Objective: To increase the thermal stability of sucrose isomerase from Erwinia rhapontici NX-5, we designed a comprehensive strategy that combines different thermostabilizing elements.Results: We identi ed 19 high B value amino acid residues for site-directed mutagenesis. An in silicoevaluation of the in uence of post-translational modi cations on the thermostability was also carried out. The sucrose isomerase variants were expressed in Pichia pastoris X33. Thus, for the rst time, we report the expression and characterization of glycosylated sucrose isomerases. The designed mutants K174Q, L202E and K174Q/L202E, showed an increase in their optimal temperature of 5 °C, while their half-lives increased 2.21, 1.73 and 2.89 times, respectively. The mutants showed an increase in activity of 20.3% up to 25.3%. The Km values for the K174Q, L202E, and K174Q/L202E mutants decreased by 5.1%, 7.9%, and 9.4%, respectively; furthermore, the catalytic e ciency increased by up to 16%.Conclusions: With the comprehensive strategy followed, we successfully obtain engineered mutants more suitable for industrial applications than their counterparts: native (this research) and wild-type from E. rhapontici NX-5, without compromising the catalytic activity of the molecule.