An improved assay method for a pseudomurein-degrading enzyme of Methanobacterium wolfei and the Protoplast formation of Methanobacterium thermoautotrophicum by the enzyme |
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| Institution: | 1. Key Laboratory of Coalbed Methane Resources and Reservoir Formation Process, Ministry of Education, China University of Mining and Technology, Xuzhou 221008, China;2. School of Resources and Geosciences, China University of Mining and Technology, Xuzhou 221116, China;1. Engineering Research Center of Technical Textiles, Ministry of Education, Donghua University, Shanghai 201620, PR China;2. College of Textiles, Donghua University, Shanghai 201620, China;3. Department of Industrial and Systems Engineering, Texas A&M University, College Station, TX 77843, USA;1. Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA;2. Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, USA;3. Energy Research Institute, Montana State University, Bozeman, MT 59717, USA;4. Department of Hydrology and Atmospheric Sciences, University of Arizona, Tucson, AZ 85721, USA;5. U.S. Geological Survey, Wyoming-Montana Water Science Center, Helena, MT 59601, USA;6. Department of Geography and Earth Sciences, University of North Carolina, Charlotte, NC 28223, USA;7. U.S. Geological Survey, Eastern Energy Resources, Reston, VA 20192, USA;1. Department of Civil and Environmental Engineering, 1230 Lincoln Drive, Southern Illinois University, Carbondale, IL 62901, USA;2. Materials Technology Center, 1230 Lincoln Drive, Southern Illinois University, Carbondale, IL 62901, USA |
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| Abstract: | An improved assay method of a pseudomurein-degrading enzyme and its properties are described. The pseudomurein-degrading enzyme purified from Methanobacterium wolfei autolysate under an anoxic condition was assayed with the cell wall of Methanobacterium thermoautotrophicum as a substrate. By this improved method the enzyme activity was measured quantitatively and reproducibly. Moreover, the cell wall substrate can be stored in a freezer and used as needed, and the time required for an assay was as short as 1 h. The optimum pH and temperature of the enzyme was pH 6.8-7.4 and 75°C, respectively. Although the enzyme lost 50% of the activity upon heating at 75°C for 10 min in the absence of the cell wall substrate, it was more stable against heat inactivation in the presence of the substrate. Furthermore the inactivated enzyme recovered some of the activity by incubating with the substrate. Although the enzyme lost most of the activity under aerobic conditions, the activity was recovered under reducing conditions with Na2S·9H2O or DTT (dithiothreitol). The enzyme was also purified under aerobic conditions retaining the same specific activity as the anoxically purified enzyme. Using the partially purified enzyme the conditions preparing protoplasts of M. thermoautotrophicum was established. |
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