Protein Engineering vol. 16 no. 9 pp. 683-690, 2003
© 2003 Oxford University Press
Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH
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
4 To whom correspondence should be addressed. e-mail: zeikus{at}msu.edu
The Thermotoga neapolitana xylose isomerase (TNXI) is extremely thermostable and optimally active at 95°C. Its derivative, TNXI Val185Thr (V185T), is the most active type II xylose isomerase reported, with a catalytic efficiency of 25.1 s–1 mM–1 toward glucose at 80°C (pH 7.0). To further optimize TNXI’s potential industrial utility, two rounds of random mutagenesis and low temperature/low pH activity screening were performed using the TNXI V185T-encoding gene as the template. Two highly active mutants were obtained, 3A2 (V185T/L282P) and 1F1 (V185T/L282P/F186S). 1F1 was more active than 3A2, which in turn was more active than TNXI V185T at all temperatures and pH values tested. 3A2 and 1F1’s high activities at low temperatures were due to significantly lower activation energies (57 and 44 kJ/mol, respectively) than that of TNXI and V185T (87 kJ/mol). Mutation L282P introduced a kink in helix 
7 of 3A2’s (/ß)8 barrel. Surprisingly, this mutation kinetically destabilized 3A2 only at pH 5.5. 1F1 displayed kinetic stability slightly above that of TNXI V185T. In 1F1, mutation F186S creates a cavity that disrupts a four-residue network of aromatic interactions. How the conformation of the neighboring residues is affected by this cavity and how these conformational changes increase 1F1’s stability still remain unclear.
Received February 21, 2003; revised June 24, 2003; accepted July 17, 2003.
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