Structural basis for the recognition of α‐1,6‐branched α‐glucan by GH13_47 α‐amylase from Rhodothermus marinus
Yuki MiyasakaKohei YokoyamaTakuma KozonoYoshikazu KitanoTakatsugu MiyazakiMasayoshi SakaguchiAtsushi NishikawaTakashi Tonozuka
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Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.Bacteriostatic experiment of twelve species of Chinese medicines and its compounds against escherichia coli K88 and hemoclatic escherichia coli had been made.The result showed that nine species of Chinese medicines had shown bacteriostasis against escherichia coli K88 and eleven species against hemoclastic escherichia coli. All compounds had shown bacteriostasis against escherichia coli Kgg and hemoclastic escherichia coli.The bacteriostatic effect of compuond 5 was superior to enrofloxacin's(1‰) against escherichia coli K88 (P0.05), and the bacteriostatic effect of compound 5 was superior to enrofloxacin's (1‰) against hemoclastic escherichia coli (P0.01 ).There was synergism between coptis root and enrofloxacin.
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Background: The level of pollution in Indonesia is still very high, consist of water pollution, air pollution and soil pollution. Mercury is one of the heavy metals that pollutes the waters of the sea, while Escherichia coli is exposed to mercury will try to defend itself by doing mercury detoxification so that it can live in an environment that contains mercury. Escherichia coli that tries to defend itself from mercury exposure in the environment will experience a change in its genes into mercury resistant Escherichia coli. In plasmids or transposons, it might also stimulate the formation of resistance genes for some antibiotics, include producing the ESBL enzyme, so that it can convert non ESBL Escherichia coli into ESBL Escherichia coli. Objective: This study aims to prove that the repeated exposure of mercury will change non ESBL-mercury sensitive Escherichia coli into ESBL- mercury resistant Escherichia coli. Method: This was an experimental study with 27 non-ESBL Escherichia coli isolates as identified from Phoenix. Non-ESBL Escherichia coli clinical isolates were tested by giving exposure to HgCl2 with concentrations of 0.02 ppm, 0.10 ppm, 0.20 ppm for 1-14 days until mercury resistant Escherichia coli was formed, and then ESBL screening was tested by giving Cefotaxime exposure to them. Results: On the first day of mercury exposure, there were 9 isolates of 0.02 ppm HgCl2 resistant Escherichia coli, 9 isolates of 0.10 ppm HgCl2 resistant Escherichia coli, 9 isolates of 0.20 ppm HgCl2 resistant Escherichia coli. Furthermore, this Escherichia coli isolate was exposed to Cefotaxim as ESBL screening. The final results of post-exposure HgCl2 0.02 ppm was obtained 3 (33.3%) isolates were still sensitive to Cefotaxime and 6 (66.7%) isolates that were resistant to Cefotaxime. The final results of post-exposure HgCl2 0.10 ppm was obtained all 9 (100%) isolates that were resistant to Cefotaxime. The final results of post-exposure HgCl2 0.20 ppm obtained 2 (22.2%) isolates were still sensitive to Cefotaxime and 7 (77.8%) isolate were resistant to Cefotaxime. Conclusion: Escherichia coli in urine had the phenotive change into mercury resistant Escherichia coli. Mercury exposure of 0.02 ppm, 0.10 ppm, 0.20 ppm for 1 day in vitro on isolates of non ESBL-mercury resistant Escherichia coli caused changes in 22 isolates of Escherichia coli in urine
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Amylolytic enzymes are one of the oldest industrial enzymes and hold the maximum market share of enzyme sales. With the advent of new frontiers in biotechnology the spectrum of the application of the enzymes have expanded to major area of industry. Among different amylolytic enzymes, α amylase, β amylase and glucoamylase are the most useful and best known enzymes. This review focuses on the sources, structure-function and some important properties of the three amylolytic enzymes. Research is focused on improving enzymes, modifying them to achieve desired properties of the three enzymes.α-amylase uses double displacement mechanism whereas β-amylase and glucoamylase use single displacement mechanism.The active site of α-amylase contains a trio of acidic group s, β-amylase contains thiol groups, glucoamylase contains tryptophan.
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Актуальність. В Україні з кожним роком відзначається збільшення кількості жінок, вагітність яких перебігає на тлі хронічних інфекційних захворювань. Escherichia coli є частим збудником бактеріальних інфекцій у жінок. Метою дослідження було виявлення морфологічних особливостей нирок плодів і новонароджених від матерів з експериментальним абдомінальним підгострим інфекційно-запальним процесом, викликаним Escherichia coli. Матеріали та методи. Авторами проведено експеримент на щурах лінії WAG, в ході якого сформовано дві групи: група I — 7 плодів та 11 новонароджених від 3 здорових самок; група II — 10 плодів і 13 новонароджених від 4 самок, яким моделювали абдомінальний інфекційно-запальний процес, викликаний Escherichia coli. Матеріалом дослідження послужили нирки плодів та новонароджених. Використовували гістологічні, гістохімічні, морфометричні та статистичні методи досліджень. Результати. Абдомінальний підгострий інфекційно-запальний процес в організмі матері, викликаний Escherichia coli, призводить до структурних змін у паренхіматозному та стромальному компонентах нирок, які наростали від плода до новонародженого. Гломерулярний апарат нирок характеризується нерівномірним розташуванням у кірковому шарі, затримкою розвитку, зміною форми, гемодинамічними змінами, розширенням сечового простору, відсутністю судинних клубочків, зменшенням кількості і компактності розташування капілярних петель в деяких молодих і зрілих ниркових тільцях; тубулярний апарат — затримкою розвитку, зміною форми та вогнищевим потовщенням базальних мембран окремих канальців, вогнищевими дистрофічними, некротичними та десквамативними змінами епітеліальної вистилки; стромальний компонент — склеротичними змінами, гемодинамічними порушеннями, більш вираженими в мозковому шарі, клітинною інфільтрацією, яка характеризується наявністю клітин фібробластичного ряду та імунних клітин. Висновки. Гістологічні та морфометричні зміни, що розвинулися в нирках плодів і новонароджених, зумовлені наявністю у матері експериментального підгострого інфекційно-запального процесу в черевній порожнині, викликаного Escherichia coli, призведуть в майбутньому до зниження функціональних можливостей нирок як гомеостатичного органу і розвитку різної нефрологічної патології у таких дітей.
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To investigate the mechanism of drug resistance of biofilm escherichia coli.The model of escherichia coli biofilm was established with the flat-board method. And the biofilm was confirmed by scanning electron microscopy. The beta-lactamase activities were quantitated, in escherichia coli, biofilm escherichia coli, biofilm escherichia coli induced by impenem or cefoxitin.The beta-lactamase activity of biofilm escherichia coli was 2.16 times as much as that of escherichia coli planctonically, and the beta-lactamase activities of biofilm escherichia coli induceded by impenem or cefoxitin were 1.30 and 1.05 times as much as those of biofilm escherichia coli, respectively.The drug resistance to antibiotics of biofilm escherichia coli was related to the production of beta-lactamase.
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Objective To investigate the mechanism of drug resistance of biofilm escherichia coli .Methods The model of escherichia coli biofilm was established with the flat board method And the biofilm was confirmed by scanning electron microscopy The β lactamase activities were quantitated in escherichia coli, biofilm escherichia coli , biofilm escherichia coli induced by impenem or cefoxitin. Results The β lactamase activity of biofilm escherichia coli was 2 16 times as much as that of escherichia coli planctonically, and the β lactamase activities of biofilm escherichia coli induceded by impenem or cefoxitin were 1 30 and 1 05 times as much as those of biofilm escherichia coli ,respectively.Conclusion The drug resistance to antibiotics of biofilm escherichia coli was related to the production of β lactamase.
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[Objective]The damage in the cell membrane of bacteria could be reflected on the leakiness of the material in bacteria and the absorption of dye in bacteria.Compared with pasteurization(63 °C,30 min),the damaging effect of pressurized CO2 on the cell membrane of Escherichia coli was studied.The aim of the study was to analyze the relationship between the death of Escherichia coli and the damage of cell membrane.[Methods]The change of cell membrane permeability and the leakiness of protein and nucleic acid in Escherichia coli were detected.The change of Escherichia coli morphology was observed by TEM.[Results]The results indicated that pressurized CO2 treatment induced the change of cell membrane permeability of Escherichia coli.Pressurized CO2 treatment induced the leakiness of protein in Escherichia coli,but the time of leakiness was lagged behind the time of 99% Escherichia coli death,so it was not the reason for death,it was only the secondary phenomenon of death.The death of Escherichia coli could be related to the leakiness of nucleic acid induced by the pressurized CO2 treatment.The death of Escherichia coli could be related to the ultrastructure change of Escherichia coli induced by the pressurized CO2 treatment.[Conclusion]There was direct relationship between the damaging effect of pressurized CO2 on cell membrane of Escherichia coli and the death of Escherichia coli.
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Host, vector, and culture conditions (including cultivation media) are considered among the three main elements contributing to a successful production of recombinant proteins. Accordingly, one of the most common hosts to produce recombinant therapeutic proteins is Escherichia coli.A comprehensive literature review was performed to identify important factors affecting production of recombinant proteins in Escherichia coli.Escherichia coli is taken into account as the easiest, quickest, and cheapest host with a fully known genome. Thus, numerous modifications have been carried out on Escherichia coli to optimize it as a good candidate for protein expression and; as a result, several engineered strains of Escherichia coli have been designed. In general; host strain, vector, and cultivation parameters are recognized as crucial ones determining success of recombinant protein expression in Escherichia coli. In this review, the role of host, vector, and culture conditions along with current pros and cons of different types of these factors leading to success of recombinant protein expression in Escherichia coli were discussed.Successful protein expression in Escherichia coli necessitates a broad knowledge about physicochemical properties of recombinant proteins, selection among common strains of Escherichia coli and vectors, as well as factors related to media including time, temperature, and inducer.
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A total of nine Escherichia coli O157:H7 isolates were originally isolated from imported Indian beef purchased from local retail markets. These nine Escherichia coli O157:H7 isolates were confirmed as Escherichia coli O157:H7 by their positive growth characteristics on Rainbow agar O157 and PCR assay for the detection of the presence of the H7 (fliC) gene unique to Escherichia coli O157:H7. Published specific primers FLICH (625 bp) of the fliC gene encoding the H7 antigen were utilized in the specific PCR assay. Three of the Escherichia coli O157:H7 isolates were selected for the amplification of their H7 (fliC) gene. The amplicon of the PCR assay were successfully cloned into pGEM-T vector and sequenced. The sequence of the Escherichia coli O157:H7 that was obtained from sequencing was analyzed. This sequence was identified using Basic Local Alignment Search Tools (Blast) program. Based on the sequence obtained, primer pairs were designed to detect the Escherichia coli O157:H7, but only Sk7 and Sk8 were found to be specific for detection of locally isolated Escherichia coli O157:H7. Sk7 and Sk8 produced an amplicon size of 520 bp and 603 bp respectively against all tested locally isolated Escherichia coli O157:H7. Both new primers were specific against Escherichia coli O157:H7, as they did not produce any amplicons from the 32 isolats representing 20 different bacterial species. DNA hybridization analysis of 10 other bacterial species, including E. coli, Salmonella and other isolates showed that the probe (SkP) reacted only with Escherichia coli O157:H7 isolates.
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Escherichia
Primer (cosmetics)
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