LOWERING THE PH OPTIMUM OF D-XYLOSE ISOMERASE - THE EFFECT OF MUTATIONS OF THE NEGATIVELY CHARGED RESIDUES

Streptomyces rubiginosus D-xylose isomerase catalyzes the reversible i somerization of D-glucose to D-fructose. The isomerization reaction is maximized in the alkaline region of pH 8.5-8.8. The amino acid residu es around two active site histidines (His-54 and His-220) and on the s urface of the enzyme were mutated to improve the catalytic efficiency at neutral pH. The mutations have been made by removing the negatively charged residues based upon the sequence comparison of other D-xylose isomerases and the hypothesis proposed by Russell anal Fersht (1987). The effects of these substitutions on kinetic parameter, pH dependenc e, and thermostability were characterized, The k(cat) values for D56N and E221A mutants on D-glucose are increased by 30-40% over that of th e wild-type enzyme at pH 7.3 and the increased activities are maintain ed between pH 6 and 7.5. However, the surface mutants D65A, D81A, and D163N/E167Q only show 40-60% of the wild-type activity over the entire pH range. The pH activity profiles of the mutants are broader than th at of the wild-type enzyme. The optimum pHs and the pK(a) values for a ll the mutants are lowered by 0.5-0.8 and 0.1-0.5 units, respectively, The small Delta(Delta G) and high T-m valles for all the mutants indi cate that there is no significant change ire the hydrogen bond network in the active site by mutations. These results indicate that D56N and E221A are possible candidates as good catalysts for High-Fructose Cor n Syrup (HFCS) production.