Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 72 , Issue 1
Showing 1-40 articles out of 40 articles from the selected issue
Analytical Chemistry Regular Paper
  • Hiroko TSUKATANI, Kazuhiro TOBIISHI, Yoshito TANAKA, Kenji SAKURAGI, T ...
    2008 Volume 72 Issue 1 Pages 149-154
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    A simple and selective method was developed for determining the concentration of hexaconazole in river and sea water samples by using liquid chromatography/tandem mass spectrometry with an electrospray ionization interface in the positive ion mode and selective reaction monitoring mode. Trace amounts of hexaconazole were collected in a Sep-Pak Plus tC18 cartridge that was eluted with methanol. The detection limit for hexaconazole was 6 ng/l. The recovery of a standard aqueous solution containing 1 μg/l was 96%. The recovery of hexaconazole in the river and sea water samples was 95% and 90%, respectively. Hexaconazole was not detected in the sea water samples. Trace peaks of hexaconazole were found in the river water samples, the concentration being less than 6 ng/l in all cases. The biological degradation of hexaconazole was tested by using river water. No degradation of hexaconazole was apparent in river water incubated at 20 °C for 3 weeks.
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Organic Chemistry Regular Papers
Biochemistry & Molecular Biology Regular Papers
  • Katsuhiko SEKIMATA, Toshiyuki OHNISHI, Masaharu MIZUTANI, Yasushi TODO ...
    2008 Volume 72 Issue 1 Pages 7-12
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    Arabidopsis thaliana (Arabidopsis) treated with the four stereoisomers of Brz220 (2RS, 4RS-1-[4-propyl-2-(4-trifluoromethylphenyl)-1, 3-dioxane-2-ylmethyl]-1H-1, 2, 4-triazole) showed a dwarf phenotype like brassinosteroid (BR) biosynthesis mutants that were rescued by treatment of BRs. The target sites of each Brz220 stereoisomer were investigated by treatment of Arabidopsis with BRs in the dark. The results suggest that the stereoisomers block the 22-hydroxylation step in BR biosynthesis. This step is catalyzed by DWF4, an Arabidopsis cytochrome P450 identified as a steroid 22-hydroxylase. The enzyme was expressed in E. coli, and the binding affinity of the stereoisomers to recombinant DWF4 was analyzed. The results indicate that in these stereoisomers there exists a positive correlation between binding affinity to DWF4 and inhibition of Arabidopsis hypocotyl growth. In this context, we concluded that DWF4 is the target site of Brz220 in Arabidopsis.
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  • Taiyong YU, Guifen LUO, Lijie ZHANG, Jiangwei WU, Haowei ZHANG, Gongsh ...
    2008 Volume 72 Issue 1 Pages 13-21
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    Leptin, a major regulator of body weight, was recently suggested to play a role in myoblasts. We conducted an experiment to determine whether leptin can influence the proliferation and differentiation of porcine skeletal myoblasts. Myoblasts occurred in non-leptin and leptin forms in various concentrations for various periods of cell states. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry assays demonstrated that leptin significantly promoted myoblast proliferation and increased cell accumulation in the S + G2/M phase, in a dose-dependent manner. Furthermore, in morphologic experiments, the formation of myotubes and the myogenic index was markedly reduced by leptin. In addition, biochemical analysis showed that leptin decreased creatine kinase (CK) activity and the amount of myogenin and myosin heavy chain (MyHC) protein. Taking all this together, our study indicated that exogenous leptin promoted proliferation but inhibited differentiation in porcine skeletal myoblasts, suggesting that leptin might be an important mediator in the regulation of the growth and development of muscle cells.
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  • Hossein HELI, Mojtaba AMANI, Ali Akbar MOOSAVI-MOVAHEDI, Ali JABBARI, ...
    2008 Volume 72 Issue 1 Pages 29-36
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    The electrochemical behavior of redox centers in the active site of amine oxidases from lentil seedlings and Euphorbia characias latex was investigated using a mercury film electrode. Tyrosine-derived 6-hydroxydopa quinone (TPQ) and copper ions in the active site are redox centers of these amine oxidases. The enzymes undergo two reduction processes at negative potentials related to the reduction of the TPQ cofactor to the corresponding hydroquinones and the reduction of copper ions, (Cu(II)→Cu(I)). Copper depleted enzymes, prepared by reduction with dithionite followed by dialysis against cyanide, undergo only one reduction process. Nyquist diagrams, recorded at potentials corresponding to the reduction of cofactors as dc-offset, represent charge transfer impedance followed by a Warburg-type line at low frequencies, indicating the occurrence of a diffusion controlled process in the rate-limiting step of the reduction process.
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  • Tomoyuki MIYASHITA, Takuo HANASHITA, Michinori TORIYAMA, Ryousuke TAKA ...
    2008 Volume 72 Issue 1 Pages 37-47
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    The bone morphogenetic proteins (BMPs) constitute a subfamily of the transforming growth factor type beta (TGF-β) supergene family. BMP-2 plays an important role not only in osteoblast differentiation but also in pattern formation during development. To determine the function of BMP-2 in Pinctada fucata development and hard tissue formation, we isolated a BMP-2 genomic DNA clone and the BMP-2 cDNA. The deduced BMP-2 sequence consisted of 447 amino acids. The BMP-2 gene was composed of three exons. The C-terminal portion (149 amino acids) had 86% and 66% identity to the Crassostrea gigas and the human BMP-2 sequence respectively. The 5′ flanking promoter region contained putative glioma transcription factor (Gli) and retinoic acid receptor (RAR) responding elements. The BMP-2 gene was expressed strongly in the inner part of the mantle tissue, corresponding to the nacreous aragonite shell layer. This finding suggests that BMP-2 has a key role in nacreous layer formation.
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  • Hiromoto HISADA, Motoaki SANO, Yoji HATA, Yasuhisa ABE, Masayuki MACHI ...
    2008 Volume 72 Issue 1 Pages 48-53
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    The catalase-encoding gene (catB) is expressed strongly in Aspergillus oryzae. To identify the transcription regulatory elements involved in strong expression, we did promoter deletion analysis using β-glucuronidase (GUS) as a reporter and an electrophoretic gel mobility shift assay (EMSA) systematically. The deletion 200-bp sequence from −1,000 to −800 in the 1,400-bp catB promoter caused a drastic decrease in GUS activity. In addition, EMSA implicated a 45-bp element from −1,000 to −956 containing cis-elements. According to detailed promoter deletion analysis, a region from −1,000 to −975, which contains putative heat shock element (HSE) and the CCAAT-box, was involved in strong expression.
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  • Yoonjung KHO, Sungchan KIM, Byung Sun YOON, Jai-Hee MOON, Bona KIM, Su ...
    2008 Volume 72 Issue 1 Pages 70-81
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFα treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IκBα. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon γ (IFNγ). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor α (TNFα) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFκB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.
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  • Koji NOMURA, Kazuhisa SUGIMOTO, Hiromi NISHIURA, Kohji OHDAN, Takahisa ...
    2008 Volume 72 Issue 1 Pages 82-87
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    Transglucosylation from sucrose to acetic acid by sucrose phosphorylase (EC 2.4.1.7) was studied. 1-O-Acetyl-α-D-glucopyranose was isolated as the main product of the enzyme reaction. We also compared the pH-dependence of transglycosylation catalyzed by sucrose phosphorylase toward carboxyl and hydroxyl groups. With hydroquinone as an acceptor molecule, the transfer ratio of glucose residue was higher at neutral pH. This pH-activity profile was similar to that of the phosphorolysis of sucrose by sucrose phosphorylase, but with acetic acid as an acceptor molecule, the transfer ratio of glucose residue was higher at low pH. These findings suggest that the undissociated carboxyl group is essential to the acceptor molecule for the transglycosylation reaction of sucrose phosphorylase. In a sensory test, the sour taste of acetic acid was markedly reduced by glucosylation. The threshold value of the sour taste of acetic acid glucosides was approximately 100 times greater than that of acetic acid.
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  • Hirofumi UCHIYAMA, Yasuyuki HASHIDOKO, Yuhki KURIYAMA, Satoshi TAHARA
    2008 Volume 72 Issue 1 Pages 116-123
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    A 7.1-kbp DNA fragment isolated from a wild strain of Klebsiella oxytoca was sequenced, leading to the identification of 10 open-reading frames (ORFs), including a 504-bp Pad gene. The Pad gene of the Gram-negative bacterium was subsequently expressed in Escherichia coli as a chimeric Pad. The deduced amino acid (AA) sequence of the Pad gene from wild-type K. oxytoca showed approximately 50% homology to those of other bacterial PADs from Gram-positive bacilli plus a coccus. These data and a genomic library search of some γ-proteobacteria, including E. coli and Vibrio sp., indicated that PAD of K. oxytoca is a member of the bacterial PAD family characteristic of Gram-negative bacteria. Using Pad-specific PCR primers designed from the Gram-negative bacterial Pad of K. oxytoca, Pad genes of two further strains of K. oxytoca, another wild isolate and JCM 1665 and two PAD-positive Enterobacter spp. were successfully amplified for specific Pad detection.
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  • Noriyuki ISHII, Fitri FITRILAWATI, Abhijit MANNA, Haruhisa AKIYAMA, Ya ...
    2008 Volume 72 Issue 1 Pages 124-131
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    The azobenzene moiety, well-known not only for its reversible cis-to-trans photoisomerization but also as a hapten, does not induce antibodies on its own, but it reacts with antibodies raised against conjugates with protein carriers. Hence we selected azobenzene dye as an indicator to assess the possibility of having gold nano-particles act as an immunological carrier instead of protein carriers. In rabbits, we confirmed an in vivo response against azobenzene dye presented on the entire surface of gold nanoparticles (azo-nanoparticles), where the gold nanoparticles appeared to play a role as a carrier for the hapten. A high yield of immunoglobulin G (IgG) against the azobenzene derivative took place in rabbits injected with azo-nanoparticles, whereas no increase in IgG was recognized in other rabbits treated solely with chemically equivalent azobenzene dye instead of azo-nanoparticles. Electron microscopy and surface plasmon resonance spectroscopy indicated that the IgG obtained specifically recognized the difference between the isomer conformations of the azobenzene moiety.
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  • Ruthada CHANKLAN, Eiji AIHARA, Saori KOGA, Hidetoshi TAKAHASHI, Masaki ...
    2008 Volume 72 Issue 1 Pages 132-138
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    Compelled activation of Ca2+ signaling by exposure of zds1Δ strain Saccharomyces cerevisiae cells to external CaCl2 leads to characteristic physiological consequences such as growth inhibition in the G2 phase and polarized bud growth. Screening of microbial metabolites for activity alleviating the deleterious physiological effects of external CaCl2 identified the Hsp90 inhibitor radicicol as an inhibitor of Ca2+-signal-dependent cell-cycle regulation in yeast. Radicicol alleviated analogous physiological effects due to the expression of a constitutively active form of calcineurin or overexpression of Swe1, the negative regulatory kinase of the Cdc28-Clb complex. Western blot analysis indicated that radicicol inhibited Ca2+-induced accumulation of Swe1 and Cln2.
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  • Youzuo ZHANG, Yoshiaki KOUZUMA, Takayuki MIYAJI, Masami YONEKURA
    2008 Volume 72 Issue 1 Pages 171-178
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0×10−9 M and 1.0×10−6 M respectively. The inhibitory activity was not affected by heating at 80 °C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.
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  • Hideyuki IWATA, Tsutomu NAKAGAWA, Yuichiro YOSHIOKA, Kazufumi KAGEI, K ...
    2008 Volume 72 Issue 1 Pages 179-185
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    The pH dependence and kinetics parameters of renin-angiotensinogen reactions were determined using wild-type and S84G mutant human renins and wild-type and H13Y mutant sheep angiotensinogens. It is explained in this report that (i) renin catalyzes acidic and basic reactions of which the optimum pHs are 5.5 and 7.5–8.2 respectively, both of which produce angiotensin I; (ii) Ser84 specific to human renin accelerates the acidic reaction by 75–110% through elevation of Vmax, and shifts the optimum pH of the basic reaction from 7.5 to 8.0–8.2; and (iii) His13 specific to sheep angiotensinogen accelerates the acidic and basic reactions by 25–42% through reduction of Km. It is concluded from these results that the coexistence of Ser84 in renin and His13 in angiotensinogen brings a pH profile of two separate peaks at pHs 5.5 and 8.2 to the reaction of human renin and sheep angiotensinogen.
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Biochemistry & Molecular Biology Notes
Food & Nutrition Science Regular Papers
  • Jeongrai LEE, Suk-Hyung KWON, Hyun-Mi KIM, Stefan N FAHEY, Derek R KNI ...
    2008 Volume 72 Issue 1 Pages 1-6
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    Colostrum is a complex mixture of bioactives that promotes neonate growth. Studies show that it contains components capable of promoting bone formation and inhibiting bone resorption. Although many colostrum-based nutritional supplements have been developed as growth promotants, few studies have investigated their functional effects. A bovine colostrum 1–30 kDa fraction, Growth Protein-Colostrum (GP-C), was administered to juvenile rats as a dietary supplement to determine effects on growth and development. GP-C enhanced the growth and mineralization of the femur as evidenced by increased serum osteocalcin and bone mineral density. Increased levels of serum growth hormone and insulin-like growth factor-1 suggest that the mechanism of enhanced growth is partially controlled by endocrine factors. GP-C was also found to increase osteoblast proliferation in vitro, a finding that indicates a possible mechanism of action of GP-C, but further studies are required. Based on our findings, we hypothesize that a colostrum-based dietary supplement enhances bone growth and development in humans.
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  • Shota TANIMOTO, Hideyuki MATSUMOTO, Kazuyoshi FUJII, Ritsushi OHDOI, K ...
    2008 Volume 72 Issue 1 Pages 22-28
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    The effect of a high-pressure carbonation treatment on the change in quality of sake during storage was investigated. Measurements of the amino acidity and isovaleraldehyde content of carbonated sake (20 MPa pressure at 40, 45 and 50 °C for 7, 21 and 33 min, respectively) as well as of heat-treated sake (reaching temperature of 65 °C and immediately cooled) were almost unchanged during storage at 3 and 20 °C. Glucose in the sake subjected to these treatments was retained at an almost constant under the same storage conditions, except for the sake carbonated at 40 °C and stored at 20 °C. In contrast, the amino acidity, and glucose and isovaleraldehyde contents of non-pasteurized (fresh) sake increased during storage at both temperatures. The sake samples subjected to the carbonation treatment and heat treatment both gave better sensory scores than the fresh sake sample after 6 month of storage at 3 and 20 °C, especially at 3 °C for the flavor. These results suggest that the high-pressure carbonation treatment is an effective new technique for preserving the quality of sake.
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  • Alessandra FERRAMOSCA, Viviana SAVY, Vincenzo ZARA
    2008 Volume 72 Issue 1 Pages 62-69
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    Diet supplementation with olive oil exerts beneficial effects on an organism, even if an increase in the level of hepatic lipids has been concomitantly observed. This study was therefore designed to investigate whether the stimulation of lipogenesis was responsible for the olive oil-induced hepatic fat accumulation. In mice fed for 8 weeks with an olive oil-enriched diet, an increase of about 2.6 fold in the level of liver triglycerides was found in comparison to animals fed with a corn oil-containing diet. Despite that, no increase in the activities of cytosolic lipogenic enzymes or of the mitochondrial tricarboxylate carrier was found; on the contrary, a decrease in the activity of carnitine palmitoyltransferase I was observed. This impairment of fatty acid oxidation, which was not apparent in corn oil-fed animals, may have had a role in the increase of hepatic lipid content found in the olive oil-fed mice.
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  • Hiroyuki SAKAKIBARA, Saki YOSHINO, Yoshichika KAWAI, Junji TERAO
    2008 Volume 72 Issue 1 Pages 94-100
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    The present study evaluated the antidepressant-like effect of the quercetin-rich vegetable, onion, by using the rat behavioral model of depression, the forced swimming test (FST). Daily administration of onion powder at a dosage of 50 mg/kg of body weight/day for 14 days significantly reduced the immobility time in FST without changing the motor dysfunction, indicating that the daily consumption of onion exerted antidepressant-like activity. The plasma corticosterone level was elevated after an FST trial, and pretreatment with onion powder did not modulate this elevation. Although the FST trial tended to increase the dopaminergic activity in the rat hypothalamus, the administration of onion powder (50 mg/kg) suppressed the increase in the turnover of this neurotransmitter. However, the same prevention was also observed with a higher dosage of onion, in which no significant antidepressant effect was apparent. The results of the present study suggest that onion exerted antidepressant-like activity in a behavioral model that acted independently of the hypothalamic-pituitary-adrenal axis.
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  • Yuji NAKAI, Hiroko HASHIDA, Koji KADOTA, Michiko MINAMI, Kentaro SHIMI ...
    2008 Volume 72 Issue 1 Pages 139-148
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    Supplementary material
    The functional balance between brown adipose tissue (BAT) and white adipose tissue (WAT) is important for metabolic homeostasis. We compared the effects of fasting on the gene expression profiles in BAT, WAT and liver by using a DNA microarray analysis. Tissues were obtained from rats that had been fed or fasted for 24 h. Taking the false discovery rate into account, we extracted the top 1,000 genes that had been differentially expressed between the fed and fasted rats. In all three tissues, a Gene Ontology analysis revealed that the lipid and protein biosynthesis-related genes had been markedly down-regulated. The whole-body fuel shift from glucose to triacylglycerol and the induction of autophagy were also observed. There was marked up-regulation of genes in the ‘protein ubiquitination’ category particularly in BAT of the fasted rats, suggesting that the ubiquitin-proteasome system was involved in saving energy as an adaptation to food shortage.
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Food & Nutrition Science Notes
Food & Nutrition Science Communication
  • Yuko YAMADA, Daiki YAMAUCHI, Megumi YOKOO, Kousaku OHINATA, Hachiro US ...
    2008 Volume 72 Issue 1 Pages 257-259
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    In this study, we found that novokinin (Arg-Pro-Leu-Lys-Pro-Trp), a potent hypotensive peptide acting through the AT2 receptor, has vasorelaxing activity in the mesenteric artery isolated from spontaneously hypertensive rats. The vasorelaxing activity was significantly blocked by PD123319, indomethacin, and CAY10441, which are an AT2 receptor antagonist, a cyclooxygenase inhibitor, and an IP receptor antagonist, respectively. NG-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, did not block the vasorelaxing activity. These results suggest that the vasorelaxing activity of novokinin, which contributes to the hypotensive effect, is mainly mediated by prostaglandin I2 (prostacyclin) and the IP receptor downstream of the AT2 receptor.
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Microbiology & Fermentation Technology Regular Papers
  • Shigekazu YANO, Nopakarn RATTANAKIT, Arata HONDA, Yuta NODA, Mamoru WA ...
    2008 Volume 72 Issue 1 Pages 54-61
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia. α-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity.
    Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to α-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with α-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.
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  • Seisuke ARAI, Taketo KAWARAI, Ritsuko ARAI, Minoru YOSHIDA, Soichi FUR ...
    2008 Volume 72 Issue 1 Pages 88-93
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
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    On the basis of our previous study concerning the effect of high hydrostatic pressure treatment (HPT) on Escherichia coli FtsZ ring (bacterial cytoskeleton) formation, we aimed to determine the effect of HPT on the growth properties of a representative eukaryotic microbe, Schizosaccharomyces pombe, in relation to the behavior of genuine cytoskeletons. Microtubules were visualized with GFP-linked α-tubulin. Actin-related cytoskeletons were fluorescently stained with rhodamine-phalloidin. We observed growth retardation of about 10 h in post growth after HPT (75 MPa, 30 min, 28 °C), which caused only a little loss of viable cells. In accordance with the period of growth retardation, cessation of cytokinesis and disappearance of the contractile ring (composed of actin, myosin II, and other proteins), directly participates in cytokinesis, continued for 18 h after HPT. On the other hand, the microtubules disappeared only for 6 h after HPT. Based on these observations, the contractile ring was the site most sensitive to HPT resulting in the cessation of cytokinesis.
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  • Sachiko YAMAMOTO, Mamoru WAKAYAMA, Takashi TACHIKI
    2008 Volume 72 Issue 1 Pages 101-109
    Published: January 23, 2008
    Released: January 23, 2008
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    γ-Glutamylmethylamide synthetase (GMAS), found in an obligate methylotroph, Methylovorus mays No. 9, can form theanine from glutamic acid and ethylamine in a mixture in which yeast sugar fermentation is coupled for ATP regeneration. The internal and N-terminal amino acid sequences of GMAS had certain similarities to putative glutamine synthetase type III (GS III) of Methylobacillus flagellatus KT. From the M. mays No. 9 genomic DNA library, a clone containing a 6.5-kbp insertional DNA fragment was selected by the PCR screening technique with oligonucleotide primers specific for the GMAS gene. The fragment had an open reading frame of the GMAS gene encoding a protein of 444 amino acids (molecular mass, 49 kDa). The deduced amino acid sequence showed significant identity with that of Met. flagellatus KT GS III (78%). The isolated gene was ligated into an expression vector (pET21a) and expressed in Escherichia coli AD494 (DE3). Enzyme productivity in the expression system was about 23-fold higher than that in M. mays No. 9. Recombinant GMAS had the same properties as intrinsic GMAS, and it formed theanine by coupling the reaction with the ATP-regeneration system of yeast sugar fermentation.
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  • Maizom HASSAN, Masahiro OKADA, Tsuyoshi ICHIYANAGI, Nobuhiro MORI
    2008 Volume 72 Issue 1 Pages 155-162
    Published: January 23, 2008
    Released: January 23, 2008
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    4-N-trimethylaminobutyraldehyde dehydrogenase from Pseudomonas sp. 13CM was purified 14-fold to apparent homogeneity by hydrophobic chromatography on a Phenyl-Toyopearl, and affinity chromatography was done on a 5′-AMP Sepharose4B in the presence of dithiothreitol. The enzyme was found to be a trimer with identical 55 kDa subunits. The isoeletric point was found to be 5.5. The optimum temperature and pH were 40 °C and pH 10.0. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters, and analog inhibition. The Km values for 4-N-trimethylaminobutyraldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were 7.4, 51, and 125 μM respectively. The enzyme was inhibited by SH reagents, and by heavy metal ions.
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  • Koki TAKAKI, Shinya FUSHINOBU, Sang-Wan KIM, Morio MIYAHARA, Takayoshi ...
    2008 Volume 72 Issue 1 Pages 163-170
    Published: January 23, 2008
    Released: January 23, 2008
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    Ralstonia pickettii K50 (strain K50) is a denitrifying bacterium that produces low levels of N2O under aerobic conditions. In this study, we found that co-culturing of strain K50 with Streptomyces griseus significantly enhanced the denitrification activity of strain K50 in an artificial wastewater (AWW) system. Most factors that enhance denitrification activity were in the high molecular weight fraction of the cell-free broth of S. griseus, and were suggested to be extracellular proteases. Further investigation revealed that the cultivation of strain K50 in protease-treated AWW medium fully enhanced denitrification, and that a shortage of amino acids in the medium limited it. Among the 20 standard amino acids tested, only histidine had a significant effect in inducing denitrification by strain K50. Our results indicatate that histidine is a novel inducer of bacterial denitrification.
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  • Mitsuo OGURA, Taku OHSAWA, Teruo TANAKA
    2008 Volume 72 Issue 1 Pages 186-196
    Published: January 23, 2008
    Released: January 23, 2008
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    The Bacillus subtilis yrkP gene encodes a response regulator of a two-component regulatory system of unknown function. A previous DNA microarray experiment suggested that multicopy yrkP greatly enhanced the expression of yrkN, the ykcBC operon, and yrkO, which encodes a putative transporter. Here, lacZ fusion analysis confirmed these results and also revealed that YrkP autoregulates the putative yrkPQR operon, indicating that yrkPQR and yrkO form a divergon structure. In addition, real-time PCR analysis revealed that transcription of yrkO, yrkN, and ykcBC was significantly reduced in the yrkP strain. Hence, YrkP positively regulates the expression of these genes. Gel retardation analyses showed that YrkP bound to the promoter regions of yrkO, yrkN, and ykcB, albeit with lower binding affinities to the latter two promoters. The in vitro binding of YrkP to the promoter region of the yrkPQR and yrkO divergon was then analyzed by DNase I footprinting analysis. This revealed that YrkP recognizes three regions containing single-motifs or a direct repeat of the ten-base sequence [T/G]TCA[T/C]AAATT. lacZ fusion analysis of deleted and mutagenized promoter regions of yrkO and yrkPQR divergon confirmed that the three YrkP-binding regions are needed for the YrkP-mediated activation of yrkO and/or yrkPQR.
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  • Takafumi SUGIHARA, Tomo-o WATSUJI, Shin KUBOTA, Kazune YAMADA, Kaori O ...
    2008 Volume 72 Issue 1 Pages 204-211
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
    JOURNALS FREE ACCESS
    We study the ecological distribution of a unique syntrophic bacterium, Symbiobacterium thermophilum, and related bacteria. In this study, we found that they were frequently obtained from seashells and several marine samples. Symbiobacterium also grew from sterilized oyster shells incubated undersea for 2 or 3 months on the coast of Shimoda, Shizuoka, Japan. 16S rRNA gene-based phylogeny of the clones obtained from the Symbiobacterium-positive cultures demonstrated the potential diversity of this bacterial group, which constitutes a distinct clade between Actinobacteria and Firmicutes. We successfully isolated two new Symbiobacterium strains from oyster shells. 16S rRNA gene-based phylogeny indicated that one belongs to S. thermophilum, and that the other is affiliated with a different species. We also isolated Ureibacillius spp., which showed activity supporting the growth of S. thermophilum.
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Microbiology & Fermentation Technology Notes
  • Sho-ichi TSUJIYAMA, Masayoshi UENO
    2008 Volume 72 Issue 1 Pages 212-215
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
    JOURNALS FREE ACCESS
    In order to utilize phenolic compounds in unused biomass resources, the metabolic pathway of ferulic acid by way of a white-rot fungus, Schizophyllum commune, was investigated. Ferulic acid was immediately degraded, and the formation of 4-vinyl guaiacol was confirmed by GC-MS analysis. The metabolic test of ferulic acid and its degradation products indicated that S. commune converted ferulic acid into 4-vinyl guaiacol by decarboxylation. This was then oxidized to vanillin and vanillic acid. This result indicates that S. commune distinguished ferulic acid from lignins and metabolized it specifically.
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  • Wayoon POONPERM, Goro TAKATA, Ken IZUMORI
    2008 Volume 72 Issue 1 Pages 231-235
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
    JOURNALS FREE ACCESS
    The conversion specificity of Bacillus pallidus Y25 for polyols, including elusive rare sugar alcohols, was investigated. B. pallidus cells showed transformation potential for several rare polyols, including allitol, L-mannitol, D/L-talitol, and D-iditol, and converted them to their corresponding ketoses. This indicates that the bacterium had two polyol dehydrogenases specific for polyols that have D-erythro and D-threo configurations. By combination with intrinsic isomerases, polyols were converted directly to various aldoses, including L-xylose, L-talose, D-altrose, and L-glucose.
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Microbiology & Fermentation Technology Communications
  • Richard HAAS, Bo JIN, Florian Tobias ZEPF
    2008 Volume 72 Issue 1 Pages 253-256
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
    JOURNALS FREE ACCESS
    There has been a considerable interest in using low cost carbon substrates for the production of Poly(3-hydroxybutyrate) (PHB). We have shown that saccharified waste potato starch can be used as a viable alternative carbon source in high cell density PHB production. Using Ralstonia eutropha NCIMB 11599 with phosphate limitation, 179 g/l biomass, 94 g/l PHB, Ybiomass/starch = 0.46 g/g, YPHB/starch = 0.22 g/g, and PHB productivity = 1.47 g/(l*h) were achieved. Residual maltose accumulated in the fed-batch reactor but caused no noticeable inhibition. Performance with saccharified starch was virtually identical to that with glucose.
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  • Emiko SHINAGAWA, Yoshitaka ANO, Osao ADACHI, Kazunobu MATSUSHITA
    2008 Volume 72 Issue 1 Pages 260-264
    Published: January 23, 2008
    Released: January 23, 2008
    [Advance publication] Released: January 07, 2008
    JOURNALS FREE ACCESS
    A novel NADH dehydrogenase (NADH-dh) involving FAD as coenzyme, distinct from NADPH dehydrogenase (NADPH-dh, old yellow enzyme, EC 1.6.99.1), was found in the same cytoplasmic fraction of Gluconobacter strains. Conventional artificial electron acceptors were more effective than molecular oxygen in the NADH-dh reaction. NADH-dh did not appear to be identical with any previously described flavoproteins, although the N-terminal amino acid sequence showed 100% similarity with a non-heme chloroperoxidase. The N-terminal amino acid sequence of NADPH-dh matched 100% a putative oxidoreductase containing the old yellow enzyme-like FMN-binding domain. NADH-dh might function to regenerate NAD coupling with NAD-dependent dehydrogenases in the cytoplasm of Gluconobacter strains.
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