2099
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      A methanol extract from the flower heads of Chrysanthemum morifolium showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide). The methanol extract was re-extracted with hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction showed a suppressive effect. Suppressive compounds in the ethyl acetate fraction were isolated by silica gel column chromatography and identified as the flavonoids acacetin (1), apigenin (2), luteolin (3), and quercetin (4) by EI-MS, IR, and 1H and 13C NMR spectroscopy. Compounds 1-4 suppressed the furylfuramide-induced SOS response in the umu test. Compounds 1-4 suppressed 60.2, 75.7, 90.0, and 66.6% of the SOS-inducing activity at a concentration of 0.70 μmol/ml. The ID50 (50% inhibitory dose) values of 1-4 were 0.62, 0.55, 0.44, and 0.59 μmol/ml. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4-nitroquinolin 1-oxide (4NQO) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), which do not require liver-metabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 (AfB1) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), which require liver-metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100.
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  • Hirosuke OKU, Naoya FUTAMORI, Kenichi MASUDA, Yumiko SHIMABUKURO, Tomo ...
    2003 Volume 67 Issue 10 Pages 2106-2114
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      It was found that the partially purified β-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for α-keto-β-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.
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  • Osao ADACHI, Nozomi YOSHIHARA, Somboon TANASUPAWAT, Hirohide TOYAMA, K ...
    2003 Volume 67 Issue 10 Pages 2115-2123
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Several bacterial strains carrying quinoprotein quinate dehydrogenase (QDH) were screened through acetic acid bacteria and other bacteria. Strong enzyme activity was found in the membrane fraction of Gluconobacter melanogenus IFO 3294, G. oxydans IFO 3292, G. oxydans IFO 3244, and some strains of Acinetobacter calcoaceticus. Interestingly, in the membrane fraction of A. calcoaceticus AC3, which is unable to produce pyrroloquinoline quinone (PQQ), fairly large amounts of apo-QDH were formed, and were converted to holo-QDH only by the addition of PQQ. It was difficult to detach PQQ from the holo-QDH by EDTA treatment, and EDTA treatment with apo-QDH prior to PQQ addition gave no significant holo-QDH. For QDH purification, Gluconobacter strains were not suitable due to the presence of huge amounts of quinohemoprotein alcohol dehydrogenase (ADH) in the same membrane, which was co-solubilized with QDH and disturbed purification of QDH. Purification of holo-QDH was done with Acinetobacter sp. SA1 instead, which contained no ADH. Apo-QDH was purified from A. aclcoaceticus AC3.

      This is the first report dealing with QDH purification, and two different criteria of QDH purification were given. A combination of two steps using butyl-Toyopearl and hydroxyapatite columns gave a highly purified holo-QDH which was monodispersed and showed enough purity, though the specific activity did not increase as much as expected. When QDH purification was done with A. calcoaceticus AC3 in the absence of PQQ, purified apo-QDH appeared to be a dimer, which was converted to the monomer on addition of PQQ. Since QDH was highly hydrophobic, one-step chromatography on a DEAE-Sepharose column was tried. Purified holo-QDH of higher specific activity was obtained with a higher yield. The molecular mass of QDH was estimated to be 88 kDa. There was no characteristic absorption spectrum with the purified QDH except for a small bump around 420 nm. QDH oxidized only quinate and shikimate so far examined. The optimal QDH activity was found at pH 6-7 when assayed with artificial electron acceptors. QDH was formed in the presence or absence of quinate in the culture medium, although stronger induction was usually observed in the presence of quinate.

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  • Riadh BEN SALAH, Nacim ZOUARI, Joseph REINBOLT, Hafedh MEJDOUB
    2003 Volume 67 Issue 10 Pages 2139-2144
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Turkey pancreatic phospholipase (TPP) has been purified from delipidated pancreases. The purification included ammonium sulfate fractionation, acidic (pH 5) treatment, followed by sequencial column chromatographies on DEAE-cellulose, Sephadex G-75, and reverse phase high pressure liquid chromatography. The purified enzyme was found to be a monomeric protein with molecular mass of 14 kDa. The optimal activity was measured at pH 8 and 37°C using egg yolk emulsion as substrate. Our results show that the enzyme (TPP) was not stable for 1 h at 60°C, and that bile salt and Ca2+ were required for the expression of the purified enzyme. The sequence of the N-terminal amino acids of the purified enzyme shows a very close similarity between TPP and all other known pancreatic phospholipases.
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  • Hirokazu NANBA, Yasuko TAKAOKA, Junzo HASEGAWA
    2003 Volume 67 Issue 10 Pages 2145-2153
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to α-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with α-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD+ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.
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  • Takashi SUZUKI, Kohki ENDO, Masaaki ITO, Hiroshi TSUJIBO, Katsushiro M ...
    2003 Volume 67 Issue 10 Pages 2167-2175
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7. About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0%. REN-7-PPO showed broad substrate specificity toward various aromatic compounds. Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase. Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70°C. The gene encoding the PPO was cloned. Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence. We constructed a high-level expression system of this gene in Escherichia coli. The properties of the recombinant enzyme were identical that of wild-type. In conclusion, this PPO is a thermostable laccase.
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  • Hisashi KIMOTO, Akira TAKETO
    2003 Volume 67 Issue 10 Pages 2203-2209
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Hemolytic streptococci are lacking in natural competence for uptake of DNA, and existing electrotransformation methods are still ineffective for most strains. By optimizing biological and electric parameters of electroporation, we established a simple, efficient, and reproducible transformation method for streptococcal cells. The major factor was an increase in the electric field strength. All tested streptococci (6 group A strains and one group C strain) were successfully transformed, and the maximal efficiency was higher than 1×107 transformants per μg of plasmid DNA. Targeted inactivation of the chromosomal genes of group A and C streptococci was achieved, using the electrotransformation method. The slo- or sagB- mutants constructed by the gene-targeting showed elevated competence for electrotransformation. Availability of the electrotransfer system for cloning and analysis of streptococcal genes is discussed.
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  • Yoshinobu KIMURA, Takeo YOSHIIE, Woo Kwan KIT, Megumi MAEDA, Mariko KI ...
    2003 Volume 67 Issue 10 Pages 2232-2239
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing α1-3 fucose and/or β1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).
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  • Tetsuya TANAKA, Sanae SATO, Haruto KUMURA, Kei-ichi SHIMAZAKI
    2003 Volume 67 Issue 10 Pages 2254-2261
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Lactoperoxidase (LPO) is a heme-containing oxidation-reduction enzyme present in milk. In this study, the gene encoding bovine lactoperoxidase (bLPO) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLPO was isolated. A bLPO-related recombinant baculovirus-expressed protein of 78 kDa was detected using anti-bLPO antibodies. After digestion with N-glycosidase F, the molecular weight of the recombinant bLPO (rbLPO) decreased. In addition, rbLPO reacted with lectin, indicating that the protein was glycosylated. The rbLPO activity and heme content in the culture supernatants increased upon addition of δ-aminolevulinic acid, which is a heme precursor. Differences in the δ-aminolevulinic acid-dependent circular dichroism spectrum and rbLPO pepsin hydrolysis were observed. These results suggest that the secondary structure and structural stability of rbLPO depends on the heme environment. Our data suggest that this bLPO expression system is useful for studying structure, catalytic mechanisms, and biological function.
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Biochemistry & Molecular Biology Notes
Biochemistry & Molecular Biology Communication
  • Hiroko KOBAYASHI, Tadashi ITAGAKI, Norio INOKUCHI, Kazuko OHGI, Tatsuy ...
    2003 Volume 67 Issue 10 Pages 2307-2310
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively. The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast. The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.
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Food & Nutrition Science Regular Papers
  • Yukihiro AOSHIMA, Yoshinori HASEGAWA, Sho HASEGAWA, Atsushi NAGASAKA, ...
    2003 Volume 67 Issue 10 Pages 2068-2074
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      After screening extensively factors in plant extracts that increase alkaline phosphatase activity, an osteoblastic differentiation marker protein in mouse calvarial osteoblast MC3T3-E1 cells, GnafC derived from Gnaphalium affine, was found to significantly enhance the alkaline phosphatase (ALPase) activity in a synergistic manner with ascorbate. GnafC was a polysaccharaide with an approximate molecular mass of 10,000 and comprised mannose, xylose, arabinose, galactose and glucose in a molar ratio of 1:2:4.3:2.5:2.7. Expression of the osteoblastic differentiation marker genes was examined by semiquantitative RT-PCR with RNAs prepared from cells at different developmental stages. With ascorbate in the culture, GnafC enhanced the expression of the ALPase and MMP13 genes from the early stage of differentiation, leading to maturation of the collagenous extracellular matrix (ECM), a prerequisite for mineralization.
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  • Hyo-seon LEE, Chun-yeon CHOI, Chungwon CHO, Youngsun SONG
    2003 Volume 67 Issue 10 Pages 2083-2090
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      This study was designed to investigate whether chlorella supplementation may ameliorate oxidative stress and nuclear factor kappa B (NFκB) activation in peritoneal macrophages and liver of C57BL/6 mice fed on an atherogenic diet. The animals were maintained on an atherogenic diet (control), or an atherogenic diet supplemented with 3% (w/w) chlorella or 5% (w/w) chlorella for 12 wks. The plasma and hepatic lipid levels were not affected by chlorella supplementation. Hepatic thiobarbituric acid-reactive substances and superoxide anion production in peritoneal macrophages were significantly lower in the 5% chlorella group (p<0.05), but the glutathione level was not altered by chlorella supplementation. The hepatic antioxidative enzyme activities of Cu, Zn-superoxide dismutase and catalase were higher in the mice fed on the 5% chlorella diet (p<0.05). The plasma aspartate aminotransferase activity was lower in the mice fed on the chlorella-containing diets (p<0.05), whereas the alanine aminotransferase activity was not affected by chlorella supplementation. The NFκB nuclear binding activities of peritoneal macrophages and liver were significantly lower in the 5% chlorella groups (p<0.05). These results suggest that chlorella supplementation may attenuate oxidative stress by reducing reactive oxygen production and increasing antioxidative processes, thus suppressing inflammatory mediator activation in peritoneal macrophages and liver.
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  • Yoko SANO, Kaori SATO, Motoharu UCHIDA, Masakazu MURATA
    2003 Volume 67 Issue 10 Pages 2100-2105
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.
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  • Noriko BANDO, Rintaro YAMANISHI, Junji TERAO
    2003 Volume 67 Issue 10 Pages 2176-2182
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      A diet containing different amounts of vitamin E (α-tocopherol; 0.5 mg, 5 mg, 10 mg or 50 mg per 100 g diet) was supplemented to BALB/c mice for 6 weeks. These mice were subcutaneously immunized twice with ovalbumin (OVA). A passive cutaneous anaphylaxis (PCA) analysis demonstrated that the mice fed on the diet containing 5 mg of vitamin E produced the highest level of the OVA-specific immunoglobulin E (IgE) antibody. A lower level of serum IgE was found in the mice supplemented with 0.5 mg, 10 mg and 50 mg of vitamin E. A sandwich ELISA analysis showed that the pattern of the total IgE antibody level among these four groups was the same as that of the allergen-specific IgE. In a separate experiment, 5 mg of vitamin E and/or 50 mg of β-carotene was supplemented to the basal diet containing vitamin E as α-tocopherol acetate (5 mg) in order to evaluate the effect of their combination on OVA-specific and total IgE production in the mice. The supplementation with β-carotene alone had no effect on OVA-specific or total IgE production. In contrast, supplementation with vitamin E plus β-carotene effectively suppressed both the antigen-specific and total IgE antibodies. The serum vitamin E and β-carotene levels were increased by supplementation with the respective compounds. These results strongly suggest that the combination of dietary vitamin E and β-carotene suppressed IgE production and would therefore help to prevent the type-I allergic reaction.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Bi-Zhen ZOU, Kiyoshi TAKEDA, Akio TONOUCHI, Shinji AKADA, Takashi FUJI ...
    2003 Volume 67 Issue 10 Pages 2059-2067
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      The number of syntrophic butyrate-degrading bacte- ria in a flooded paddy field soil was 1.7×103 MPN/g dry soil. Butyrate was degraded to acetate and methane when paddy soils were incubated anaerobically with the addition of butyrate. However, butyrate degradation was completely suppressed by the addition of the specific inhibitor of methanogenesis, 2-bromoethanesulfonate (BES) to the soil. A hydrogen-using methanogen, strain TM-8, was isolated from flooded paddy field soil. Strain TM-8 was identified as Methanobacterium formicicum based on its physiology and phylogeny. Syntrophic butyrate-degrading bacteria were enumerated and isolated using strain TM-8. A syntrophic butyrate-degrading bacterium, strain TB-6, was isolated in coculture with strain TM-8 from paddy soil. The strain was Gram-negative, had curved rods, and grew on crotonate. Sulfate was not used as an electron acceptor. Strain TB-6 was closely related to S. wolfei subsp. wolfei. The relation between strain TB-6 and the members of Syntrophomonas are discussed.
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  • Osao ADACHI, Somboon TANASUPAWAT, Nozomi YOSHIHARA, Hirohide TOYAMA, K ...
    2003 Volume 67 Issue 10 Pages 2124-2131
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      3-Dehydroquinate production from quinate by oxidative fermentation with Gluconobacter strains of acetic acid bacteria was analyzed for the first time. In the bacterial membrane, quinate dehydrogenase, a typical quinoprotein containing pyrroloquinoline quinone (PQQ) as the coenzyme, functions as the primary enzyme in quinate oxidation. Quinate was oxidized to 3-dehydroquinate with the final yield of almost 100% in earlier growth phase. Resting cells, dried cells, and immobilized cells or an immobilized membrane fraction of Gluconobacter strains were found to be useful biocatalysts for quinate oxidation. 3-Dehydroquinate was further converted to 3-dehydroshikimate with a reasonable yield by growing cells and also immobilized cells. Strong enzyme activities of 3-dehydroquinate dehydratase and NADP-dependent shikimate dehydrogenase were detected in the soluble fraction of the same organism and partially fractionated from each other. Since the shikimate pathway is remote from glucose in the metabolic pathway, the entrance into the shikimate pathway from quinate to 3-dehydroquinate looks advantageous to produce metabolic intermediates in the shikimate pathway.
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  • Soo Jeong CHO, Woo Jin LIM, Su Young HONG, Sang Ryeol PARK, Han Dae YU ...
    2003 Volume 67 Issue 10 Pages 2132-2138
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270-1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 107 cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.
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  • SURYANI, Tetsuya KIMURA, Kazuo SAKKA, Kunio OHMIYA
    2003 Volume 67 Issue 10 Pages 2160-2166
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      The α-galactosidase gene aga36A of Clostridium stercorarium F-9 was cloned, sequenced, and expressed in Escherichia coli. The aga36A gene consists of 2,208 nucleotides encoding a protein of 736 amino acids with a predicted molecular weight of 84,786. Aga36A is an enzyme classified in family 36 of the glycoside hydrolases and showed sequence similarity with some enzymes of family 36 such as Geobacillus (formerly Bacillus) stearothermophilus GalA (57%) and AgaN (52%). The enzyme purified from a recombinant E. coli is optimally active at 70°C and pH 6.0. The enzyme hydrolyzed raffinose and guar gum with specific activities of 3.0 U/mg and 0.46 U/mg for the respective substrates.
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  • Kazuki NAGAMINE, Kenji MURASHIMA, Taku KATO, Hitoshi SHIMOI, Kiyoshi I ...
    2003 Volume 67 Issue 10 Pages 2194-2202
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      Aspergillus kawachii produces two kinds of α-amylase, one is an acid-unstable α-amylase and the other is an acid-stable α-amylase. Because the quality of the shochu depends strongly on the activities of the α-amylases, the culture conditions under which these α-amylases are produced were examined. In liquid culture, acid-unstable α-amylase was produced abundantly, but, acid-stable α-amylase was not produced. The acid-unstable α-amylase was produced significantly when glycerol or glucose was used as a carbon source, similarly to the use of inducers such as starch or maltose. In liquid culture, A. kawachii assimilated starch at pH 3.0, but no α-amylase activity was recognized in the medium. Instead, the α-amylase was found to be trapped in the cell wall. The trapped form was identified as acid-unstable α-amylase. Usually, acid-unstable α-amylase is unstable at pH 3.0, so its stability appeared to be due to its immobilization in the cell wall. In solid-state culture, both kinds of α-amylase were produced. The production of acid-stable α-amylase seems to be solid-state culture-specific and was affected by the moisture content in the solid medium.
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  • Yoshiaki OHASHI, Takashi INAOKA, Koji KASAI, Yasuhiro ITO, Susumu OKAM ...
    2003 Volume 67 Issue 10 Pages 2245-2253
    Published: 2003
    Released: October 28, 2003
    JOURNALS FREE ACCESS
      A DNA microarray technique was used to demonstrate global changes in the transcription pattern of translation-associated genes that encode fifty-four ribosomal proteins including a putative ribosomal gene, and eleven translation factors in sporulating B. subtilis. We found that the mRNA levels of nine genes involved in the translation system, which include the genes for three ribosomal proteins (rpmA, rpmGB, and ctc) and two translation factors (efp, and frr), were maintained at a high level at the onset of sporulation. The ypfD gene, which encodes the ribosomal protein S1 homologue, was also found to be expressed significantly during the early sporulation stage. In order to demonstrate the significance of these genes for sporulation, mutants were constructed using the pMutinT3 disruption vector. We detected an impaired sporulation in the mutants of rpmA (gene for the ribosomal protein L27), efp (elongation factor P), frr (ribosome recycling factor), and ypfD. The effect was especially pronounced in the efp mutant, sporulation of which was entirely abolished without affecting growth. The reduced expression of rpmGB (ribosomal protein L33) resulted in an impaired sporulation only at a high temperature (47°C). Only the rplI mutant, which encodes the ribosomal protein L9, could not be obtained, implying that its function is essential for viability. Thus, we successfully demonstrated the significance of several translation-associated genes in sporulation by using the results of the gene expression profiling.
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Microbiology & Fermentation Technology Notes
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