Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 64 , Issue 5
Showing 1-36 articles out of 36 articles from the selected issue
Organic Chemistry Regular Papers
  • Hiroshi SHIBA, Nobuko KIMURA, Seiji TAKAYAMA, Kokichi HINATA, Akinori ...
    2000 Volume 64 Issue 5 Pages 1016-1024
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Self-incompatible (SI) Brassica rapa (syn. B. campestris) was transformed with an antisense SLG gene by using SLG8 cDNA isolated from the B. campestris S8 homozygote. Two transformed lines were obtained and analyzed. Northern blot and Western blot analyses revealed that endogenous SLG and SRK were greatly reduced of the transcriptional and translational levels in the transformant. Pollination experiments confirmed that their SI phenotype had broken down. In addition, the progeny with the antisense SLG gene, resulting from self- or cross-pollination of the transgenic plant, also showed the self-compatible phenotype. The breakdown of SI in the tranformants was due to the change in property of the stigma and not of the pollen. These results provide strong evidence that SLG and/or SRK is implicated in the pollen-stigma recognition of SI and that they act only as stigmatic factors.
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  • Atsushi ISHIHARA, Naoki KAWATA, Tetsuya MATSUKAWA, Hajime IWAMURA
    2000 Volume 64 Issue 5 Pages 1025-1031
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Both N-p-coumaroyl- and N-feruloyltyramine accumulated in response to wounding in leaf segments of maize. The amount of N-hydroxycinnamoyltyramines started to increase 3-6 h after wounding and peaked at 12 h. Thereafter, the amount of N-p-coumaroyltyramine decreased rapidly, while the N-feruloyltyramine content remained at a high level. The accumulation of N-hydroxycinnamoyltyramines was accompanied by an increase in the tyramine N-hydroxycinnamoyltransferase (THT) activity. This increase was initially detected 3 h after wounding and reached a maximum at 36 h, the level of activity being 40 and 11 times that in the leaves before wounding and in the control leaves, respectively. Partial purification of THT from wounded leaves by (NH4)2SO4 precipitation and subsequent two steps of anion-exchange chromatography resulted in a 12.5-fold increase in specific activity. Kinetic studies with this partially purified enzyme revealed that the best substrates were tyramine and feruloyl-CoA, although tryptamine and sinapoyl-CoA also efficiently served as substrates. The apparent native molecular weight of the enzyme was determined by gel filtration as 40 kDa.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Sang Ryeol PARK, Soo Jeong CHO, Han Dae YUN
    2000 Volume 64 Issue 5 Pages 925-930
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      The phytopathogenic bacterium Erwinia chrysanthemi secretes multiple isozymes of plant cell wall disrupting enzymes such as pectate lyase and endoglucanase. We cloned genomic DNA from Erwinia chrysanthemi PY35. One of the E. coli XL1-Blue clones contained a 5.1-kb BamHI fragment and hydrolyzed carboxymethyl cellulose and polygalacturonic acid. By subsequent subcloning, we obtained a 2.9-kb fragment (pPY100) that contained the pel gene responsible for CMCase and pectate lyase activities. The pel gene had an open reading frame (ORF) of 1,278 bp encoding 425 amino acids with a signal peptide of 25 amino acids. Since the deduced amino acid sequence of this protein was very similar to that of PelL of E. chrysanthemi EC16, we concluded that it belonged to the pectate lyase family EC 4.2.2.2, and we designated it PelL1. Sequencing showed that the PelL1 protein contains 400 amino acids and has a calculated pI of 7.15 and a molecular mass of 42,925 Da. The molecular mass of PelL1 protein expressed in E. coli XL1-Blue, as analyzed by SDS-PAGE, appeared to be 43 kDa. The optimum pH for its enzymatic activity was 9, and the optimum temperature was about 40°C.
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  • Hyekyeong PARK, Nanae YAMANAKA, Anita MIKKONEN, Isao KUSAKABE, Hideyuk ...
    2000 Volume 64 Issue 5 Pages 931-939
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained non-covalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.
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  • Takahiro KANEKO, Saori TAKAHASHI, Kyuichi SAITO
    2000 Volume 64 Issue 5 Pages 940-947
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      The glucose isomerase from Streptomyces olivaceoviridis E-86 was purified by chromatographic procedures, showing one single protein band in the SDS-PAGE. The enzyme had high acid stability, and there was no loss in enzyme activity at pH 5.0 after incubation at 60°C for 30 hr. The enzyme had sufficients activity at 60°C, pH 5.5, (which is the reaction condition for a single-step process with a glucoamylase from A. niger), and at 58°C, pH 6.0, (condition with a glucoamylase from R. niveus). By using this acid-stable glucose isomerase, a single-step process to produce high-fructose corn sweetener (HFCS) from liquefied starch was formed without any reductant or other reagents for enzyme stabilization. The HFCS produced was about fifty percent fructose and less than 1.5% unknown oligosaccharides.
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  • Hiroko KOBAYASHI, Fumi KUMAGAI, Tadashi ITAGAKI, Norio INOKUCHI, Takas ...
    2000 Volume 64 Issue 5 Pages 948-957
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      The fruit bodies of Lentinus edodes produce two acid nucleases, nucleases Le1 and Le3, both of which are thought to be candidates for the enzymes producing a tasty substance, 5′-GMP. To obtain the basic information on the mechanism of production of 5′-GMP, and structure-function relationship of these nucleases, the primary structure of nuclease Le1 was estimated by both protein chemistry and gene cloning. Nuclease Le1 is a glycoprotein and consists of 290 amino acid residues, and about 2 and 6 residues of hexosamine and neutral sugar, respectively. The nucleotide sequence of cDNA and genomic DNA encoding nuclease Le1 indicated the presence of 20 amino acid residues of a signal peptide.
      Nuclease Le1 has 115 and 108 residues of identical amino acid residues with nucleases P1 and S, respectively. The amino acid residues concerning the coordination with Zn2+ in nuclease P1 are all conserved in nuclease Le1. Nuclease Le1 contains 8 half-cystine residues and 4 of them are located at the same places as those of nucleases P1 and S.
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  • Hisashi KAWASAKI, Megumi SHIMAOKA, Yoshihiro USUDA, Takashi UTAGAWA
    2000 Volume 64 Issue 5 Pages 972-979
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized. The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer. A pI of 6.0 was obtained by isoelectric focusing. In addition to ATP, it was found that deoxyadenosine 5′-triphosphate, UTP, and CTP could serve as phosphate donors. The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine. Maximum activity was attained at an ATP/Mg2+ concentration ratio of 0.5. In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP. Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released. Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.
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  • Sachiko MACHIDA, Setsuko NIIMI, Xiaohua SHI, Yoshiji ANDO, Yong YU
    2000 Volume 64 Issue 5 Pages 985-994
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      The design of amphipathic peptides resulted in a novel peptide with a selective ability to destabilize lipid bilayers of acidic liposomes. The newly synthesized peptide, termed mast 21, is a 21-residue long amino acid chain and can only act effectively on acidic liposomes lacking cholesterol. Moreover, mast 21 killed Gram-positive and Gram-negative bacteria, and it had no hemolytic activity. The antimicrobial and hemolytic activities paralleled the results of membrane destabilizing activity using liposomes. Circular dichroism and Trp-fluorescence emission spectra showed changes in the peptide conformation and circumstances around the peptide during interaction with liposomes. These changes were consistent with an increased α-helical content and a less polar environment for the tryptophan residue of the peptide. Mast 21 was observed under dark-field microscopy in real time attacking liposomes. Acidic liposomes were attacked, which resulted in peeling of the lipid bilayer with its subsequent destruction.
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  • Tadao NAKANO, Toshio JOH, Kazumasa NARITA, Toshiro HAYAKAWA
    2000 Volume 64 Issue 5 Pages 995-1003
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Phytases are the primary enzymes responsible for the hydrolysis of phytic acid, myo-inositol-1, 2, 3, 4, 5, 6-hexakisphosphate (InsP6). The pathway of hydrolysis of InsP6 by phytase from wheat bran of Triticum aestivum L. cv. Nourin #61 is proved in this study. Structures of the intermediates were established by a variety of nuclear magnetic resonance techniques (1H-, two-dimensional 1H-1H coupling-correlation spectra and two-dimensional 31P-1H correlation spectra), gas chromatography, and bioassay. On the basis of the structures identified, initial hydrolysis of the phosphate ester occurs at the D/L-4 position of InsP6 to yield D/L-Ins (1, 2, 3, 5, 6) P5. After the dephosphorylation, the pathway of dephosphorylation is divided into two routes. The main route proceeds via D/L-Ins (1, 2, 5, 6) P4, D/L-Ins (1, 2, 6) P3 and D/L-Ins (1, 2) P2, while the minor route proceeds via D/L-Ins (1, 2, 3, 6) P4, Ins (1, 2, 3) P3 and D/L-Ins (1, 2) P2. D/L-Ins (1, 2) P2 is hydrolyzed at the D/L-1 or 2-position, and finally myo-inositol is produced.
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Biochemistry & Molecular Biology Notes
Biochemistry & Molecular Biology Preliminary Communication
  • Yuji YAMAMOTO, Masako HIGUCHI, Leslie B. POOLE, Yoshiyuki KAMIO
    2000 Volume 64 Issue 5 Pages 1106-1109
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Alkyl hydroperoxide reductase in Streptococcus mutans consists of two components, Nox-1 and AhpC. Deletion of nox-1 and ahpC in a double mutant as well as the wild-type of Streptococcus mutans can form colonies in the presence of air to the same extent. The evidence suggested the presence of some other antioxidant system(s) independent of the Nox-1/AhpC system in the bacterium. Here we identified a new antioxidant gene (dpr) and the gene product (Dpr) which complements the defect of peroxidase activity caused by the deletion of nox-1 and ahpC in S. mutans. The dpr-disruption mutant of S. mutans could form colonies anaerobically but not aerobically.
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Food & Nutrition Science Regular Papers
  • Meng-Tsan CHIANG, Hsien-Tsung YAO, Hsing-Chen CHEN
    2000 Volume 64 Issue 5 Pages 965-971
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      To investigate the effect of dietary chitosan on lipid metabolism, male SD (Sprague-Dawley) rats were fed a cholesterol-enriched diet containing 5% cellulose (CE), 5% chitosan (CCS; high viscosity), or 5% chitosan (FCS; low viscosity) for 4 weeks. The two types of chitosan with a comparable degree of deacetylation had a different molecular weight and intrinsic viscosity. Significantly (p<0.05) lower plasma total cholesterol, LDL-cholesterol and VLDL-cholesterol concentrations were observed in the rats fed on the chitosan diets. In addition, chitosan significantly increased the fecal cholesterol and triglyceride contents. Although no significant difference in body weight was found among the dietary groups, the rats fed on the chitosan diets had lower relative liver weight when compared with those fed on the cellulose diet. Both of the chitosan groups had significantly lower liver total lipid and total cholesterol contents compared to the cellulose group, although the FCS group was less effective. The plasma and liver thiobarbituric acid reactive substances (TBAR) values were similar in the CE and FCS groups, while the CCS group had increased liver TBAR values. Although a significant increase in liver glucose-6-phosphate dehydrogenase activity was observed in the CCS group, no significant change was found in the FCS group. The observed influence of chitosans with different viscosity on the plasma lipid level, liver lipids and lipid peroxidation suggests that, while the hypocholesterolemic action of chitosans with different viscosity was similar, changes in the liver lipids and liver peroxidation status depended on their molecular weight when the deacetylation degree was comparable.
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  • Takashi NAGASAWA, Hiromichi HAYASHI, Naoko FUJIMAKI, Naoyuki NISHIZAWA ...
    2000 Volume 64 Issue 5 Pages 1004-1010
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      The oxidative stress produced by electrical stimulation-induced muscle contraction was examined in the skeletal muscle proteins of rats that had been fed on the dietary flavonoid, (-)-epigallocatechin gallate (EGCg). Electrical stimulation of the rat leg muscle every second day for a two-week period resulted in an increased (p<0.05) muscle weight and accumulation of oxidatively induced modified proteins. Similar stimulation conducted every day for only one week had no effect on the muscle weight or protein oxidation, although the rate of protein degradation increased. Rats fed on a 20% casein diet supplemented with 0.1% EGCg for 2 weeks responded to the electrical stimulation of muscle contraction by reducing the increased muscle protein carbonyl content when compared to their counterparts fed on a control diet. There was no change in activity of antioxidative enzymes in muscle tissue of the EGCg-fed rats receiving electrical stimulation. The results of this study show that the antioxidative property of EGCg was effective for suppressing oxidative modification of the skeletal muscle protein induced by electrical stimulation. This finding demonstrates that EGCg has a beneficial effect in vivo on the free radical-mediated oxidative damage to muscle proteins.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Shigeru OITA, Mayumi OHNISHI-KAMEYAMA, Tadahiro NAGATA
    2000 Volume 64 Issue 5 Pages 958-964
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      An antimicrobial peptide termed BCP-2 was purified from barley grain by chitin-affinity treatment and HPLC. The results of amino acid analysis and mass spectrometry of BCP-2 indicate that the peptide is very similar to barley α-thionin. BCP-2 and wheat α1-thionin were also bound to β-glucan but not to starch. The binding of BCP-2 to laminarin (β-1,3-1,6-glucan) and laminarioligosaccharides was supported by fluorescence polarization data. This is the first report on the binding of α-thionins to polysaccharide containing chitin and β-1,3-glucan, which construct fungal cell walls.
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  • Osamu AKITA, Chiharu NISHIMORI, Toshihiro SHIMAMOTO, Tsutomu FUJII, Ha ...
    2000 Volume 64 Issue 5 Pages 980-984
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Pyruvate uptake in Saccharomyces cerevisiae was not observed at 0°C and was prevented by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The initial uptake rate of S. cerevisiae kyokai No. 901 was maximum at pH 6 and Km=4.1 mM. It seemed that lactate inhibited the pyruvate uptake competitively from the results of the Lineweaver-Burk plots. The inhibition constant (Ki) in the presence of 3 mM lactate was 1.6 mM. The pyruvate uptake was inhibited by D-glucose and deoxyglucose, but not by L-glucose, acetate or ethanol.
      Mutants of laboratory strain No. 5022 ((a) his(2,6), ura3) deficient in pyruvate uptake were isolated from fluoropyruvate resistant mutants. Transformation of the mutant with a yeast genomic library allowed the isolation of the gene JEN1 (YKL217w), which restored pyruvate uptake. Disruption of JEN1 abolished the uptake of pyruvate and gained the resistance against fluoropyruvate. The results indicate that no other monocarboxylate permease is able to efficiently transport pyruvate in S. cerevisiae.
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  • Hiroyuki SHIBASAKI, Tsuneo YAMANE
    2000 Volume 64 Issue 5 Pages 1011-1015
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      To reduce the freezing point of sesame oil, the lipase-catalyzed interesterification of sesame oil in a solvent free system was studied. The lipase was immobilized on Celite and refined sesame oil was used as the only substrate for the reaction. After interesterification, the oil did not solidify at 0°C even after 24 h, and even longer storage at 2-4°C did not result in solidification. The change of physical behavior was investigated with a differential scanning calorimeter and X-ray diffraction, and reduction in the thermodynamic and crystallographic stability of the interesterified oil was demonstrated. The change in triacylglycerol species composition after the reaction was analyzed, showing that content of trisaturated acylglycerol was decreased.
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Microbiology & Fermentation Technology Notes
  • Toshihiro TAKAHASHI, Yasukazu NAKAKITA, Junji WATARI, Ken SHINOTSUKA
    2000 Volume 64 Issue 5 Pages 1032-1037
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      We set up the original operating conditions of the MicroStarTM-Rapid Microbe Detection System (RMDS) to suppress false positives, which have kept this system from practical. The detection limit of our system was between 6.3×10-16 mol and 3.1×10-16 mol in terms of the amount of ATP, which is approximately equal to the ATP content of one yeast cell or 50 lactic acid bacteria cells. The detection time and the detection count were compared between the RMD method and the conventional plate count method (C.P.C. method) using 23 test samples of beer-spoilage Lactobacillus brevis. Judging from the detection time and detection count, 16-24 hours of cultivation for the RMD method corresponded to 40-96 hours of cultivation for the C.P.C. method. The RMD method reached a useful level for our practical use at the point of sensitivity.
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  • Kenzo KOIKE, Mikio TAKAIWA, Yoshiharu KIMURA, Shigeo INOUE, Susumu ITO
    2000 Volume 64 Issue 5 Pages 1064-1066
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.
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  • Mamoru KOH, Hiroshi HANAGATA, Shogo EBISU, Kazuyuki MORIHARA, Hiroaki ...
    2000 Volume 64 Issue 5 Pages 1079-1081
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      A synthetic gene encoding a single chain human insulin precursor [B-chain (1-29)-A-chain] linked to the C-terminal lysine of human epidermal growth factor (1-28) (EGF-SCI) was constructed. This gene was expressed using Bacillus brevis. EGF-SCI was isolated from the supernatant of the culture broth. Treatment of EGF-SCI with lysyl endopeptidase resulted in the formation of des-B30 human insulin. The identification of the formed des-B30 human insulin was made by the measurement of molecular weight and amino acid analysis. The binding coefficient to anti-human insulin antibody was comparable to that of human insulin.
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  • Miho IKEGAMI, Hiroko TAKAHASHI, Kazuei IGARASHI, Yoshimi KAKINUMA
    2000 Volume 64 Issue 5 Pages 1088-1092
    Published: 2000
    Released: February 15, 2005
    JOURNALS FREE ACCESS
      Enterococcus hirae grows in a broad pH range from 5 to 11. An E. hirae mutant 7683 lacking the activities of two sodium pumps, Na+-ATPase and Na+/H+ antiporter, does not grow in high Na+ medium at pH above 7.5. We found that 7683 grew normally in high Na+ medium at pH 5.5. Although an energy-dependent sodium extrusion at pH 5.5 was missing, the intracellular levels of Na+ and K+ were normal in this mutant. The Na+ influx rates of 7683 and two other strains at pH 5.5 were much slower than those at pH 7.5. These results suggest that Na+ elimination of this bacterium at acid pH is achieved by a decrease in Na+ entry and a normal K+ uptake.
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