Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 63 , Issue 10
Showing 1-37 articles out of 37 articles from the selected issue
Review
  • Tanetoshi KOYAMA
    1999 Volume 63 Issue 10 Pages 1671-1676
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Multiple alignments of primary structures of many kinds of prenyltransferases that participate in the most fundamental prenyl-chain backbone synthesizing process in isoprenoid biosynthesis showed seven conserved regions in the primary structures of (E)-prenyl diphosphate synthases. However, no information has been available about the structures of (Z)-prenyl diphosphate synthases until our recent isolation of the gene for the undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26.
      The amino acid sequence of the (Z)-prenyl diphosphate synthase is totally different from those of (E)-prenyl chain elongating enzymes. Protein data base searches for sequences similar to that of the undecaprenyl diphosphate synthase yielded many unknown proteins which have not yet been characterized. Two of the proteins have recently been identified as the undecaprenyl diphosphate synthase of Escherichia coli and the dehydrodolichyl diphosphate synthase of Saccharomyces cerevisiae, indicating that there are three highly conserved regions in the primary structure of (Z)-prenyl chain elongating enzymes.
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Organic Chemistry Regular Papers
  • Masahiro MIYASHITA, Miki AKAMATSU, Hideki UENO, Yoshiaki NAKAGAWA, Kei ...
    1999 Volume 63 Issue 10 Pages 1684-1690
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      The activities of a series of RGD mimetics, which contained a variety of cationic structures, for the inhibition of platelet aggregation and fibrinogen-receptor binding were measured. The stability of the coulombic ion-pairing complex of the model compounds with the acetate anion as a model for the receptor was calculated in terms of the ionic interaction energy. The results suggest that stability is one of the significant factors which govern the inhibitory potency of fibrinogen-receptor binding. The distance between cationic and anionic groups might also affect the potency. A compound which contained an amidinophenyl structure as the cationic moiety showed exceptionally high inhibitory activity, suggesting that some other factors, in addition to coulombic interaction and the distance, affect the potency.
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  • Kensuke NABETA, Masateru YAMAMOTO, Hiroyuki KOSHINO, Hiroki FUKUI, Yuk ...
    1999 Volume 63 Issue 10 Pages 1772-1776
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      The absolute configurations of the rare sesquiterpenes, kelsoene and prespatane, were determined to be (1S, 2R,5S, 6S, 7R, 8R)-2,8-dimethyl-6-(1-methylethenyl)tricyclo[5.3.0.02,5] decane (IUPAC name) and (1R, 2S, 5R, 6R, 7R, 8S)-1,5-dimethyl-8-(1-methylethenyl) tricyclo-[5.3.0.02,6] decane, respectively, on the basis of the observed chemical shifts and NOEs in NOESY and NOEDS experiments after conversion with the chiral reagent, 2′-methoxy-1,1′-binaphthalene-2-carbohydroximoyl chloride (MBCC). The absolute configurations of kelsoene and prespatane thus determined suggest that initial cyclization of FPP from the si-face at C-10 to form a 10R-germacradienyl cation leads to kelsoene, while that from the re-face leads to prespatane via the 10S-germacradienyl cation.
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  • Mariko SHIBATANI, Yasuyuki HASHIDOKO, Satoshi TAHARA
    1999 Volume 63 Issue 10 Pages 1777-1780
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Isohemigossypolone (1) and 2-O-methylisohemigossypolone (2), major fungitoxins of Pachira aquatica, were found to accumulate locally in the outer bark of the swollen trunk, whereas the inner bark and heartwood contained only a trace amount of them. From P. aquatica that was infected with a phytopathogenic bacterium, we detected significant amounts of 1 and 2 from browned inner tissues of the swollen trunk. According to a quantitative analysis by a gas-chromatograph, the concentration of 1 in the diseased inner tissues was calculated to be approximately 780 μg/g f.w., which was the same level as that in the outer bark of healthy individuals. These findings suggest that the inner tissues inducibly produced and accumulated antifungals 1 and 2 during infection events, as do many plants with phytoalexins. 11-Nor-2-O-methylisohemigossypolone (3), showing approximately equivalent fungitoxic activity to that of 1 and 2, was also isolated from the infected inner tissues. We screened pathogenic bacteria from the infected tissue, and isolated a rod-shaped bacterium that was tentatively identified as Pseudomonas sp. which promoted tissue-browning on sectioned disks of P. aquatica trunks.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Kenichi HAMAYASU, Koki FUJITA, Kozo HARA, Hitoshi HASHIMOTO, Toshiko T ...
    1999 Volume 63 Issue 10 Pages 1677-1683
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Novel heterobranched cyclodextrins (CDs), N-acetylglucosaminyl-cyclodextrins (GlcNAc-CD), were synthesized from a mixture of GlcNAc and α, β, or γCD by the reverse reaction of N-acetylhexosaminidase from jack bean. Optimum pH and temperature for the production of GlcNAc-αCD by N-acetylhexosaminidase were pH 4.9 and 50-70°C, respectively. The maximum yield of GlcNAc-αCD was 17.5% (mol/mol) at the concentration of 1 M GlcNAc and 0.25 M αCD. The reverse reaction product, GlcNAc-αCD, was separated into two peaks by HPLC analysis on the ODS column. Their structures were identified as 6-O-β-D-N-acetylglucosaminyl-αCD and 2-O-β-D-N-acetylglucosaminyl-αCD by FAB-MS and NMR spectroscopies. N-Acetylhexosaminidase from jack bean also synthesized N-acetylgalactosaminyl-αCD from N-acetylgalactosamine and αCD.
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  • Tang DAHAI, Shoji ANDO, Yozo TAKASAKI, Jutaro TADANO
    1999 Volume 63 Issue 10 Pages 1703-1707
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Site-directed mutagenesis by inverse PCR was done on the HindIII gene. Target residues to be mutated were chosen according to (i) the fact that a mutant obtained by sodium nitrite treatment showed almost no HindIII activity, where Asp-123 was replaced with Asn, and (ii) the model proposed by Stahl et al. (Stahl, F., Wende, W., Jeltsch, A. and Pingoud, A. Biol. Chem. 379, 467-473 (1998)). Sevenkinds of mutants were obtained by the PCR, and their enzymatic and biochemical properties were examined. Threemutants, P50S, D108L, and D123N, showed fairly low HindIII activity. On the other hand, the other four, P84Q, E86K, V106E, and K125N, retained the activity. In particular, E86K showed higher activity than the wild enzyme. This fact was confirmed when activities of the purified wild and E86K enzymes were assayed. These results coincided fairly well with data using E. coli strains that carry the respective mutant plasmids, on their resistance to phage T7 and on growth rate. We conclude that the PE motif at residues 50 and 51, and DXK motif at residues 108-110, are responsible for the enzymic reaction of HindIII.
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  • Kunihiko MORIYOSHI, Takashi OHMOTO, Tatsuhiko OHE, Kiyofumi SAKAI
    1999 Volume 63 Issue 10 Pages 1708-1713
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      An esterase catalyzing the hydrolysis of acetyl ester moieties in cellulose acetate was purified 1,110-fold to electrophoretic homogeneity from the culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The purified enzyme was a monomeric protein with a molecular mass of 40 kDa and the isoelectric point was 5.3. The pH and temperature optima of the enzyme were 8.0-8.5 and 45°C. The enzyme catalyzed the hydrolysis of acetyl saccharides, p-nitrophenyl esters of short-chain fatty acids, and was slightly active toward aliphatic and aromatic esters. The Km and Vmax for cellulose acetate (degree of substitution, 0.88) and p-nitrophenyl acetate were 0.0162% (716 μM as acetyl content in the polymer) and 36.0 μM, and 66.8 and 39.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, which indicated that the enzyme was a serine esterase.
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  • Shin-ichi KASHIWABARA, Sosaku OGAWA, Naoko MIYOSHI, Masayo ODA, Yuzuru ...
    1999 Volume 63 Issue 10 Pages 1736-1748
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      The gene that coded for a cellular pullulanase of type I (α-dextrin 6-glucanohydrolase, EC 3.2.1.41) in Bacillus flavocaldarius KP1228 (FERM-P9542) cells growing at 51 to 82°C was expressed in Escherichia coli MV1184. The enzyme had a half-life of 10 min at 107°C. Purification of the enzyme and its characterization showed that the enzyme was identical with the native one. Its primary structure of 475 residues with a molecular weight of 53,856 deduced from the gene was 15-21% and 43% identical to the corresponding C-terminal regions in the sequences of 2 plant and 6 bacterial pullulanases of type I, and of Bacillus stearothermophilus TRS40 neoplullulanase, respectively.
      Sequence analysis showed that B. flavocaldarius pullulanase comprised 3 domains, i.e., one catalytic (β/α)8-barrel domain, one domain made of the region protruding from the barrel between the third β-strand and the third α-helix, and one β-stranded domain attached to the C-end of the barrel domain, but that the pullulanase lacked the β-stranded domain commonly found in addition to the 3 domains in the neopullulanase and all other pullulanases, and attached to the N-end of the barrel domain.
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  • Keiji NAKAJIMA, Yoko OSHITA, Masahiko KAYA, Yasuyuki YAMADA, Takashi H ...
    1999 Volume 63 Issue 10 Pages 1756-1764
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      In the biosynthesis of tropane alkaloids, two tropinone reductases (TRs) catalyze reduction of tropinone to different stereoisomers, tropine and pseudotropine. Two TRs from Hyoscyamus niger have 64% of identical amino acids and hence a common evolutionary origin. In this study, genomic clones of TRs were isolated from H. niger. Their sequence comparison showed that although they have the same exon/intron organization, sequence similarity was restricted to the coding regions. In H. niger transgenic hairy roots, both TR promoters activated transcription of the reporter genes in endodermis and pericycle of the roots. A quantitative reporter assay and a nuclear run-on experiment indicated that the two genes are transcribed at a similar rate. The results indicate that although different activity levels have been observed for the TR enzymes in the H. niger root, the TR genes per se conserve similar tissue-specific expression pattern and transcriptional rate.
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  • Masae HORINOUCHI, Takako YOSHIDA, Hideaki NOJIRI, Hisakazu YAMANE, Tos ...
    1999 Volume 63 Issue 10 Pages 1765-1771
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      In our previous study, the multicomponent monooxygenase DsoABCDEF in Acinetobacter sp. strain 20B was cloned based on its ability to oxidize dimethyl sulfide (DMS) to dimethyl sulfoxide (DMSO) in E. coli cells, which had high sequence similarity with phenol hydroxylase MopKLMNOP in Acinetobacter calcoaceticus NCIB8250, DmpKLMNOP in Pseudomonas sp. CF600 and some other multicomponent monooxygenases. In this study, DsoB, C, D, E, and F were found to be needed for DMS-oxidizing activity in polypeptide requirement experiments, while DsoA was not necessary for it. It was also found that complementation of the deletion mutants lacking DsoC or F with the corresponding Dmp polypeptides supported the DMS-oxidizing activity, while complementation of the deletion mutants lacking any of the oxygenase subunits (DsoB, D, or E) with the corresponding Dmp polypeptides reduced or nullified the activity.
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Biochemistry & Molecular Biology Notes
Biochemistry & Molecular Biology Preliminary Communications
  • Yumiko TAKATSUKA, Toshio TOMITA, Yoshiyuki KAMIO
    1999 Volume 63 Issue 10 Pages 1843-1846
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Lysine decarboxylase (LDC, EC 4.1.1.18) from Selenomonas ruminantium has decarboxylating activities towards both L-lysine and L-ornithine with similar Km and Vmax. Here, we identified four amino acid residues that confer substrate specificity upon S. ruminantium LDC and that are located in its catalytic domain. We have succeeded in converting S. ruminantium LDC to an enzyme with a preference in decarboxylating activity for L-ornithine when the four-residue of LDC were replaced by the corresponding residues of mouse ornithine decarboxylase (EC 4.1.1.17).
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  • Yoshikatsu MATSUBAYASHI, Yoshinori TAKAHATA, Akiko MORITA, Keisuke ATS ...
    1999 Volume 63 Issue 10 Pages 1847-1849
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      We report the preparation of biotinylated analogs of phytosulfokine-α (Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln; PSK-α), an endogenous peptide growth factor in plants. Because the modification of the N-terminal amino group leads to significant loss of the activities, a Lys residue was incorporated in the C-terminal region of PSK-α, and its e amino group was reacted with biotinylation reagent. Results of the binding assay showed that [Nε-(biotinyl)Lys5]PSK-α retained the same binding activity and mitogenic activity as that of native PSK-α. Insertion of a single or double 6-aminohexanoic acid spacer between the ε amino group of Lys5 and the carboxyl group of biotin did not significantly alter the activities of biotinylated [Lys5]PSK-α. Structure-activity information obtained here would be useful for the detection and isolation of PSK-α receptors.
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Food & Nutrition Science Regular Papers
  • Mari OHTA, Kaoru NAKAMURA, Hiroji TSUCHIYA, Kozo TAKAMA, Tetsuya SUZUK ...
    1999 Volume 63 Issue 10 Pages 1691-1696
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      To examine the detoxification effect of mineral-encaged zeolites on cells impaired by pollutant-intoxication, we used a bioassay system involving Euglena gracilis Z as the model organism and TBTCl as a pollutant. TBTCl exposure causes Euglena cells to quickly change shape from a spherical to spindle form, with the process being reversible by detoxification. Taking advantage of this morphological characteristic, we examined the restoration of motility by water containing zeolites encaging different minerals. TBTCl-intoxicated Euglena cells were incubated in processed water with different types of mineral-encaging zeolites for up to 3 hours. The restoration of motility was evaluated by observing the number of motile cells with a video microscope. Remarkable recovery was observed in the incubation systems with water containing Fe-, Zn-, and Mn-encaging zeolites. However, the effect was suppressed when the water species were treated with the chelator, Chelex-100®. An equivalent concentration of FeCl3 to that in the Fe-encaging zeolite processed water did not show significant restoration effect.
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  • Jiong-Yan GU, Yoko WAKIZONO, Yousuke SUNADA, Phum HUNG, Michiko NONAKA ...
    1999 Volume 63 Issue 10 Pages 1697-1702
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      The immunoregulatory effects of dietary α-tocopherol (Toc) and tocotrienols (T-3) on humoral and cell-mediated immunity and cytokine productions were examined in Brown Norway rats. We found that the IgA and IgG productivity of spleen and mesenteric lymph node (MLN) lymphocytes was significantly enhanced in the rats fed on Toc or T-3, irrespective of concanavalin A (Con A) stimulation of the lymphocytes. On the contrary, the IgE productivity of lymphocytes from the rats fed on Toc or T-3 was less without Con A stimulation, but was greater in the presence of Con A, especially in the T-3 group. Toc or T-3 feeding significantly decreased the proportion of CD4+ T cells and the ratio of CD4+/CD8+ in both spleen and MLN lymphocytes of the rats fed on Toc or T-3. The interferon-γ productivity of MLN lymphocytes was higher in the rats fed on Toc or T-3 than in those fed on a control diet in the presence of Con A, while that of spleen lymphocytes was lower in the rats fed on Toc or T-3. In addition, T-3 feeding decreased the productivity of tumor necrosis factor-α of spleen lymphocytes, while it enhanced the productivity of MLN lymphocytes. These results suggest that oral administration of Toc and T-3 affects the proliferation and function of spleen and MLN lymphocytes.
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  • Tadayuki TSUKATANI, Kiyoshi MATSUMOTO
    1999 Volume 63 Issue 10 Pages 1730-1735
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      L-Tartrate in wines and grapes was enzymatically quantified by using the secondary activity of D-malate dehydrogenase (D-MDH). NADH formed by the D-MDH reaction was monitored spectrophotometrically. Under the optimal conditions, L-tartrate (a 1.0 mM sample solution) was fully oxidized by D-MDH in 30 min. A linear relationship was obtained between the absorbance difference and the L-tartrate concentration in the range of a 0.02-1.0 mM sample solution with a correlation coefficient of 0.9991. The relative standard deviation from ten measurements was 1.71% at the 1.0 mM sample solution level. The proposed method was compared with HPLC, and the values determined by both methods were in good agreement.
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  • Naohito AOKI, Yumiko YAMAGUCHI, Sachiyo OHIRA, Tsukasa MATSUDA
    1999 Volume 63 Issue 10 Pages 1749-1755
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
    “High Fat Feeding of Lactating Mice Causing a Drastic Reduction in Fat and Energy Content in Milk without Affecting the Apparent Growth of Their Pups and the Production of Major Milk Fat Globule Membrane Components MFG-E8 and Butyrophilin” by Naohito AOKI, Yumiko YAMAGUCHI, Sachiyo OHIRA, and Tsukasa MATSUDA, Bioscience, Biotechnology, and Biochemistry , Vol.63, No.10, pp. 1749-1755, October 1999.
    This manuscript was canceled on October16, 2013 because it was found containing a number of serious ethical problems.
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Food & Nutrition Science Notes
  • Achmad SUBAGIO, Hiroko WAKAKI, Naofumi MORITA
    1999 Volume 63 Issue 10 Pages 1784-1786
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Stabilities of lutein and its myristate esters against heat and UV light were studied by HPLC response. Free lutein (FL) was very unstable against heat; lutein monomyristate (LM), slightly stable; and lutein dimyristate (LD), very stable. Both LM and LD were more stable toward UV light than FL. These results suggest that esterification of the OH group with a fatty acid might stabilize lutein against heat and UV-light degradation.
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  • Akihiko NAGAO, Michiko SEKI, Hidetaka KOBAYASHI
    1999 Volume 63 Issue 10 Pages 1787-1790
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Various dietary flavonoids were evaluated in vitro for their inhibitory effect on xanthine oxidase, which has been implicated in oxidative injury to tissue by ischemia-reperfusion. Xanthine oxidase activity was determined by directly measuring uric acid formation by HPLC. The structure-activity relationship revealed that the planar flavones and flavonols with a 7-hydroxyl group such as chrysin, luteolin, kaempferol, quercetin, myricetin, and isorhamnetin inhibited xanthine oxidase activity at low concentrations (IC50 values from 0.40 to 5.02 μM) in a mixed-type mode, while the nonplanar flavonoids, isoflavones and anthocyanidins were less inhibitory. These results suggest that certain flavonoids might suppress in vivo the formation of active oxygen species and urate by xanthine oxidase.
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  • Yasuhisa NARUMI, Suguru TAKATSUTO
    1999 Volume 63 Issue 10 Pages 1800-1802
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Trace sterols in the seeds of foxtail millet (Setaria italica Beauv.) were investigated by GC-MS. Eleven of these trace sterols, i.e., brassicasterol, episterol, 24-methyllathosterol, 24-ethyllathosterol, avenasterol, 24-methylenecholesterol, fucosterol, isofucosterol, 24-methyl-5α-cholest-24(28)-en-3β-ol, 24-ethyl-5α-cholest-24(28)Z-en-3β-ol, and 24-ethyldesmosterol, were identified, suggesting that the previously reported high content of sitostanol was possibly contaminated with a small amount of isofucosterol.
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  • Fumiaki YOSHIZAWA, Taketoshi KIDO, Takashi NAGASAWA
    1999 Volume 63 Issue 10 Pages 1803-1805
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      The effect of dietary protein on p70S6k phosphorylation was examined in rats starved for 18 h and then fed either a 20% casein diet (20C) or a protein-free diet (0C). Refeeding the 20C diet, but not the 0C diet, increased p70S6k phosphorylation in both the skeletal muscle and liver. The plasma insulin concentrations were the same after refeeding the 20C or 0C diet, suggesting that a combination of dietary protein and insulin may be required to stimulate p70S6k phosphorylation.
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  • Akira MURAKAMI, Miyuki TAKAHASHI, Suratwadee JIWAJINDA, Koichi KOSHIMI ...
    1999 Volume 63 Issue 10 Pages 1811-1812
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Zerumbone was isolated from the rhizomes of Zingiber zerumbet Smith as a potent inhibitor of tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation. The IC50 value of zerumbone (0.14 μM) is noticeably lower than those of the anti-tumor promoters we have hitherto obtained. Interestingly, α-humulene lacking the carbonyl group at the 8-position in zerumbone was inactive (IC50>100 μM), while 8-hydroxy-α-humulune was markedly active (IC50=0.95 μM).
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  • Masakuni TAKO, Taketo NAKADA, Fujiya HONGOU
    1999 Volume 63 Issue 10 Pages 1813-1815
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Fucoidan was isolated from Nemacystus decipiens KUCKUCK which is commercially cultured at the rate of about 2,000 t/year in fields of the Okinawa Islands, in Japan. The yield of fucoidan extracted by 0.1 M HCl was 0.5% (W/W) based on the wet algae. The total carbohydrate, sulfuric acid, ash, and moisture in fucoidan was 65.8, 30.8, 22.3, and 3.8%, respectively. Fucose and galactose were identified by liquid chromatography. The molar ratio of the sugar residues in the fucoidan was estimated to be L-fucose:D-galactose:sulfuric acid=1.0:0.05:1.0. The optical rotation of the fucoidan (0.4%) showed a value of −0.315° at 60°C, then it decreased a little with decreasing temperature. The infrared spectrum of isolated fucoidan is in agreement with that of standard fucoidan. The molecular mass of fucoidan was estimated to be 2.4×105. The 1H- and 13C-NMR spectral data suggest that fucoidan is mainly composed of L-fucopyranose, 2-mono-O-sulfo-L-fucopyranose, 4-mono-O-sulfo-L-fucopyranose, and 2,4-di-O-sulfo-L-fucopyranose.
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  • Jun KAYASHITA, Iwao SHIMAOKA, Misao NAKAJOH, Naoko KISHIDA, Norihisa K ...
    1999 Volume 63 Issue 10 Pages 1837-1839
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Female rats were examined for the effects of feeding buckwheat protein extract (BWPE) on the development of mammary tumor caused by administration of 7,12-dimethylbenz[α]anthracene. The percentage of rats with palpable mammary tumors and serum estradiol were lower in the BWPE-fed animals than the casein-fed ones, implying that BWPE intake retarded the mammary carcinogenesis by lowering serum estradiol.
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Microbiology & Fermentation Technology Regular Papers
  • Il KWON, Keisuke EKINO, Masatoshi GOTO, Kensuke FURUKAWA
    1999 Volume 63 Issue 10 Pages 1714-1720
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      Endoglucanase I (EGI) secreted from Trichoderma viride HK-75 has a unique transglycosylation activity. The genomic and cDNA clones encoding EGI (egl1) of T. viride HK-75 were isolated and characterized. The coding region of egl1, composed of 1392 bp, was found to encode a polypeptide of 464 amino acids that has extensive similarity (93.8%) with EGI of T. reesei. Expression of the egl1 gene in E. coli as a fusion protein (with N-terminal thioredoxin and C-terminal histidine tag) led to a large production of a nonglycosylated protein of 62.5 kDa. However, it formed an insoluble inclusion body. Upon denaturation with 8 M urea followed by dialysis and successive purification, the enzymatically active recombinant EGI (rEGI) was obtained at a level as high as 18.3 mg/l of 1,000 ml of culture. The rEGI had 67.8% activity for carboxymethyl cellulose (CMC), compared to native EGI (nEGI). The optimum pH and optimum temperature of rEGI were lower than those of nEGI by 0.5 and 5°C, respectively. The rEGI also had narrower CMCase ranges than nEGI in pH and temperature stabilities. However, the catalytic and transglycosylation abilities against cellotriose of rEGI were comparable to those of nEGI. These results suggest that the glycosylation is important for the stabilities of EGI but not critical for the essential enzymatic capacity.
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  • Sheng-Xue XIE, Jun OGAWA, Sakayu SHIMIZU
    1999 Volume 63 Issue 10 Pages 1721-1729
    Published: 1999
    Released: March 10, 2005
    JOURNALS FREE ACCESS
      An NAD+-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The Km and Vmax for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 μmol/min/mg, and 0.33 mM and 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The Km and Vmax for 2-hexanone reduction were 2.5 mM and 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.
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Microbiology & Fermentation Technology Note
Microbiology & Fermentation Technology Preliminary Communication
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