ABSTRACT
The toxicity and biodegradability of the main hydrolysis products of chemical warfare agents were investigated under methanogenic conditions. Among the tested substances, only MPhA does not have any toxic effect with regard to the aceticlastic methanogenic activity. The toxicity of other compounds varied between moderate (TDG, mercaptoethanol) to strong (ethanolamine, diisobutyl ester of MPhA). Biodegradability tests showed that all the products of chemical detoxification of mustard gas (ethanolamine, ethylene glycol, TDG, mercaptoethanol) can be biomineralized under methanogenic conditions. On the contrary, phosphorus-containing compounds from the chemical detoxification of nerve warfare agents (Sarin, Soman, Vx-gases) are quite persistent under these conditions.
Subject(s)
Bacteria, Anaerobic/metabolism , Chemical Warfare Agents/metabolism , Biodegradation, Environmental , Chemical Warfare Agents/toxicity , HydrolysisABSTRACT
The endoglucanase (EG5) has been isolated from the recombinant strain of E. coli TG1 carrying a plasmid with C. thermocellum F7 chromosomal DNA insertion. Using high performance ion-exchange chromatography and chromatofocusing, the enzyme was 98-fold purified with a 27% yield. The enzyme has a molecular mass of 35 kDa (SDS-PAGE data) and is represented by three isoforms with pI 4.4-4.8 (isoelectrofocusing data). The activity of EG5 was determined by chromatography on carboxymethylcellulose, amorphous cellulose, xylan, lichenan and avicel. The optimal conditions for these substrates hydrolysis are: pH 6.0-6.5, 80 degrees C (60 degrees C for avicel).
Subject(s)
Cellulase/isolation & purification , Clostridium/enzymology , Escherichia coli/genetics , Cellulase/metabolism , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Plasmids , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Endoglucanase 5 (EG5) has been isolated from the strain of E. coli TG1 harboring recombinant plasmid pCU108, which contains the cel5 gene of C. thermocellum. The enzyme has been produced with 98-fold purification and a final yield of 27% by using subsequent twofold high performance ion-exchange chromatography on Mono Q and high performance chromatofocusing on Mono P. The protein has a mol mass of 35 kDa and includes 3 multiple forms with pI 4.4-4.8 as evidenced by analytical gel isoelectrofocusing. EG5 cleaves CMC (Km = 0.097 g/L, Vmax = 8.2 mg/min.mg of protein), amorphous cellulose, xylan, lichenan as a substrate with an optimum temperature of 80 degrees C and pH 6.0 and Avicel (Km = 18.2 g/L, Vmax = 0.035 mg/min.mg of protein) with an optimum temperature of 60 degrees C and pH 6.0. Cellobiose in concentrations up to 200 micrograms/mL do not inhibit the hydrolysis of CMC by EG5, but 10-30 micrograms/mL of glucose significantly decrease the activity of this enzyme. The stimulating role of calcium chloride and concentration of protein in the system has been demonstrated for Avicel hydrolysis by EG5.
Subject(s)
Cellulase/isolation & purification , Clostridium/enzymology , Escherichia coli/genetics , Cellobiose/pharmacology , Cellulase/chemistry , Cellulase/metabolism , Cellulose/metabolism , Chromatography, Ion Exchange , Clostridium/genetics , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Escherichia coli/enzymology , Glucose/pharmacology , Hydrogen-Ion Concentration , Isoelectric Point , Molecular Weight , Plasmids/genetics , Recombinant Proteins/biosynthesis , Substrate Specificity , TemperatureABSTRACT
The kinetics of growth and metabolism of Clostridium thermosaccharolyticum DSM 571 has been studied at varying initial pH and glucose concentration. A weak inhibitory effect of excess glucose on the specific growth rate has been shown. The effect of antibiotics of various classes on culture growth and hydrogen evolution has been studied. Streptomycin and kanamycin resistance of this culture has been revealed as well as the phenomenon of increased hydrogen production in the presence of the above antibiotics. New plasmids, pNB1 (4.9 kb) and pNB2 (2.0 kb), were isolated from C. thermosaccharolyticum DSM 571. The restriction analysis of pNB1 and pNB2 has been performed.
