Purification and characterization of two novel proteolytic enzymes in membranes of Escherichia coli. Protease IV and protease V.
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MI06Escherichia coli cells contain two membrane-bound esterases, one of which is localized in both inner and outer membranes and the other present only in the inner membrane (Pacaud, M. (1982) J. Bacteriol.149, in press).These two enzymes have been purified to homogeneity and shown to be capable of hydrolyzing a mixture of E. coli proteins.The inner membrane esterase has been called protease N ; the other esterase has been designated protease V. Protease N migrates as a single band on sodium dodecyl sulfate gels with an apparent Mr = 34,000.However, this enzyme displays an apparent M, = 660,000 on Ultrogel columns in the presence of Emulphogen BC-720.The large difference in size of the native enzyme appears to be due to its association with the detergent.The molecular weight of protease V could not be determined because of the interactions of this enzyme with detergents.ProteaseKeywords:
Proteolytic enzymes
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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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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The methods of determining the activities of proteolytic enzymes used in Leather making, such us Folin method, Lhlein-Volard method (LVU) and the method of transforming coefficient of trypsinase to casein, were introduced. The above three methods were applied to determine 21 proteolytic enzymes analysis,which are used in leather making processes, including high content trypsinases, microorganic proteolytic enzymes and some industrial leather enzymes such as soaking enzymes, liming enzymes , bating enzymes as well. The results show that the activity values of the Folin method and the LVU method reacting under the same pH and temperature (pH8.2, temperature 37℃) are almost the same.
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Ascaris suum
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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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The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.
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