Thermostability can be increased by introducing prolines at suitable sites in target proteins. Two single (G138P, G247D) mutants and one double (G138P/G247D) mutant of xylose isomerase from Streptomyces diastaticus No.7, strain M1033 have been constructed by site-directed mutagenesis. With respect to the wild-type enzyme, G138P showed about a 100% increase in thermostability, and G247D showed an increased catalytic activity. Significantly, the double mutant, G138P/G247D displayed even higher activity than G247D and better heat stability than G138P. Its half life was about 2.5-fold greater than the wild-type enzyme, using xylose as a substrate. Molecular modelling suggested that the introduction of a proline residue in the turn of a random coil may cause the surrounding conformation to be tightened by reducing the backbone flexibility. The change in thermostability can, therefore, be explained based on changes in the molecular rigidity. Furthermore, the improvements in the properties of the double mutant indicated that the advantages of two single mutants can be combined effectively.