Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 63 , Issue 12
Showing 1-40 articles out of 40 articles from the selected issue
Organic Chemistry Regular Papers
Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Hiromasa IMAISHI, Yuka MATSUMOTO, Utako ISHITOBI, Hideo OHKAWA
    1999 Volume 63 Issue 12 Pages 2082-2090
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The cDNA clone of novel cytochrome P450 CYP703A1 from petunia floral buds was isolated by RT-PCR. The nucleotide sequences of this cDNA clone contained the open reading frame that has been predicted to encode polypeptides consisting of 539 amino acid residues. A significantly high level of the transcript of the cyp703A1 gene was found in the early stage of petunia flower buds, but not in the leaves, stems and roots. The 1041bp 5′-flanking sequences of the cyp703A1 gene contained the conserved motifs of ATHB-1, AGAMOUS, MYB.Ph3, P and SBF-1 binding boxes. CYP703A1 cDNA was expressed in yeast Saccharomyces cerevisiae AH22 cells under the control of an alcohol dehydrogenase I promoter and terminator. The recombinant yeast microsome containing the CYP703A1 hemoprotein were found to metabolize lauric acid. Based on these results, CYP703A1 was specifically expressed in the early stage of flower development and appeared to participatie in the monooxygenation of fatty acids.
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  • Jun-Ho KIM, Yang Sun KIM
    1999 Volume 63 Issue 12 Pages 2130-2136
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both Aα and Bβ subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60°C and the maximum fibrinolytic activity was at 55°C.
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  • Akira OHNISHI, Toshihiko OOI, Shinichi KINOSHITA, Hirotaka TOMATSURI, ...
    1999 Volume 63 Issue 12 Pages 2157-2162
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were located in the cleft from the X-ray crystallographic analysis of FI-CMCase, endo-1,4-β-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No. F-50. To identify the catalytic residue of the FI-CMCase, these residues were mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118 and Glu202 by site-directed mutagenesis, and totally 8 single mutant enzymes experssed in Escherichia coli were prepared: D97E, D97S, D101E, D101S, E118D, E118S, E202D, and E202S. Mutant enzymes E118S and E202S were not shown to have any detectable activity. Kinetic parameters of other mutant enzymes were measured after purification. The Km of mutant enzymes were not much different from that of wild type FI-CMCase, while the Vmax of mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type FI-CMCase, respectively. From these results we concluded that Glu118 and Glu202 were most probable candidates for a catalytic pair of acidic amino acids in FI-CMCase.
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  • Tetsuya OGUMA, Toshiko KUROKAWA, Kouichiro TOBE, Satoshi KITAO, Mikihi ...
    1999 Volume 63 Issue 12 Pages 2174-2182
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was cloned by using a combination of gene walking and probe methods and expressed on the recombinant plasmid pGD8, which was constructed with pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique open reading frame of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and 6 internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggested the enzyme was translated from mRNA as a secretory precursor with a signal peptide of 28 amino acids residues. The deduced amino acids sequence of the mature enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 daltons. The deduced sequence showed about 38% identity to that of the glucoamylase from Clostridium sp. G0005. The glucodextranase activity of transformant harboring pGD8 was about 40 mU/ml at 30°C for a 16-h culture. Although the GDase that was produced from the transformant was shorter than authentic GDase by 2 amino acid residues at the N-terminal end side, its enzymatic properties were almost same as the authentic one.
      Two kinds of genes, dex1 and dex2, for endo-dextranases from A. globiformis T-3044 were also cloned into Escherichia coli cells. The N-terminal of the purified endo-dextranase from A. globiformis T-3044 agreed with the deduced amino acid sequence, after the 33rd alanine residue, of only the dex1 gene for endo-dextranase. This result suggests that the endo-dextranase is translated from mRNA as a secretory precursor with a signal peptide of 32 amino acids residues. The deduced sequence of endo-dextranase 1 and endo-dextranase 2 showed about 93% and 65% identity with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively.
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Biochemistry & Molecular Biology Notes
  • Yoichiro SHIMURA, Qiong WANG, Yoshiyuki SAKANO
    1999 Volume 63 Issue 12 Pages 2199-2201
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      We estimated the subsite structure of α-amylase II (TVA II) from Thermoactinomyces vulgaris R-47 expressed in Escherichia coli. TVA II has eight subsites, and the catalytic site is between the 5th and 6th subsite from the non-reducing end side. The subsite affinities, A-5, A-4, A-3, A-2, (A-1+A+1), A+2, and A+3, were calculated to be -0.35, 0.93, 0.55, 2.56, 1.18, 1.71, and 0.01 kcal mol-1, respectively.
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  • Young-Ok KIM, Jung-Kee LEE, Byung-Chul OH, Tae-Kwang OH
    1999 Volume 63 Issue 12 Pages 2205-2207
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      An efficient expression system was developed in Bacillus subtilis for the large scale production of phytase. The phytase gene with a native promoter derived from Bacillus amyloliquefaciens was cloned in the Bacillus expression vector pJH27 under a strong BJ27 promoter and its expression was optimized. The expression of the phytase gene occurred during late exponential growth and the extracellular phytase production was 2.0 units/ml, which constituted over 90% of the total protein. The yield was 100-fold higher than wild type Bacillus amyloliquefaciens DS11.
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  • Jiu-Cun WANG, Mikio SAKAKIBARA, Michiko MATSUDA, Nobuya ITOH
    1999 Volume 63 Issue 12 Pages 2216-2218
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Phenylacetaldehyde reductase (PAR) with a unique and wide substrate range from styrene-assimilating Corynebacterium sp. strain ST-10, which is a useful biocatalyst producing chiral alcohols, has been found to belong to a family of zinc-containing, long-chain alcohol dehydrogenases (ADHs) on the basis of the primary structure similarity. The enzyme contains 2 moles of zinc per mole of subunit. The amino acid residues assumed to be three catalytic and four structural zinc-binding ligands were characterized by site-directed mutagenesis, compared with other zinc-containing, long-chain ADHs. Sixteen PAR mutants gave measurable but rather low activities toward phenylacetaldehyde, n-hexyl aldehyde, and 2-heptanone, although they maintained the activities of 8 to 16% of that of wild-type PAR for an acetophenone substrate except that the D153N mutant showed quite low activity. The results suggested that the seven residues present in PAR were probably zinc-binding ligands, and mutation in these residues caused a change in activities for some substrates.
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  • Kuniaki ADACHI, Yasusi WATANABE, Kuniyo INOUYE
    1999 Volume 63 Issue 12 Pages 2219-2221
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Activity of glucose-6-phosphate 1-dehydrogenase (G6PDH) in human hair follicles was measured. A good relationship has been demonstrated between the activity and the ratio of the number of the anagen hairs to that of all the plucked hairs in the frontal-parietal region of the scalp with male-pattern alopecia. As the ratio becomes lower so that the advancing degree of alopecia is higher, the G6PDH activity becomes lower.
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  • Tomohisa KUZUYAMA, Takayuki SEKI, Seiji KOBAYASHI, Tomomi HIDAKA, Haru ...
    1999 Volume 63 Issue 12 Pages 2222-2224
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The fosfomycin resistance gene, fosC, has been cloned from the fosfomycin-producing organism, Pseudomonas syringae PB-5123. Sequence analysis upstream of this gene found a new ORF showing significant homology to 2-hydroxypropylphosphonic acid epoxidase from fosfomycin-producing Streptomyces wedmorensis. The purified recombinant protein of this ORF converted 2-hydroxypropylphosphonic acid to fosfomycin. This result clearly showed the ORF to encode 2-hydroxypropylphosphonic acid epoxidase in PB-5123.
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  • Naoki ICHIKAWA, Saori USHIDA, Miho KAWABATA, Yukiko MASAZUMI
    1999 Volume 63 Issue 12 Pages 2225-2227
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Mitochondrial ATP synthase (F1Fo-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, cDNA coding the human homolog of the inhibitor protein was isolated and sequenced. The deduced protein sequence shows that the protein was composed of 106 amino acids and had a molecular weight of 12248. The structural features of the protein show that the cDNA isolated in this study codes the human ATPase inhibitor.
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  • Toru YASUDA, Kazuo SHISHIDO
    1999 Volume 63 Issue 12 Pages 2228-2231
    Published: 1999
    Released: March 08, 2005
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      A fruiting-body-specific mfbA cDNA derived from Lentinus edodes FMC2 has been shown to encode a high-molecular-weight protein, MFBA, containing the cell-adhesion-promoting Arg-Gly-Asp (RGD) sequence. Southern-blot analysis showed that all L. edodes strains tested have the mfbA gene (homologue). Nucleotide sequence analysis of the 1-kb mfbA fragments containing the RGD-coding sequence showed that each L. edodes strain has two types of mfbA homologues. It was found in FMC2 that two mfbA homologues are derived from different nuclei and these mfbA alleles are transcribed with similar frequencies in the fruiting bodies.
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  • Fumiyo SAITOH, Shunsuke KAWAMURA, Nobuyuki YAMASAKI, Isao TANAKA, Mako ...
    1999 Volume 63 Issue 12 Pages 2232-2235
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      DNA-binding protein HU (BstHU) from Bacillus stearothermophilus is a homodimeric protein which binds to DNA in a sequence-nonspecific manner. In order to identify the Arg residues essential for DNA binding, four Arg residues (Arg-53, Arg-55, Arg-58, and Arg-61) within the β-arm structure were replaced either by Gln, Lys, or Glu residues, and the resulting mutants were characterized with respect to their DNA-binding activity by a filter-binding analysis and surface plasmon resonance analysis. The results indicate that three Arg residues (Arg-55, Arg-58, and Arg-61) play a crucial role in DNA binding as positively charged recognition groups in the order of Arg-55>Arg-58>Arg-61 and that these are required to decrease the dissociation rate constant for BstHU-DNA interaction. In contrast, the Arg-53 residue was found to make no contribution to the binding activity of BstHU.
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  • Dong-Sun LEE, Sang-Han LEE, Jae-Geun NOH, Soon-Duck HONG
    1999 Volume 63 Issue 12 Pages 2236-2239
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Cryptotanshinone and dihydrotanshinone I, constituents of a medicinal plant, Salvia miltiorrhiza Bunge, had antibacterial activity against a broad range of Gram positive bacteria. These compounds generated superoxide radicals in Bacillus subtilis lysates. A recombination-deficient mutant strain of B. subtilis was 2- to 8-fold more sensitive than a wild strain, and this hypersensitivity was reduced in the presence of dithiothreitol as an antioxidant. DNA, RNA, and protein syntheses in B. subtilis were non-selectively inhibited by these compounds. These results suggest that superoxide radicals are important in the antibacterial actions of the agents.
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  • Seiyei YAMAKAWA, Yoshikatsu MATSUBAYASHI, Youji SAKAGAMI, Hiroshi KAMA ...
    1999 Volume 63 Issue 12 Pages 2240-2243
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      In order to investigate the function of the peptidyl plant growth factor, phytosulfokine-α (PSK-α), in plants, we examined the effect of PSK-α on the growth and chlorophyll content of Arabidopsis seedlings under high night-time temperature conditions. Although exposure to high night-time temperatures markedly reduced the fresh weight and chlorophyll content of the seedlings, these parameters in the plants supplied with PSK-α remained at the same levels as those of non-treated controls. These effects were not apparent when [2-5]PSK, Tyr-SO3H and kinetin were similarly supplied. The results suggest that PSK-α not only promotes cell proliferation, but may aid plants in their tolerance of heat stress.
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  • Eiji TAKAHASHI, Kiyoshi ITO, Tadashi YOSHIMOTO
    1999 Volume 63 Issue 12 Pages 2244-2247
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The L-amino acid degrading enzyme gene from Proteus vulgaris was cloned and the nucleotide sequence of the enzyme gene was clarified. An open reading frame of 1,413 bp starting at an ATG methionine codon was found, which encodes a protein of 471 amino acid residues, the calculated molecular weight of which is 51,518. The amino acid sequence of P. vulgaris was 58.6% identical with the L-amino acid deaminase of P. mirabilis. A significantly conserved sequence was found around the FAD-binding sequence of flavo-proteins. The partially purified wild and recombinant enzymes had the same substrate specificity for L-amino acids to form the respective keto-acids, however not for D-amino acids.
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Food & Nutrition Science Regular Papers
  • Hiroshi TAKII, Kengo ISHIHARA, Takashi KOMETANI, Shigetaka OKADA, Tohr ...
    1999 Volume 63 Issue 12 Pages 2045-2052
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      We investigated the ergogenic effect in mice of administering highly branched cyclic dextrin (HBCD), a new type of glucose polymer, on the swimming endurance in an adjustable-current swimming pool. Male Std ddY mice were administered a HBCD, a glucose solution or water via a stomach sonde 10 min before, 10 min after or 30 min after beginning swimming exercise, and were then obliged to swim in the pool. The total swimming period until exhaustion, an index of the swimming endurance, was measured. An ergogenic effect of HBCD was observed at a dose of 500 mg/kg of body weight, whereas it had no effect at a dose of 166 mg/kg of body wt (p<0.05). The mice administered with the HBCD solution 10 min after starting the exercise were able to swim significantly longer (p<0.05) than the mice who had ingested water or the glucose solution. The rise in mean blood glucose level in the mice administered with HBCD, which was measured 20 min after starting swimming, was significantly lower (p<0.05) than that in the mice administered with glucose, although it was significantly higher (p<0.05) than that in the mice administered with water. The mean blood insulin rise in the mice given HBCD was significantly lower (p<0.05) than that in the mice given glucose. The mice administered with HBCD 30 min after starting the exercise swam significantly longer (p<0.05) than the mice who had ingested water, although the enhancement of swimming time was similar to that of the glucose-ingesting mice. The gastric emptying rate of the HBCD solution was significantly faster (p<0.05) than that of the glucose solution. However, this glucose polymer must have spent more time being absorbed because it has to be hydrolyzed before absorption, reflecting a lower and possibly longer-lasting blood glucose level. We conclude that the prolongation of swimming endurance in mice administered with HBCD depended on its rapid and longer-lasting ability for supplying glucose with a lower postprandial blood insulin response, leading to a delayed onset of fatigue.
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  • Hidehiko IZUMI, Minoru SUGIYAMA, Tsukasa MATSUDA, Ryo NAKAMURA
    1999 Volume 63 Issue 12 Pages 2059-2063
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The 16-kDa rice allergen, RA17, belonging to the α-amylase/trypsin inhibitor family was isolated from rice seed and structurally characterized by identifying cystine-containing peptides and predicting the secondary structure and hydrophobic regions. Eight peptides, which constitute three sets of cystine-containing peptides, were purified by HPLC from a thermolytic digest of RA17 and identified by their amino acid sequence and composition, indicating five intramolecular disulfide bridges: Cys34-Cys94, Cys26-(Cys50 or Cys51)-Cys110 and Cys12-(Cys62 or Cys64)-Cys122. Analyses of the CD spectrum and the Chou-Fasman prediction suggested that RA17 had some helical- and sheet-structure regions. Based on these experimental and predicted data, RA17 is proposed to be a globular molecule with a small hydrophobic core having folding restricted by five intramolecular disulfide bridges.
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  • Mi-Ryung KIM, Sang-Yun CHOI, Chan-Shick KIM, Chan-Wha KIM, Shigeru UTS ...
    1999 Volume 63 Issue 12 Pages 2069-2074
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The cDNA encoding A1aB1b proglycinin was expressed in E. coli, for the efficient isolation of a single peptide responsible for the bitterness. The 55-kD proglycinin was highly purified, hydrolyzed, and further purified through a series of chromatographic steps to yield fractions with the major bitter peptides. The most bitter-tasting fractions contained peptides with average molecular weights lower than 1,700 Da. An analysis of the amino acid sequences indicated that many small bitter peptides (<1,000 Da) are composed of uncharged polar amino acids as well as hydrophobic amino acids, with a charged residue often being present at either end. This suggests the involvement of a certain structural requirement in taste perception.
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  • Hitoshi FURUTA, Junko TOBE, Ryosuke KIWATA, Hirokazu MAEDA
    1999 Volume 63 Issue 12 Pages 2075-2081
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      We studied the rheological properties of a heat-reversible gel formed with water-soluble soybean polysaccharides (RG-SSPS) which had been extracted from purified polysaccharides of soybean cotyledon meal at pH 2 and 80°C for 90 min. It was found that the gelling point and melting point of this heat-reversible gel were at approximately 51°C and 46°C, respectively. Gelation was observed in aqueous solutions with a concentration of over 3%, but not at 1%, at 5°C. The influence of pH on the gel strength of RG-SSPS was strong. Especially on the acidic side, the G′ and G″ values, which were highest at pH 7, were remarkably low and no gel was formed at pH 5. The addition of EDTA lowered the G′ and G″ values, while the addition of Ca raised them. These findings and the analytical results for the fractions obtained from RG-SSPS after reheating at pH 5 and 120°C for 90 min, under which conditions the non-gel-forming polysaccharides were extracted, suggest that gelation was due to weak bonding and cross-linking between polyvalent cations and low-methoxyl uronic acid regions in the homogalacturonic or rhamnogalacturonic main chain of the RG-SSPS structure. The results also suggest that the heat reversibility was due to weakness of the bonding which did not conform to the egg-box model, like the mechanism for low-methoxyl pectin or sodium alginate, because the neutral sugar side chains of the RG-SSPS structure are thought to limit their mutual proximity.
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  • Akira ASAI, Kiyotaka NAKAGAWA, Teruo MIYAZAWA
    1999 Volume 63 Issue 12 Pages 2118-2122
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Phospholipid hydroperoxides (PLOOH) in the plasma, red blood cells (RBC) and liver of mice were measured after dietary supplementation for one week (1% w/w of diet) with a turmeric extract (curcuminoid), hexane extract of rosemary, and supercritical CO2-extracted capsicum pigment (supplemented with α-tocopherol to prevent fading). A lower PLOOH level was found in RBC of the spice extract-fed mice (65-74% of the non-supplemented control mice). The liver lipid peroxidizability induced with Fe2+/ascorbic acid was effectively suppressed by dietary supplementation with the turmeric and capsicum extracts to mice. While no difference in the plasma lipids was observed, the liver triacylglycerol concentration of the turmeric extract-fed mice was markedly reduced to one-half of the level in the control mice. These findings suggest that these spice extracts could act antioxidatively in vivo by food supplementation, and that the turmeric extract has the ability to prevent the deposition of triacylglycerols in the liver.
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  • Michiko SHIMODA, Yuri INOUE, Norihiro AZUMA, Choemon KANNO
    1999 Volume 63 Issue 12 Pages 2123-2129
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      To understand local antibody production to dietary protein antigens in the gut, the reactivity of the monoclonal antibodies (mAbs) from Peyer’s patches of BALB/c mice raised against orally administered hen egg lysozyme (HEL) was studied. These mAbs were of IgG1 (7 clones), IgA (5 clones) and IgM (13 clones) isotypes. Some of the HEL-binding mAbs preferentially reacted with reduced, carboxy-methylated HEL, rather than with native HEL. MAbs of the IgA and IgM isotypes had cross-reactivity with other unrelated environmental antigens such as E. coli, single-strand DNA, and soluble components of mouse food. In contrast, the IgG1 mAbs did not cross-react with these antigens. The average of the Kd values for HEL of these mAbs was in the order of 10-6 M, which is moderately higher than those of mAbs from the preimmune repertoire. These results suggest that, under normal physiological conditions, orally administered dietary proteins predominantly induce the local production of polyreactive IgA/IgM antibodies cross-reacting with environmental luminal antigens.
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  • Koji TAKAHASHI, Yoshikadzu NAKATA, Kenji SOMEYA, Makoto HATTORI
    1999 Volume 63 Issue 12 Pages 2144-2149
    Published: 1999
    Released: March 08, 2005
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      Pepsin-solubilized elastin (PSE)-conjugated collagen film was prepared from a collagen matrix with PSE by drying it and crosslinking the constituents with water-soluble carbodiimide or microbial transglutaminase to improve the physical properties of the collagen film. The crosslinking reduced the solubility and improved the thermal stability, the thermal transition properties, and the elasticity of the control film in water. In particular, water-soluble carbodiimide strongly influenced these properties. The PSE-conjugated collagen film showed good permeation by water-soluble tasting substances such as oligosaccharides and amino acids, but poor permeation by polysaccharide, protein, and hydrophobic substances such as retinol and cholesterol.
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  • Koji YAMADA, Yoko TOKUNAGA, Atsushi IKEDA, Ken-ichi OHKURA, Soichi MAM ...
    1999 Volume 63 Issue 12 Pages 2163-2167
    Published: 1999
    Released: March 08, 2005
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      The dietary effect of the water-soluble dietary fibers (WSDF), guar gum, partially hydrolyzed guar gum (PHGG), glucomannan, highly methoxylated (HM) pectin, on the serum lipid level and immunoglobulin (Ig) production of Sprague-Dawley rats was compared with that of water-insoluble cellulose. Although serum total cholesterol and triglyceride levels were significantly lower in the rats fed with WSDF than in those fed with cellulose, a decrease in the level of phospholipids was only observed in the rats that had been fed on guar gum or glucomannan. In addition, all WSDF feeding enhanced IgA productivity in the spleen and mesenteric lymph node lymphocytes, although the increase in serum IgA level was only observed in the rats fed on WSDF, and not on PHGG. When mesenteric lymph node lymphocytes were cultured in the presence of various concentrations of guar gum or glucomannan, no significant increase in Ig production was apparent. These data suggest that WSDF indirectly enhanced the Ig production of lymphocytes, and that serum lipid reduction and IgA production-enhancing activities of WSDF were dependent on their molecular sizes.
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  • Minji KIM, Takashi KOMETANI, Shigetaka OKADA, Makoto SHIMUZU
    1999 Volume 63 Issue 12 Pages 2183-2188
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The intestinal permeability to hesperidin glycosides was investigated by using a cultured monolayer of Caco-2 as a model for the small intestinal epithelium. Hesperidin glycosides were added to the apical side of the monolayer, and the substances that permeated to the basolateral side were determined by HPLC. Whereas hesperidin did not permeate across the Caco-2 monolayer, probably owing to its low solubility, the hesperidin glycosides did permeate. The transepithelial transport of hesperidin glycosides occurred in time- and dose-dependent manners. The transport was observed to be energy-independent, and was inversely correlated with the transepithelial electrical resistance (TEER) of the monolayer. These results suggest that hesperidin glycosides permeate across the Caco-2 cell monolayer via the paracellular pathway.
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Food & Nutrition Science Notes
Food & Nutrition Science Preliminary Communication
Microbiology & Fermentation Technology Regular Papers
  • Ssang-Goo CHO, Yong-Jin CHOI
    1999 Volume 63 Issue 12 Pages 2053-2058
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Catabolite repression of the Bacillus stearothermophilus No. 236 xynA gene, encoding an extracellular xylanase, was investigated in this work. Expression of the xynA gene in the B. stearothermophilus strain was found to be subject to glucose catabolite repression, and the level of repression was about 50-fold when the relative amounts of xynA transcript synthesized on different carbon sources were analyzed. The experiments with the B. subtilis MW15 strains carrying plasmids containing the xynA::aprA fusion gene showed that the cloned xynA gene did not require any specific carbon source for its induction. Nevertheless, the expression of the cloned gene was repressed by the presence of glucose. From the nucleotide sequence of the cloned xynA gene, we found two potential catabolite responsive elements (cre) within its reading frame region (cre-1: nucleotides +160 to +173 and cre-2: +173 to +186). Furthermore, by using various deletion derivatives of the xynA::aprA fusion plasmid (pMGW23), we suggested that only the cre-2 element might play a role in the glucose catabolite repression. Repression level of the xynA gene expression in the recombinant B. subtilis strain was estimated to be about 3-fold by analysis of the amounts of xynA transcript.
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  • Kuniho NAKATA, Ryuichiro KURANE
    1999 Volume 63 Issue 12 Pages 2064-2068
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Klebsiella pneumoniae H12 produced a newly identified extracellular polysaccharide in an ethanol medium with a yield of 3.0 g/l. The molar composition of the polysaccharide was 56.04% galactose, 25.92% glucose, 10.92% galacturonic acid, 3.71% mannose, and 3.37% glucuronic acid. The addition of 0.5%-1.5% NaCl increased production. The polysaccharide flocculated with kaolin clay in suspension at the concentration of 1 ppm in a 300-ppm solution of CaCl2. Almost all bacterial species cells aggregated in the polysaccharide solution. The ability to flocculate with kaolin clay changed with the pH and with the concentrations of coexisting cation and anion species. The polysaccharide flocculant may participate in in vivo bacterial aggregation or adherence to host organisms.
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  • Yoshie HASEGAWA, Tai TOKUYAMA, Hiroaki IWAKI
    1999 Volume 63 Issue 12 Pages 2091-2096
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      N,N-Dimethylformamidase (DMFase) from Alcaligenes sp. strain KUFA-1, a bacterium that can grow on N,N-dimethylformamide (DMF) as the sole carbon and nitrogen source, catalyzes the first step of the DMF degradation. The DMFase gene dmfA1A2 was cloned in Escherichia coli, and its nucleotides were sequenced. The deduced amino acid sequence of the enzyme consisted of two α- and two β-subunits with 132 and 762 amino acids, respectively, and had little similarity to sequences in protein databases, including various amidases. The protein may be a new kind of amidase. DMFase activity was detected in E. coli cells transformed with an expression plasmid of the cloned DMFase gene. The properties of recombinant DMFase purified from E. coli were identical to those of Alcaligenes DMFase.
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  • Lan JIANG, Yonghua YANG, Shampa CHATTERJEE, Bertolt SEIDEL, Gerald WOL ...
    1999 Volume 63 Issue 12 Pages 2097-2101
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      The expression of the proUK gene was improved by the coexpression of the argU gene cloned in a moderate copy number vector. As the proUK gene contains 2% AGG/AGA condons, which is much higher than the normal frequency in E. coli, about 0.14%-0.21%, the argU gene cloned in a multicopy plasmid was coexpressed with the proUK expression vector in our experiments. In E. coli strain BL21(DE3), IPTG is known to induce the expression of T7 RNA polymerase gene and this enzyme can transcribe the proUK gene under the control of the T7 promoter leading to expression of proUK. To replace IPTG by a cheaper alternative on a large scale, we constructed a plasmid in which the vgb promoter—which is known to be activated by the onset of hypoxic conditions—controls the T7RNA polymerase gene expression. Low oxygen conditions were then used to activate the vgb promoter causing T7RNA polymerase gene expression and finally leading to the expression of proUK as inactive inclusion bodies. Our experiments on a large scale in a bioreactor show that the expression of proUK accounts for about 30% of total protein after about 6 h of anaerobic cultivation, so the presented model represents an economical alternative to IPTG induction.
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  • Akihiko SAEKI, Kazunobu MATSUSHITA, Seiki TAKENO, Mariko TANIGUCHI, Hi ...
    1999 Volume 63 Issue 12 Pages 2102-2109
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      Several acetic acid bacteria of the genus Acetobacter oxidize much acetate oxidation, which is not desired in vinegar manufacturing. Acetobacter rancens SKU 1111, a strong acetate oxidant, grew rapidly with a biphasic growth curve while consuming acetate in the second growth phase. Acetobacter aceti IFO 3284 did not show extensive acetate oxidation. Addition of glycerol to the culture medium of Acetobacter rancens SKU 1111 increased acetate oxidation and resulted in more biomass in the second growth phase than when glycerol was not added. Enzyme activities of acetyl-CoA synthetase and phosphotransacetylase in the organism were high during acetate oxidation. The activity of phosphoenolpyruvate carboxylase was most stimulated by a trace amount of acetyl-CoA among the enzymes of glycerol catabolism. Phosphoenolpyruvate carboxylase in A. rancens SKU 1111 showed a sigmoidal saturation curve with acetyl-CoA. This finding suggested that strong acetate oxidation caused by acetyl-CoA synthetase or phosphotransacetylase activity, together with phosphoenolpyruvate carboxylase, increased the biomass.
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  • Osao ADACHI, Yoshitaka ANO, Duangtip MOONMANGMEE, Emiko SHINAGAWA, Hir ...
    1999 Volume 63 Issue 12 Pages 2137-2143
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      NADPH-Dependent L-sorbose reductase (SORD, synonimously NADP-dependent D-srobitol dehydrogenase) was purified and crystallized for the first time from the cytosolic fraction of Gluconobacter melanogenus IFO 3294. The enzyme catalyzed oxidoreduction between D-sorbitol and L-sorbose in the presence of NADP or NADPH. Affinity chromatography by a Blue-dextran Sepharose 4B column was effective for purifying the enzyme giving about 770-fold purification with an overall yield of more than 50%. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 3.8 s. Gel filtration on a Sephadex G-75 column gave the molecular mass of 60 kDa to the enzyme, which dissociated into 30 kDa subunit on SDS-PAGE, indicating that the enzyme is composed of 2 identical subunits. Reduction of L-sorbose to D-sorbitol predominated in the presence of NADPH with the optimum pH of 5.0-7.0. Oxidation of D-sorbitol to L-sorbose was observed in the presence of NADP at the optimum pH of 7.0-9.0. The relative rate of L-sorbose reduction was more than seven times higher to that of D-sorbitol oxidation. NAD and NADH were inert for both reactions. D-Fructose reduction in the presence of NADPH did not occur with SORD. Since the reaction rate in L-sorbose reduction highly predominated over D-sorbitol oxidation over a wide pH range, the enzyme could be available for direct enzymatic measurement of L-sorbose. Even in the presence of a large excess of D-glucose and other substances, oxidation of NADPH to NADP was highly specific and stoichiometric to the L-sorbose reduced. Judging from the enzymatic properties, SORD would contribute to the intracellular assimilation of L-sorbose incorporated from outside the cells where L-sorbose is accumulated in huge amounts in the culture medium.
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  • Tadao OIKAWA, Mayumi WATANABE, Hidemi MAKIURA, Hitoshi KUSAKABE, Kazuh ...
    1999 Volume 63 Issue 12 Pages 2168-2173
    Published: 1999
    Released: March 08, 2005
    JOURNALS FREE ACCESS
      We studied production of D-glutamate from L-glutamate using a bioreactor consisting of two columns of sequentially connected immobilized glutamate racemase (EC 5.1.1.3, from Bacillus subtilis IFO 3336) and L-glutamate oxidase (EC 1.4.3.11, from Streptomyces sp. X119-6): L-glutamate was racemized by the glutamate racemase column, and then L-glutamate was oxidized by the L-glutamate oxidase column. Consequently only D-glutamate remained, and was easily separated from the α-ketoglutarate formed by anion-exchange chromatography. Both enzymes were highly stabilized by immobilization. The pH and temperature optima of immobilized glutamate racemase (pH 8, 40°C) were similar to those of immobilized L-glutamate oxidase (pH 7, 50°C). Accordingly, we connected the two columns tandemly to do both enzyme reactions under the same conditions. Actually 4.5 μmol of D-glutamate was produced and isolated from 10 μmol of L-glutamate, about 90% of the theoretical yield.
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