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91.
Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation 总被引:1,自引:0,他引:1
Wakayama M Yamagata T Kamemura A Bootim N Yano S Tachiki T Yoshimune K Moriguchi M 《Journal of industrial microbiology & biotechnology》2005,32(9):383-390
Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was
estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that
the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA.
Besides l-glutamine, which was hydrolyzed with the highest specific activity (100%), l-asparagine (74%), d-glutamine (75%), and d-asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme
was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About
70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed
high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher
salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese
soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation. 相似文献
92.
Fisher DJ Miyamoto K Harrison B Akimoto S Sarker MR McClane BA 《Molecular microbiology》2005,56(3):747-762
Clostridium perfringens type A isolates carrying an enterotoxin (cpe) gene are an important cause of human gastrointestinal diseases, including food poisoning, antibiotic-associated diarrhoea (AAD) and sporadic diarrhoea (SD). Using polymerase chain reaction (PCR), the current study determined that the cpb2 gene encoding the recently discovered beta2 toxin is present in <15% of food poisoning isolates, which typically carry a chromosomal cpe gene. However, >75% of AAD/SD isolates, which usually carry a plasmid cpe gene, tested cpb2(+) by PCR. Western blot analysis demonstrated that >97% of those cpb2(+)/cpe(+) AAD/SD isolates can produce CPB2. Additional PCR analyses, sequencing studies and pulsed field gel electrophoresis experiments determined that AAD/SD isolates carry cpb2 and cpe on the same plasmid when IS1151 sequences are present downstream of cpe, but cpb2 and cpe are located on different plasmids in AAD/SD isolates where IS1470-like sequences are present downstream of cpe. Those analyses also demonstrated that two different CPB2 variants (named CPB2h1 or CPB2h2) can be produced by AAD/SD isolates, dependent on whether IS1470-like or IS1151 sequences are present downstream of their cpe gene. CPB2h1 is approximately 10-fold more cytotoxic for CaCo-2 cells than is CPB2h2. Collectively, these results suggest that CPB2 could be an accessory toxin in C. perfringens enterotoxin (CPE)-associated AAD/SD. 相似文献
93.
Komichi D Tazuma S Nishioka T Hyogo H Chayama K 《Free radical biology & medicine》2005,39(11):1418-1427
Bile acids have been suggested to be involved in biliary carcinogenesis, although the underlying mechanisms are yet to be established. The aim of this study was to investigate the carcinogenic effect of bile acids in the biliary tract in relation to oxidative stress. Immortalized mouse cholangiocytes were incubated with various bile acids, followed by measurement of reactive oxygen species (ROS) and the glutathione (GSH) level. As a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG) expression in cholangiocytes was analyzed by flow cytometry. Then the expression of oxidative DNA repair enzymes in cholangiocytes was examined by real-time PCR. In addition, the long-term effect of bile acid-induced oxidative DNA damage on cholangiocytes was investigated using a mouse oligo DNA microarray. It was found that glycochenodeoxycholate (GCDC) induced the generation of ROS and the depletion of GSH. In contrast, no marked changes were induced by the other bile acids. The percentage of 8-OHdG-positive cells was also increased by GCDC, but the expression of oxidative DNA repair enzymes was not up-regulated. DNA microarray analysis showed marked changes of various genes associated with carcinogenesis (genes related to cell proliferation, angiogenesis, invasion, and metastasis). In conclusion, the long-term effect of oxidative DNA damage due to GCDC may promote carcinogenesis in the biliary tract. Furthermore, accumulation of 8-OHdG due to GCDC might contribute to the dysfunction of oxidative DNA repair enzymes. 相似文献
94.
A microtubule-beta-cyclodextrin conjugate was prepared on a kinesin-adsorbed glass surface by chemical and biochemical means. Fluorescence microscope observation and a motility assay indicated that the conjugate simultaneously expressed an inherent motor function and an inclusion property. 相似文献
95.
96.
Yoshimune K Galkin A Kulakova L Yoshimura T Esaki N 《Extremophiles : life under extreme conditions》2005,9(2):145-150
Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures. 相似文献
97.
98.
Muraoka N Shum L Fukumoto S Nomura T Ohishi M Nonaka K 《Birth defects research. Part A, Clinical and molecular teratology》2005,73(12):956-965
BACKGROUND: Cleft lip with or without cleft palate is the most common congenital anomaly in the craniofacial region. Knowledge of the molecular mechanisms behind normal lip fusion can contribute to better intervention and improved functional clinical outcome. Transforming growth factor-beta3 (TGF-beta3) has been implicated in lip morphogenesis. Therefore, we hypothesized that TGF-beta3 functions during lip fusion through regulation of angiogenesis and mesenchymal cell cycle progression during early developmental stages. METHODS: To test this hypothesis we used the CL/Fraser mouse model, which has a high incidence of cleft lip. Lips isolated from embryonic day (ED) 11.5 mouse embryos were allowed to develop in serum-free organ cultures in the presence or absence of TGF-beta3. The lips that developed in these cultures fused in 2 days. RESULTS: During normal development, we detected positive immunoreactions for TGF-beta3 at the site of fusion. We also detected mesenchymal cells that were immunopositive for Flk-1 and CD31, which are markers for endothelial cell precursors. Exogenous TGF-beta3 accelerated lip fusion in culture. This enhancement was associated with an increase in the number of capillary blood vessels in the lips cultured in the presence of TGF-beta3, in comparison with controls. In tandem, TGF-beta3 increased the level of expression of both Flk-1 and CD31. Our data suggest that an elevated level of TGF-beta3 may promote angiogenesis in developing lips that is mediated by increased Flk-1 and CD31 expression. We also detected increased cyclin D1 expression (a marker for cell proliferation) in the presence of TGF-beta3, which suggests that TGF-beta3 promoted cell proliferation. CONCLUSIONS: TGF-beta3 promoted cell proliferation and angiogenesis in lip mesenchymal tissues. These events led to enhanced lip fusion in the presence of TGF-beta3. 相似文献
99.
Karasawa K Shirakura M Harada A Satoh N Yokoyama K Setaka M Inoue K 《Journal of biochemistry》2005,138(4):509-517
Although red blood cells account for about 30% of total PAF-AH activity found in the blood, the physiological function of this enzyme is unknown. To understand the role and regulatory mechanism of this enzyme, we purified it from easily obtainable pig red blood cells. PAF-AH activity was mainly found in the soluble fraction of the red blood cells. Two peaks of enzyme activity appeared with increasing concentration of imidazole on column chromatography on nickel-nitroacetic acid (Ni-NTA) resin. We called these peaks of small and large enzyme activities fractions X and Y, respectively, and then further purified the enzymes by sequential chromatofocusing on Mono P and gel filtration on TSK G-3000. In the final preparation from fraction Y, two proteins bands corresponding to 26 kDa and 28 kDa were related to enzyme activity. Determination of the partial amino acid sequences of the proteins of 26 kDa and 28 kDa revealed that these proteins were identical to alpha(1) and alpha(2), respectively, both of which are catalytic subunits of Type I intracellular PAF-AH. On Western analysis, the 26 kDa and 28 kDa protein bands cross-reacted with specific monoclonal antibodies to alpha(1) and alpha(2), respectively. Since the apparent molecular weight of the natural enzyme was estimated to be about 60 kDa, the enzyme activity in fraction Y was thought to be that of a heterodimer consisting of alpha(1) and alpha(2). On the other hand, the enzyme activity in fraction X was thought to be that of a homodimer consisting of alpha(2). Other blood cells such as polymorphonuclear leukocytes and platelets only contained the alpha(2)/alpha(2) homodimer. It has been reported that the alpha(1)/alpha(2) heterodimer is poorly expressed in adult animals except for in the spermatogonium. Taken altogether, these results suggest that high expression of the alpha(1)/alpha(2) heterodimer is important for the physiological function of mature red blood cells. 相似文献
100.
Nagaoka Y Otsu K Okada F Sato K Ohba Y Kotani N Fujii J 《Biochemical and biophysical research communications》2005,331(1):215-223
Singlet oxygen is a causal factor in light-induced skin photoaging and the cytotoxic process of tumor cells in photodynamic chemotherapy. To develop a better understanding of the functional consequences of protein modification by singlet oxygen, the effects of naphthalene endoperoxide on lysosomal protease, cathepsin, were examined. When the soluble fraction of normal human fetal skin fibroblast cells was treated with the endoperoxide, the activities of cysteine proteases, cathepsins B and L/S, were inhibited, but that of aspartate protease, cathepsin D/E, was not. The reduction of the endoperoxide-treated soluble fractions by treatment with dithiothreitol barely recovered the activities. Cathepsin B, purified from normal human liver, exhibited similar profiles to that in cytosol. These data suggest that singlet oxygen oxidatively modifies an amino acid residue essential for catalysis and consequently results in the irreversible inactivation of cysteine protease-type cathepsin. 相似文献