Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 63 , Issue 1
Showing 1-46 articles out of 46 articles from the selected issue
Review
  • Takashi OHSHIRO, Yoshikazu IZUMI
    1999 Volume 63 Issue 1 Pages 1-9
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Sulfur removal from petroleum is important from the standpoint of the global environment because the combustion of sulfur compounds leads to the production of sulfur oxides, which are the source of acid rain. As the regulations for sulfur in fuels become more stringent, the existing chemical desulfurizations are coming inadequate for the “deeper desulfurization” to produce lower-sulfur fuels without new and innovative processes. Biodesulfurization is rising as one of the candidates. Several microorganisms were found to desulfurize dibenzothiophene (DBT), a representative of the organic sulfur compounds in petroleum, forming a sulfur-free compound, 2-hydroxybiphenyl. They are promising as biocatalysts in the microbial desulfurization of petroleum because without assimilation of the carbon content, they remove only sulfur from the heterocyclic compounds which is refractory to conventional chemical desulfurization. Both enzymological and molecular genetic studies are now in progress for the purpose of obtaining improved desulfurization activity of organisms. The genes involved in the sulfur-specific DBT desulfurization were identified and the corresponding enzymes have been investigated. From the practical point of view, it has been proved that the microbial desulfurization proceeds in the presence of high concentrations of hydrocarbons, and more complicated DBT analogs are also desulfurized by the microorganisms. This review outlines the progress in the studies of the microbial desulfurization from the basic and practical point of view.
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Analytical Chemistry Note
Organic Chemistry Regular Paper
Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Sonia Salgueiro MACHADO, Ute WANDEL, Jaap A. JONGEJAN, Adrie J. J. STR ...
    1999 Volume 63 Issue 1 Pages 10-20
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Resting cells of Acetobacter pasteurianus LMG 1635 (ATCC 12874) show appreciable enantioselectivity (E=16-18) in the oxidative kinetic resolution of racemic 2,3-epoxy-1-propanol, glycidol. Distinctly lower values (E=7-9) are observed for the ferricyanide-coupled oxidation of glycidol by the isolated quinohemoprotein alcohol dehydrogenase, QH-ADH, which is responsible for the enantiospecific oxidation step in whole cells. The accuracy of E-values from conversion experiments could be verified using complementary methods for the measurement of enantiomeric ratios. Effects of pH, detergent, the use of artificial electron acceptors, and the presence of intermediate aldehydes, could be accounted for. Measurements of E-values at successive stages of the purification showed that the drop in enantioselectivity correlates with the separation of QH-ADH from the cytoplasmic membrane. It is argued that the native arrangement of QH-ADH in the membrane-associated complex favors the higher E-values. The consequences of these findings for the use of whole cells versus purified enzymes in biocatalytic kinetic resolutions of chiral alcohols are discussed.
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  • Masayuki HARA, Jun MIYAKE, Yasuo ASADA, Hideo OHKAWA
    1999 Volume 63 Issue 1 Pages 21-28
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 μM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30°C.
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  • Hironori IWASAKI, Hirosuke OKU, Takayoshi TODA, Tetsuo NASU, Koji ODA, ...
    1999 Volume 63 Issue 1 Pages 29-34
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Apolipoprotein A-I (apo A-I) has an important role in the transport of cholesterol. This study describes the complete nucleotide and deduced amino acid sequence for apo A-I of LAP quail. A full length apo A-I cDNA clone for hyperlipidemia atherosclerosis prone (LAP) quail was isolated from a λgt10 liver cDNA library. The DNA sequence of LAP apo A-I cDNA was similar to that of normal Japanese quail. The deduced amino acid sequence of LAP apo A-I was hence identical to that of normal Japanese quail. LAP apo A-I mRNA is about 1.4 kilobases in length and expressed in a variety of tissues including small intestine, liver, lung, breast muscle, testis, and heart. Although the tissue distribution of apo A-I was similar between strains, LAP quail expressed more apo A-I mRNA than normal Japanese quail in all tissues examined. This tendency was pronounced with the small intestine. Although the concentration of serum apo A-I did not correlate with the tissue expression of mRNA, the observation may suggest that the increased apo A-I expression in LAP strain had some relevance to the susceptibility of this strain to the experimental atherosclerosis.
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  • Nirianne Q. PALACPAC, Yoshinobu KIMURA, Kazuhito FUJIYAMA, Toshiomi YO ...
    1999 Volume 63 Issue 1 Pages 35-39
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      The structures of N-linked sugar chains of glycoproteins expressed in tobacco BY2 cultured cells are reported. Five pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the glycoproteins by reversed-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were identified by two-dimensional PA-sugar chain mapping, ion-spray MS/MS analysis, and exoglycosidase digestions. The five structures fell into two categories; the major class (92.5% as molar ratio) was a xylose containing-type (Man3Fuc1Xyl1GlcNAc2 (41.0%), GlcNAc2Man3Fuc1Xyl1GlcNAc2 (26.5%), GlcNAc1Man3Fuc1Xyl1GlcNAc2 (21.7%), Man3Xyl1GlcNAc2 (3.3%)), and the minor class was a high-mannose type (Man5GlcNAc2 (7.5%)). This is the first report to show that α(1→3) fucosylation of N-glycans does occur but β(1→4) galactosylation of the sugar chains does not in the tobacco cultured cells.
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  • Osamu HATAMOTO, Hiroshi SEKINE, Eiichi NAKANO, Keietsu ABE
    1999 Volume 63 Issue 1 Pages 58-64
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      We cloned and analyzed the nucleotide sequence of a cDNA that encodes polyphenol oxidase (laccase) from the white-rot basidiomycete Schizophyllum commune. The nucleotide sequence of the full-length cDNA contained a 1554-base open reading frame that encoded a polypeptide of 518 amino acid residues, including a putative signal peptide of 16 residues. It contained four highly similar regions that are conserved in the deduced amino acid sequences of other laccases, including the region thought to be involved in copper binding. Aspergillus sojae strain 1860 (which has low protease levels) was transformed with the plasmid lacAL/pTPT, which contained the laccase gene under the control of the tannase promoter from Aspergillus oryzae. Laccase was secreted into the medium when transformants A1 and A2 were cultured in tannic acid-containing medium.
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  • So-Young KIM, Seung-Ho OHK, Dong-Hoon BAI, Ju-Hyun YU
    1999 Volume 63 Issue 1 Pages 73-77
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      To find a novel lytic enzyme against cariogenic Streptococci, strains showing strong lytic activity have been screened from soil using Streptococcus mutans. A strain identified as Bacillus licheniformis secreted two kinds of lytic enzymes, which were purified by methanol precipitation, CM-cellulose chromatography, gel filtration, and hydroxyapatite chromatography. The molecular weights of these two enzymes, L27 and L45, were 27,000 and 45,000, respectively. Optimum pH and temperature of both enzymes for lytic activity were pH 8 and 37°C. L27 and L45 digest the peptide linkage between L-Ala and D-Glu in peptidoglycan of Streptococcus mutans. The lytic activity was highly specific for Streptococcus mutans, suggesting their potential use as a dental care product.
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  • Hideki OISHI, Takahiro MORIMOTO, Yasuo WATANABE, Youichi TAMAI
    1999 Volume 63 Issue 1 Pages 83-90
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Phospholipase B (PLB) from the yeast Kluyveromyces lactis was purified to homogeneity from culture medium. The enzyme was highly glycosylated with apparent molecular mass of 160-250 kDa, and had two pH optima, at pH 2.0 and pH 7.5. At acidic pH the enzyme hydrolyzed all phospholipid substrates tested here without metal ion. On the other hand, at alkaline pH the enzyme showed substrate specificity for phosphatidylcholine and lysophosphatidylcholine and required Ca2+, Fe3+, or Al3+ for the activity. The alkaline activity was increased more than 20-fold in the presence of Al3+ compared to that in the presence of Ca2+.
      cDNA sequence of PLB (KlPLB) was analyzed by a combination of several PCR procedures. KlPLB encoded a protein consist of 640 amino acids and the deduced amino acid sequence showed 66.7% similarity with the T. delbrueckii PLB. The amino acid sequence contained the lipase consensus sequence (G-X-S-X-G) and the catalytic aspartic acid motif. Replacement of Arg-112 or Asp-406 with alanine caused loss of the enzymatic actiivity at both pH. These results suggested that PLB activity are dependent on a catalytic mechanism similar to that of cytosolic phospholipase A2.
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  • Yasuhiro IKENAKA, Hirokazu NANBA, Kazuyoshi YAJIMA, Yukio YAMADA, Masa ...
    1999 Volume 63 Issue 1 Pages 91-95
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      For the improvement of N-carbamyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of DCase from Agrobacterium sp. KNK712 was improved through various combinations of thermostability-related mutations. The thermostable temperature (defined as the temperature on heat treatment for 10 min that caused a decrease in the DCase activity of 50%) of the enzyme which had three amino acids, H57Y, P203E, and V236A, replaced was increased by about 19°C. The mutant DCase, designated as 455M, was purified and its enzymatic properties were studied. The enzyme had highly increased stability against not only temperature but also pH, the optimal temperature of the enzyme being about 75°C. The substrate specificity of the enzyme for various N-carbamyl-D-amino acids was changed little in comparison with that of the native enzyme. Enzymochemical parameters were also measured.
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  • Junshi SAKAMOTO, Akiko HAYAKAWA, Tomoko UEHARA, Shunsuke NOGUCHI, Nobu ...
    1999 Volume 63 Issue 1 Pages 96-103
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      The Bacillus stearothermophilus ctaA gene, which is required for heme A synthesis, was found upstream of the ctaBCDEF/caaEABCD gene cluster as in B. subtilis and B. firmus. The deduced protein sequence indicate that CtaA is a 35-kDa intrinsic membrane protein with seven hydrophobic segments. Alignment of CtaA sequences showed conserved residues including histidines that may be involved in heme B binding and substrate binding. Expression of ctaA in E. coli resulted in increased formation of a membrane-bound b-type cytochrome, heme A production, and severe growth inhibition. Furthermore, B. stearothermophilus CtaA produced in E. coli was found to catalyze the conversion of heme O to heme A in vitro.
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  • Aiko TSUZUKI, Tomoko TATEISHI, Naohito OHNO, Yoshiyuki ADACHI, Toshiro ...
    1999 Volume 63 Issue 1 Pages 104-110
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      It has been suggested that the immunopharmacological activity of soluble (1→3)-β-D-glucan depends on its conformation in mice. In this study, we examined the relationship between the conformation of Sonifilan (SPG) and hematopietic responses in cyclophosphamide (Cy)-induced leukopenic mice. SPG, a high molecular weight (1→3)-β-D-glucan, has a triple helical conformation in water, and it was changed by treatment with aqueous sodium hydroxide to the single helical conformer (SPG-OH). The effects of SPG or SPG-OH on hematopoietic responses in cyclophosphamide induced leukopenic mice were investigated by monitoring i) gene expression of cytokines by RT-PCR, ii) protein synthesis of interleukin 6 (IL-6) by ELISA and iii) colony formation of bone marrow cells (BMC). The mice administered Cy and SPG or SPG-OH expressed and produced higher levels of IL-6 mRNA and protein than the mice administered only Cy. Gene expression of NK1.1 was also induced by Cy/SPG (or SPG-OH) treatment. Induced gene expression of stem cell factor (SCF) and macrophage-colony stimulating factor (M-CSF) by SPG/SPG-OH were also found in in vitro culture of BMC from Cy treated mice. These results strongly suggested that conformation of the glucans, single and triple helix, are independent of the hematopietic response.
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  • Norio INOKUCHI, Shigeru SAITOH, Hiroko KOBAYASHI, Tadashi ITAGAKI, Tak ...
    1999 Volume 63 Issue 1 Pages 141-145
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5′-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3′-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U>C and A≥G>C>U, and A≥G, U>C and G>U>A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site.
      pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges.
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  • Zhifeng CUI, Dai HIRATA, Tokichi MIYAKAWA
    1999 Volume 63 Issue 1 Pages 162-167
    Published: 1999
    Released: March 19, 2005
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      The yeast gene SNQ2, which encodes a multidrug resistance ABC superfamily protein, is required for resistance to the mutagen 4-nitroquinoline N-oxide (4-NQO). The expression of the SNQ2 gene is under the control of a regulatory network that involves the transcription factor Yrr1p, as well as Pdr1p/Pdr3p (Cui et al., Mol. Microbiol., 29, 1307-1315 (1998)). By 5′-deletion analysis of the promoter by using SNQ2-lacZ fusion constructs, four regions: -745 to -639 (region I), -639 to -578 (region II), -548 to -533 (region III) and -533 to -485 (region IV) were found to be important for SNQ2 expression. Genetic analysis suggested that the site in region IV was responsible for the Yrr1p-mediated SNQ2 expression. A consensus motif known for the binding of Pdr1p/Pdr3p (PDRE) was not found in region IV.
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  • Koshiki MINO, Tsuyoshi YAMANOUE, Takaharu SAKIYAMA, Naoki EISAKI, Asah ...
    1999 Volume 63 Issue 1 Pages 168-179
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Incubation of serine acetyltransferase (SAT) from Escherichia coli at 25°C in the absence of protease inhibitors yielded a truncated SAT. The truncated SAT was much less sensitive to feedback inhibition than the wild-type SAT. Analyses of the N- and C-terminal amino acid sequences found that the truncated SAT designated as SATΔC20 was a resultant form of the wild-type SAT cleaved between Ser 253 and Met 254, deleting 20 amino acid residues from the C-terminus. Based on these findings, we constructed a plasmid containing an altered cysE gene encoding the truncated SAT. SATΔC20 was produced using the cells of E. coli JM70 transformed with the plasmid and purified to be homogeneous on an SDS-polyacrylamide gel. Properties of the purified SATΔC20 were investigated in comparison with those of the wild-type SAT and Met-256-Ile mutant SAT, which was isolated by Denk and Böck but not purified (J. Gen. Microbiol., 133, 515-525 (1987)). SATΔC20 was composed of four identical subunits like the wild-type SAT and Met-256-Ile mutant SAT. Specific activity, optimum pH for reaction, thermal stability, and stability to reagents for SATΔC20 were similar to those for the wild-type SAT and Met-256-Ile mutant SAT. However, SATΔC20 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A), a counterpart of the cysteine synthetase and did not reduce OASS activity in contrast to the wild-type SAT and Met-256-Ile mutant SAT.
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Biochemistry & Molecular Biology Notes
Food & Nutrition Science Regular Papers
  • Takehito MIURA, Yasuyoshi TAKAYAMA, Masuo NAKANO
    1999 Volume 63 Issue 1 Pages 40-45
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Fossil shellfish powder (FS) and Ezo giant scallop shell powder (EG) were rendered soluble with lactate and citrate under decompression (FSEx and EGEx, respectively) and we examined the effects of lactate-citrate solubilization of FS and EG on mineral absorption, tissue mineral contents, serum biochemical indices and bone mineral density (BMD) in ovariectomized (OVX) rats. The apparent absorption ratios of minerals tended to be high in the rats fed with the solubilized mineral sources, those in the FSEx group being significantly higher than in the FS group. There was no significant difference in the tibia mineral content among the OVX groups. BMD at the distal femoral diaphysis was significantly increased by FSEx and EGEx feeding. It is suggested that solubilization with lactate and citrate under decompression increased the solubility and bioavailability of calcium from such natural sources of shellfish calcium as FS and EG.
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  • Noriko MIYAKE, Miok KIM, Tadao KURATA
    1999 Volume 63 Issue 1 Pages 54-57
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      The effects of superoxide dismutase (SOD) and catalase on the autoxidation rate of L-ascorbic acid (ASA) in the absence of metal ion catalysts were examined. The stabilization of ASA by SOD was confirmed, and the enzyme activity of SOD, which scavenges the superoxide anion formed during the autoxidation of ASA, contributed strongly to this stabilization. The stabilization of ASA by catalase was observed for the first time; however, the specific enzyme ability of catalase would not have been involved in the stabilization of ASA. Such proteins as bovine serum albumin (BSA) and ovalbumin also inhibited the autoxidation of ASA, therefore it seems that non-specific interaction between ASA and such proteins as catalase and BSA might stabilize ASA and that the non-enzymatic superoxide anion scavenging ability of proteins might be involved.
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  • Hiroyuki SUGANUMA, Takahiro INAKUMA
    1999 Volume 63 Issue 1 Pages 78-82
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      The effects of dietary ingestion of tomato were studied in mice that had been made hypercholesterolemic by feeding atherogenic diets. Mice which had been fed on the atherogenic diet without tomato for 4 months had significantly increased plasma lipid peroxide, and the vaso-relaxing activity in the aorta induced by acetylcholine (ACh) was harmed when compared with mice fed on a common commercial diet. On the other hand, mice which had been fed on the atherogenic diet containing 20% (w/w) lyophilized powder of tomato showed less increase in the plasma lipid peroxide level, and ACh-induced vaso-relaxation was maintained at the same level as that in normal mice. These results indicate that tomato has a preventive effect on atherosclerosis by protecting plasma lipids from oxidation.
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  • Yosef ADAN, Kenichi SHIBATA, Masao SATO, Ikuo IKEDA, Katsumi IMAIZUMI
    1999 Volume 63 Issue 1 Pages 111-119
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Exogenously hypercholesterolemic (ExHC) rats were fed on an atherogenic diet supplemented with 1% each of either ethyl ester docosahexaenoic acid [EE-DHA, 22:6(n-3)], ethyl ester eicosapentaenoic acid [EE-EPA, 20:5(n-3)] or safflower oil (SO) for 6 months. The rats fed on the diets containing EE-EPA or EE-DHA, compared with those fed on SO, had lower serum cholesterol and triacylglycerol levels, less aggregation of platelets and slower progress of intimal thickening in the ascending aorta. Relative to the SO-fed rats, both of the (n-3) fatty acid-fed rats had a significantly reduced proportion of arachidonic acid in the platelet and aortic phospholipids, and lower production of thromboxane A2 by platelets and of prostacyclin by the aorta. These results suggest that EPA and DHA are similarly involved in preventing atherosclerosis development by reducing hypercholesterolemia and modifying the platelet functions.
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  • Pham HUNG, Shihoko KAKU, Shin-ichi YUNOKI, Ken-ichi OHKURA, Jiong-Yan ...
    1999 Volume 63 Issue 1 Pages 135-140
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      The dietary effect of fish oils (FOs) rich in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) on the immune function of Sprague-Dawley rats was compared with that of safflower oil. After 3 weeks of feeding at the 10% level of a dietary fat, the IgG and IgM production by splenocytes and IgG production by mesenteric lymph node (MLN) lymphocytes were significantly higher in the FO-fed rats, while no significant difference was found in IgA or IgE productivity by both the spleen and MLN lymphocytes. In the FO-fed rats, peritoneal exudate cells released a lower amount of LTB4, reflecting their lower arachidonic acid level, and a higher amount of LTB5, reflecting their higher EPA level in phospholipids. On these EPA-rich FO exerted a stronger effect than DHA-rich FO immune functions.
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  • Tai-Boong UHM, Mi Sun CHUNG, Sun Hee LEE, Françoise GOURRONC, I ...
    1999 Volume 63 Issue 1 Pages 146-151
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Endoinulinase from Aspergillus ficuum, which catalyzes the hydrolysis of inulin via an endo-cleavage mode, was purified by chromatography from Novozym 230 as a starting commercial enzyme mixture on CM-Sephadex and DEAE-Sepharose, and by preparative electrophoresis under native conditions. The enzyme was estimated to be pure on the basis of its I/S ratio, whose value was infinite in our assay conditions. Two forms separated by using this method. SDS gel electrophoresis showed the two purified forms to respectively exhibit molecular weights of 64,000±500 and 66,000±1,000. The results of deglycosylation indicated that the two forms were originally the same protein but with different sugar contents. A molecular weight of 54,800±1,500 was found by gel filtration of the native enzyme, indicating the native functional protein to be a monomer. The enzyme showed nearly absolute substrate specificity towards inulin and inulooligosaccharides, and acted via an endo-attack to produce mainly inulotriose during the late stage of the reaction. The apparent Km and Vmax values for inulin hydrolysis were 8.1±1.0 mM and 773±60 U/mg, respectively. The internal peptides of the enzyme showed sequence homology to the endoinulinase of Penicillium purpurogenum.
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Food & Nutrition Science Notes
  • Hye-Young KIM, Jong Hwan OH
    1999 Volume 63 Issue 1 Pages 184-188
    Published: 1999
    Released: March 19, 2005
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      Naturally occurring substances which can prevent and treat diabetic complications were sought by examining ethanol extracts prepared from Korean forest plants for their inhibitory effects on rat lens aldose reductase activity in vitro. Among the plants examined, Acer ginnala, Illicium religiosum and Cornus macrophylla exerted the most strong inhibitory activity on aldose reductase.
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  • Michihiro FUKUSHIMA, Tetsu OHASHI, Mitsuo SEKIKAWA, Masuo NAKANO
    1999 Volume 63 Issue 1 Pages 202-205
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      The hypocholesterolemic efficacy of various animal oils was compared in rats given a cholesterol-enriched diet. After acclimatization for one week, male F344 DuCrj rats (8 weeks of age) that had been fed with a conventional diet were assigned to diets containing 5% of oil from emu (Dromaius), Japanese Sika deer (Cervus nippon yesoensis, Heude), sardine, beef tallow, or lard with 0.5% cholesterol for 6 weeks. After this feeding period, the concentrations of serum total cholesterol and of very-low-density lipoprotein+intermediate-density lipoprotein+low-density lipoprotein-cholesterol in the sardine oil group were significantly lower than those in the other groups. The serum high-density lipoprotein-cholesterol concentration in the Japanese Sika deer oil group was significantly higher than that in the other groups. The atherosclerotic index and liver cholesterol concentration in the sardine oil and Japanese Sika deer oil groups were significantly lower than those in the other groups. The fecal cholesterol excretion by the Japanese Sika deer oil group was significantly higher than that of the other groups, except for the sardine oil group, and the fecal bile acid excretion by the sardine oil group was significantly higher than that of the other groups, except for the lard group. These results suggest that Japanese Sika deer oil reduced the atherosclerotic index and liver cholesterol concentration in the presence of excess cholesterol in the diet as well as sardine oil did by increasing the excretion of cholesterol from the intestines of rats.
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  • Kenjiro TADERA, Kazutaka ODA, Chikako NAKAHARA
    1999 Volume 63 Issue 1 Pages 213-215
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      A type of vitamin B6 conjugates (B6X), which liberates free vitamin B6 by alkaline and successive β-glucosidase hydrolyses, is known to occur in rice bran and wheat bran. Conflicting experimental results, however, have been reported about the occurrence of B6X in soybeans. This study afforded evidence for B6X occuring in soybeans: certainly a highly purified B6X preparation from whole soybeans liberated pyridoxine when it was treated with alkali followed by β-glucosidase hydrolysis, and 5′-O-(β-D-glucopyranosyl)pyridoxine by alkali treatment alone. The B6X content varied with cultivars, of which a certain kind contained no B6X.
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  • Yoshio OZAWA, Yasushi UDA, Hiroki MATSUOKA, Masako ABE, Shunro KAWAKIS ...
    1999 Volume 63 Issue 1 Pages 216-219
    Published: 1999
    Released: March 19, 2005
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      Stereoisomers of the tetrahydro-β-carboline derivative, 1-(2′-pyrrolidinethione-3′-yl)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (PTCC), were formed from L-tryptophan with 4-methylthio-3-butenyl isothiocyanate, and their mutagenic properties and contents in different types of the radish products were studied. The isomers were identified as (1S*, 3S*, 3′R*)- and (1R*, 3S*, 3′R*)-PTCCs; the former was found as the major compound but had no mutagenic activity, while the latter was mutagenic toward Salmonella typhimurium TA 98 in the presence of a rat microsomal fraction. Both (1S*, 3S*, 3′R*)- and (1R*, 3S*, 3′R*)-PTCC were detected in a ratio of about 4:1 in a product fermented for 8 months, but only a trace was apparent in products manufactured within a few weeks.
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  • Hitoshi KUMAGAI, Tomohide MATSUNAGA, Tomoaki HAGIWARA
    1999 Volume 63 Issue 1 Pages 223-225
    Published: 1999
    Released: March 19, 2005
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      The fractal dimension, Df, of aggregates in a dilute BSA system with added salt was evaluated by static light scattering (SLS). A fractal structure was observed for the system with NaCl addition. The values of Df increased with increasing heating time and ionic strength. The values of Df were larger than those (Df=1.8 or 2.1) predicted by the conventional cluster-cluster aggregation model, probably due to a “restructuring” of aggregates during the aggregation process. On the other hand, a fractal structure was not apparent for the system with added CaCl2.
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Food & Nutrition Science Preliminary Communication
Microbiology & Fermentation Technology Regular Papers
  • Akira ASAKURA, Tatsuo HOSHINO
    1999 Volume 63 Issue 1 Pages 46-53
    Published: 1999
    Released: March 19, 2005
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      Gluconobacter oxydans DSM 4025 effectively oxidizes L-sorbose to 2-keto-L-gulonic acid (2 KGA), an industrial precursor of vitamin C. From this microorganism, we purified the enzyme involved in this oxidation reaction. The enzyme is a unique quinoprotein dehydrogenase catalyzing not only the conversion of L-sorbose to L-sorbosone, but also that of L-sorbosone to 2 KGA. The molecular weight of the enzyme was about 135,000, consisting of two subunits with molecular weights of 64,500 and 62,500. As its prosthetic group, non-covalently bound PQQ was found. The dye-linked spectrophotometric enzyme assay showed that the optimum enzyme activity occurred in the pH range about 7.0-9.0, and the enzyme activity was inhibited by EDTA or EGTA. The enzyme showed extremely broad substrate specificity for primary and secondary alcohols, aldehydes, aldoses, ketoses, and other sugar alcohols, but not for methanol or formaldehyde. The cytochrome c obtained from the soluble fraction of this strain was found to act as a physiological electron acceptor of the enzyme.
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  • Tohru KOBAYASHI, Kenzo KOIKE, Tadashi YOSHIMATSU, Norihiko HIGAKI, Ats ...
    1999 Volume 63 Issue 1 Pages 65-72
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73S. It was a basic protein with an isoelectric point of pH 10.3, and the α-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55°C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.
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  • Noriyuki KITAMOTO, Hideki OKADA, Shoko YOSHINO, Kunio OHMIYA, Norihiro ...
    1999 Volume 63 Issue 1 Pages 120-124
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      A gene (pmeA) encoding pectin methylesterase was isolated from a shoyu koji mold, Aspergillus oryzae KBN616, and characterized. The structural gene comprised 1,370 bp with six introns. The PMEA protein consisted of 331 amino acids with a putative signal peptide of 17 amino acids. The deduced amino acid sequence was very similar to those of Aspergillus niger PMEA and Aspergillus aculeatus PME1. The pmeA gene was efficiently expressed under control of the A. oryzae TEF1 gene promoter for purification and characterization of the ezymatic properties. PMEA had a molecular mass of 38.5 kDa, a pH optimum of 5.0, and a temperature optimum of 55°C.
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  • Ivo FRÉBORT, Kazunobu MATSUSHITA, Hirohide TOYAMA, Karel LEMR, ...
    1999 Volume 63 Issue 1 Pages 125-134
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.
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  • Junichi KATO, Tetsuya NAKAMURA, Akio KURODA, Hisao OHTAKE
    1999 Volume 63 Issue 1 Pages 155-161
    Published: 1999
    Released: March 19, 2005
    JOURNALS FREE ACCESS
      Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC3 and PC4, were selected by the swarm plate method after N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. These mutants were not complemented by the P. aeruginosa cheY and cheZ genes, which had been previously cloned (Masduki et al., J. Bacteriol., 177, 948-952, 1995). DNA sequences downstream of the cheY and cheZ genes were able to complement PC3 but not PC4. Sequence analysis of a 9.7-kb region directly downstream of the cheZ gene found three chemotaxis genes, cheA, cheB, and cheW, and seven unknown open reading frames (ORFs). The predicted translation products of the cheA, cheB, and cheW genes showed 33, 36, and 31% amino acid identity with Escherichia coli CheA, CheB, and CheW, respectively. Two of the unknown ORFs, ORF1 and ORF2, encoded putative polypeptides that resembled Bacillus subtilis MotA (40% amino acid identity) and MotB (34% amino acid identity) proteins, respectively. Although P. aeruginosa was found to have proteins similar to the enteric chemotaxis proteins CheA, CheB, CheW, CheY, and CheZ, the gene encoding a CheR homologue did not reside in the chemotaxis gene cluster. The P. aeruginosa cheR gene could be cloned by phenotypic complementation of the PC4 mutant. This gene was located at least 1,800 kb away from the chemotaxis gene cluster and encoded a putative polypeptide that had 32% amino acid identity with E. coli CheR.
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Microbiology & Fermentation Technology Notes
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