Increasing the thermostability of D-xylose isomerase by introduction of a proline into the turn of a random coil

Thermostability can be increased by introducing prolines atsuitable sites in target proteins. Two single (G138P, G247D)mutants and one double (G138P/G247D) mutant of xylose isomerasefrom Streptomyces diastaticus No.7, strain M1033 have been constructedby site-directed mutagenesis. With respect to the wild-typeenzyme, G138P showed about a 100% increase in thermostability,and G247D showed an increased catalytic activity. Significantly,the double mutant, G138P/G247D displayed even higher activitythan G247D and better heat stability than G138P. Its half lifewas about 2.5-fold greater than the wild-type enzyme, usingxylose as a substrate. Molecular modelling suggested that theintroduction of a proline residue in the turn of a random coilmay cause the surrounding conformation to be tightened by reducingthe 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 doublemutant indicated that the advantages of two single mutants canbe combined effectively.