Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH |
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| Authors: | Sriprapundh, Dinlaka Vieille, Claire Zeikus, J.Gregory |
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| Affiliation: | 1Department of Food Science and Human Nutrition and
2Department of Biochemistry and Molecular Biology, Michigan State University, 410 Biochemistry Building, East Lansing, MI 48824, USA
3Present address: Department of Pharmaceutical Chemistry and Biochemistry/Biophysics, University of California, San Francisco, CA 94143, USA |
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| Abstract: | The Thermotoga neapolitana xylose isomerase (TNXI) is extremelythermostable and optimally active at 95°C. Its derivative,TNXI Val185Thr (V185T), is the most active type II xylose isomerasereported, with a catalytic efficiency of 25.1 s1 mM1toward glucose at 80°C (pH 7.0). To further optimize TNXIspotential industrial utility, two rounds of random mutagenesisand low temperature/low pH activity screening were performedusing the TNXI V185T-encoding gene as the template. Two highlyactive mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S).1F1 was more active than 3A2, which in turn was more activethan TNXI V185T at all temperatures and pH values tested. 3A2and 1F1s high activities at low temperatures were dueto significantly lower activation energies (57 and 44 kJ/mol,respectively) than that of TNXI and V185T (87 kJ/mol). MutationL282P introduced a kink in helix |
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