Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 68 , Issue 5
Showing 1-31 articles out of 31 articles from the selected issue
Review
  • Jun KANEKO, Yoshiyuki KAMIO
    2004 Volume 68 Issue 5 Pages 981-1003
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    Staphylococcal γ-hemolysin (Hlg), leukocidin (Luk), and Panton–Valentine leukocidin (PVL) are two-component and hetero-oligomeric pore-forming cytolytic toxins (or cytolysin), that were first identified in bacteria. No information on the existence of hetero-oligomeric pore-forming cytolytic toxins in bacteria except for staphylococcal strains is available so far. Hlg (Hlg1 of 34 kDa/Hlg2 of 32 kDa) effectively lyses erythrocytes from human and other mammalian species. Luk (LukF of 34 kDa/LukS of 33 kDa) is cytolytic toward human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes, and PVL (LukF-PV of 34 kDa/LukS-PV of 33 kDa) reveals cytolytic activity with a high cell specificity to leukocytes. Hlg1 is identical to LukF and that the cell specificities of the cytolysins are determined by Hlg2 and LukS. Based on the primary and 3-dimensional structures of the toxin components, Hlg, Luk, and PVL are thought to form a family of proteins. In the first chapter of this article, we describe the molecular basis of the membrane pore-forming nature of Hlg, Luk, and PVL. We also describe a requirement of the phosphorylation of LukS and LukS-PV by protein kinase for their leukocytolytic activity besides their pore formation on human leukocytes.
    Recently, the assembly mechanism of the LukF and Hlg2 monomers into pore-forming hetero-oligomers of Hlg on human erythrocyte membranes has been clarified for the first time by our study using a single-molecular fluorescence imaging technique. We estimated 11 sequential equilibrium constants for the assembly pathway which includes the beginning with membrane binding of monomers, proceeds through single pore oligomerization, and culminates in the formation of clusters of the pores. In the second chapter of this article, we refer to an assembly mechanism of LukF and Hlg2 on human erythrocytes as well as the roles of the membranes of the target cells in pore formation by Hlg.
    The LukF, LukS, and Hlg2 proteins are derived from the Hlg locus (hlg), and have been found in 99% of clinical isolates of Staphylococcus aureus. In contrast, LukF-PV and LukS-PV are derived from the PVL locus (pvl) which is distinct from the hlg locus, and only a small percentage of clinically isolated S. aureus strains carries pvl. Recently, we discovered pvl on the genome of lysogenic bacteriophages, φPVL, and determined the entire gene of the phage. We also demonstrated the phage conversion of S. aureus leading to the production of PVL through the discovery of a PVL-carrying temperate phage, φSLT, from a clinical isolate of S. aureus. In the third chapter of this article, we discuss genetic analyses of the Hlg, Luk, and PVL genes. We also discuss the current status of knowledge of the genetic organization of PVL-converting phages in order to achieve an understanding of their molecular evolution.
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Organic Chemistry Regular Papers
  • Shinya UESUSUKI, Bunta WATANABE, Shuji YAMAMOTO, Junko OTSUKI, Yoshiak ...
    2004 Volume 68 Issue 5 Pages 1097-1105
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    Brassinosteroids containing various side chain moieties were synthesized and their activity was determined as the reciprocal logarithm of the ED50 (50% effective dose per plant in moles) in the rice lamina inclination assay using synergist indole-3-acetic acid (IAA). The introduction of a hydroxyl group in the α-position to the carbonyl group of the ester structure significantly enhanced the activity. 2α,3α-Dihydroxy-17β-[(2R,3S)-2-hydroxy-3-methylpentanoyl]oxy-B-homo-7-oxa-5α-androstan-6-one showed the highest activity, for which the pED50 was determined to be 10.5 under synergistic conditions with IAA. Under identical conditions, the pED50 values of brassinolide and castasterone were determined to be 13.6 and 12.3 respectively. With respect to the α-carbon of the acyl moiety, the R-form was 10 times more potent than the corresponding S-form. Substituting the terminal structure (Et) of the side chain to that of the most potent compound, brassinolide (i-Pr), did not increase the activity.
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  • Naoto TAJIMA, Manabu NUKINA, Nobuo KATO, Takeshi SASSA
    2004 Volume 68 Issue 5 Pages 1125-1130
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    Our search for new 3-hydroxyfusicoccins structurally related to cotylenin A from a culture of Phomopsis amygdali Niigata 2-A resulted in the isolation of novel 3-hydroxy fusicoccins, called fusicoccins R and S, and the fusicoccin S aglycon, called phomopsiol, together with known 3α-hydroxyfusicoccin J. The structure of phomopsiol was identified as that of O-demethyl-3-epicotylenol based on spectroscopic evidence. The structures of fusicoccins R and S were also determined to be those of 3′-deacetyl-3α-hydroxyfusicoccin A and 3β-hydroxy-3-epifusicoccin H. The lettuce seed germination-stimulating activity of fusicoccins R and S, phomopsiol and 3α-hydroxyfusicoccin J was examined in the presence of ABA; fusicoccin R and 3α-hydroxyfusicoccin J were highly active, while fusicoccin S and phomopsiol were inactive. The possible biosynthetic relationships among these novel fusicoccins having a 3α- or 3β-hydroxy group in their diterpene moiety are briefly discussed.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Kenji YAMAGISHI, Toshiyuki KIMURA, Masahiro SUZUKI, Hiroshi SHINMOTO, ...
    2004 Volume 68 Issue 5 Pages 1017-1026
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    In many fungi, the heterotrimeric G protein alpha subunits, and/or small G protein (RAS) control intracellular cAMP levels. But it is not clear which types of G proteins modulate cAMP levels in homobasidiomycete (mushrooms). To explain the mechanism, we expressed dominant active RAS (a homolog of S. cerevisiae RAS1) in homobasidiomycete Schizophyllum commune and compared the cAMP levels in the transformed clones with those of clones expressing dominant active heterotrimeric G protein alpha subunits ScGP-A, B, and C. The results demonstrated that the dominant active ScGP-A and C elevated the intracellular cAMP levels. In contrast, the dominant active S. commune RAS gene did not affect the cAMP levels, even though colony growth and formation of fruiting bodies were apparently repressed. These data suggest that the heterotrimeric G protein alpha subunits are involved in the mechanism of cAMP regulation, and that RAS modulates another signal-transduction pathway regulating cell growth and differentiation.
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  • Yasuhiro MIHARA, Kohki ISHIKAWA, Ei-ichiro SUZUKI, Yasuhisa ASANO
    2004 Volume 68 Issue 5 Pages 1046-1050
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5′-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5′-inosinic acid (5′-IMP) productivity and lower 5′-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5′-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.
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  • Kiyotaka FUJITA, Hideto TAKAMI, Kenji YAMAMOTO, Kaoru TAKEGAWA
    2004 Volume 68 Issue 5 Pages 1059-1066
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    The genome sequencing project on alkaliphilic Bacillus halodurans C-125 revealed a putative endo-β-N-acetylglucosaminidase (Endo-BH), which consists of a signal peptide of 24 amino acids, a catalytic region of 634 amino acids exhibiting 50.1% identity with the endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A), and a C-terminal tail of 220 amino acids. Transformed Escherichia coli cells carrying the Endo-BH gene exhibited endo-β-N-acetylglucosaminidase activity. Recombinant Endo-BH hydrolyzed high-mannose type oligosaccharides and hybrid type oligosaccharides, and showed transglycosylation activity. On deletion of 219 C-terminal amino acid residues of Endo-BH, the wild type level of activity was retained, whereas with deletions of the Endo-A homolog domain, the proteins were expressed as inclusion bodies and these activities were reduced. These results suggest that the enzymatic properties of Endo-BH are similar to those of Endo-A, and that the C-terminal tail does not affect the enzyme activity. Although the C-terminal tail region is not essential for enzyme activity, the sequence is also conserved among endo-β-N-acetylglucosaminidases of various origins.
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  • Yukari OHTA, Yuichi NOGI, Masayuki MIYAZAKI, Zhijun LI, Yuji HATADA, S ...
    2004 Volume 68 Issue 5 Pages 1073-1081
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    A gene, agaA, for a novel β-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542T. The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37–66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type β-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 °C. The optimal pH and temperature for activity were around 7.0 and 55 °C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).
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  • Yasuyuki SASAKI, Naoki TAKAYA, Akira NAKAMURA, Hirofumi SHOUN
    2004 Volume 68 Issue 5 Pages 1106-1112
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    A flavocytochrome protein was isolated from the actinomycete Streptomyces antibioticus. The purified protein contained protoheme and FAD, and its Mr was estimated to be 52,000. The absorption spectra in its resting oxidized, dithionite-reduced, carbon monoxide-bound, and oxygenated (O2-bound) forms were characteristic of those of flavohemoglobin (Fhb). The N-terminal amino acid sequence showed high identities to those of other Fhb’s. Furthermore, the actinomycete flavocytochrome scavenged nitric oxide in the presence of NADH. These results demonstrated that the flavocytochrome is the first Fhb purified from actinomycetes. The actinomycete Fhb was produced in S. antibioticus cells in large amounts without any external nitric oxide (NO) stress, which is indicative of a physiological function of Fhb other than detoxification of NO.
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  • Li YING, Motomitsu KITAOKA, Kiyoshi HAYASHI
    2004 Volume 68 Issue 5 Pages 1113-1118
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    The function of the non-homologous region of a family 3 β-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region. Five truncated mutants named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 having deletions of 4, 31, 62, 89, and 119 amino acid residues starting from Phe417, respectively, were expressed in Escherichia coli and purified. All the mutants exhibited β-glucosidase activity, indicating that the non-homologous region was not essential for the activity. The truncation caused thermal instability, decrease in pKa of the proton donor residue (Glu616), and deficient transglycosylation activity. The thermal stability and the pKa of Glu616 were partially recovered with longer truncation, suggesting that the truncation perturbed the structure and that their presence in the region was not essential. The main role of the non-homologous region could be formation of a hydrophobic atmosphere at the acceptor site to make the enzyme suitable for hydrolyzing hydrophobic glucosides.
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Biochemistry & Molecular Biology Notes
Biochemistry & Molecular Biology Communication
  • Abul Kalam AZAD, Yoshihiro SAWA, Takahiro ISHIKAWA, Hitoshi SHIBATA
    2004 Volume 68 Issue 5 Pages 1170-1174
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 °C, only the holo-type enzyme preparation acted at 5 °C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.
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Food & Nutrition Science Regular Papers
  • Norihiro SHIGEMATSU, Yasuhide OKUHARA, Takuya SHIOMI, Fusao TOMITA, Hi ...
    2004 Volume 68 Issue 5 Pages 1011-1016
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    The effects of difructose anhydride III (DFAIII) on stimulating calcium absorption was investigated in humans. We studied changes in the time-course of characteristics urinary calcium excretion in 12 healthy men given 0.3, 1.0 or 3.0 g of DFAIII and 300 mg of calcium as calcium carbonate. In addition, urinary excretion and urine concentrations of creatinine and deoxypyridinoline were determined. Urine calcium excretion every 2 hours after the intake were higher over than that of the control subjects. The total amount of urinary calcium excretion for 10 hours was significantly greates in the subjects given 1.0 g or 3.0 g of DFAIII than that of the control subjects. However, there were no differences in the urine concentrations of creatinine and deoxypyridinoline between the subjects given DFAIII and the control subjects. These findings suggests that low dose of DFAIII had a stimulating effect on calcium absorption in humans.
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  • Hidetoshi MORITA, Hiroshi YOSHIKAWA, Takehito SUZUKI, Shin HISAMATSU, ...
    2004 Volume 68 Issue 5 Pages 1027-1034
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6,000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron–nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.
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  • Jale BALKAN, Serdar ÖZTEZCAN, Aydan HATIPOGLU, Ugur ÇEVIKB ...
    2004 Volume 68 Issue 5 Pages 1035-1039
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    We studied whether taurine has any regressive effect on existing atherosclerotic lesions and lipid peroxidation in rabbits fed on a high-cholesterol (HC) diet. The cholesterol, triglyceride, malondialdehyde (MDA) and diene conjugate (DC) levels, as well as the aortic histopathological findings were examined in rabbits that had been fed on a cholesterol-containing diet for 8 months [0.5% cholesterol (w/w) for 3 months and subsequently 0.25% cholesterol (w/w) for 5 months], and then for a further 4 months on a normal diet with or without taurine treatment [1% (w/v) in the drinking water]. High levels of lipid and lipid peroxide induced by the HC diet were observed to decline in the plasma, liver and aorta of atherosclerotic rabbits, as well as a slight retardation in aortic atherosclerotic lesions during the regression period. Although no significant differences in the lipid and lipid peroxide levels in the plasma and aorta were found between the regressed groups with or without the taurine treatment, the extent of atherosclerotic lesions in the aorta was less in the taurine-treated regressed group than in the non-treated regressed group. However, the liver MDA and DC levels were lower in the regressed rabbits with the taurine treatment in the non-treated group. These results indicate that the taurine treatment may accelerate the regression of cholesterol-induced atherosclerotic lesions in rabbits without having any effect on the plasma and aorta lipid and lipid peroxide levels.
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  • Yoon-Bok LEE, Hyong Joo LEE, Kang Sung KIM, Jae-Yong LEE, Sang-Yoon NA ...
    2004 Volume 68 Issue 5 Pages 1040-1045
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    To examine a potential role for soybean phytoestrogens in postmenopausal bone loss, twenty-four 12-week-old Sprague-Dawley rats were divided randomly into 4 groups and given controlled diets for 16 weeks. The treatment groups were as followed: sham operated, ovariectomized (OVX) control, OVX + isoflavone extract (6.25 g/kg), and OVX + 17β-estradiol (4 mg/kg). OVX treatments reduced femoral and fourth lumbar vertebral bone density and mineral content (p<0.01), decreased uterine weight (p<0.01), accelerated body weight increases (p<0.05), and increased the activities (p<0.01) of both serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Supplementation with isoflavone prevented the losses of bone density and mineral content caused by OVX (p<0.01). Although both isoflavone and 17β-estradiol exhibited similar bone-sparing ability on the OVX-induced bone loss, the effect of isoflavone was not the same as that of 17β-estradiol on the serum ALP and TRAP, body weight increase, and uterine weight change. We concluded that dietary supplementation with soybean isoflavone can prevent postmenopausal bone loss via a different mechanism of estrogen in OVX rats.
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  • Jang Hoon SUNG, Seung Jun CHOI, Soon Won LEE, Kwan Hwa PARK, Tae Wha M ...
    2004 Volume 68 Issue 5 Pages 1051-1058
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    3-Hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase is the rate-limiting enzyme in the biosynthesis of cholesterol in mammals. Some microbial metabolites have been found to be HMG-CoA reductase inhibitors. Korean soybean paste is a unique food fermented by many microorganisms. The enzymatic method using the catalytic domain of Syrian hamster HMG-CoA reductase was employed for the screening of HMG-CoA reductase inhibitors. Soybean paste extract was fractionated by vacuum liquid chromatography. Fractions showing relatively high HMG-CoA reductase inhibition were further purified through Sephadex LH-20 column chromatography and C18 preparative HPLC, and the inhibitory compounds were identified as genistein, daidzein, and glycitein.
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  • Naoto HASHIMOTO, Hiroshi HARA
    2004 Volume 68 Issue 5 Pages 1067-1072
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    Regulators for pancreatic amylase were examined. Rats were fed ad libitum a 20% amino acid (AA) mixture diet (Con), a 60% AA diet (HA), a branched-chain amino acid (BCAA)-rich diet (BC), or a diet supplemented with AA other than BCAA (OA) for 7 d, or fed the Con, HA, BC diets or diets supplemented with individual BCAA. Activity and mRNA levels of pancreatic amylase in the BC and HA groups were lower than those in the Con and OA groups. Leucine and isoleucine contributed to these effects of the BC diet. The mRNA levels correlated with individual pancreatic BCAA concentrations but not with plasma insulin level. In conclusion, dietary BCAA, especially leucine and isoleucine, may reduce amylase mRNA and activity in rats.
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  • Bin Mohamed Salleh MOHAMAD RAMLAN, Nobuyuki MARUYAMA, Koji TAKAHASHI, ...
    2004 Volume 68 Issue 5 Pages 1091-1096
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    The effects of protein concentration, and heating temperature and time on the gelling properties of soybean β-conglycinin (7S globulins) lacking the α or α′ subunit were compared with those of 7S containing all three subunits (α, α′, and β) to determine whether each subunit contributes equally. In most of the conditions, the relative order of gel hardness was α′-lacking > 7S > α-lacking. From Fourier transform infrared studies, the secondary structure change after heating was very similar among the three samples; thus, the secondary structural change is not the reason for the differences in gel hardness. By using scanning electron microscopy, we observed differences in strand thickness and the density of the gel network among the three samples. These differences correlated well with the differences in gel hardness.
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  • Chinatsu KASAMATSU, Sumiko KIMURA, Mieko KAGAWA, Keiko HATAE
    2004 Volume 68 Issue 5 Pages 1119-1124
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    The high molecular weight protein connectin (also called titin) in Japanese common squid (Todarodes pacificus) mantle muscle was identified by western blotting analysis with 3B9, the mouse anti-chicken skeletal muscle connectin monoclonal antibody. Similarly to vertebrate samples, there exists connectin in invertebrate squid mantle muscle, and the amino acid sequences are assumed to resemble those present in the A band of vertebrate connectin, judging by the specificity of 3B9. Moreover, the connectin in squid muscle migrated in this study as a closely spaced doublet of α and β (titins 1 and 2). Between 5 and 7 h post-mortem, the SDS PAGE patterns of the squid sample indicated a change of the doublet bands into a single β-connectin band. Simultaneously, the rheological properties of the squid muscle changed substantially. This degradation of α-connectin into β-connectin in the muscle can explain the critical change that occurs during the post-mortem tenderization of squid muscle.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Hideaki UCHIDA, Kenji FUJITANI, Yasushi KAWAI, Haruki KITAZAWA, Akira ...
    2004 Volume 68 Issue 5 Pages 1004-1010
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcα1-3(Fucα1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.
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  • Xiqian LAN, Naomi OZAWA, Naohide NISHIWAKI, Ritsuko KODAIRA, Mitsuo OK ...
    2004 Volume 68 Issue 5 Pages 1082-1090
    Published: 2004
    Released: May 23, 2004
    JOURNALS FREE ACCESS
    A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A β-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-β-D-glucosaminide and pNP-N-acetyl-β-D-galactosaminide. A gene coding for the purified β-N-acetylglucosaminidase was isolated. The ORF identified is 2,661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial β-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.
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Microbiology & Fermentation Technology Notes
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