RESUMO
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 identified 19 high B value amino acid residues for site-directed mutagenesis. An in silico evaluation of the influence of post-translational modifications on the thermostability was also carried out. The sucrose isomerase variants were expressed in Pichia pastoris X33. Thus, for the first 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 efficiency 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.
Assuntos
Glucosiltransferases , Sacarose , Glucosiltransferases/metabolismo , Temperatura , Mutagênese Sítio-Dirigida , Estabilidade Enzimática , Cinética , Sacarose/químicaRESUMO
We present theoretical and experimental evidence of an anomalous surface corrugation behavior in He-KCl(001) for incidence along ⟨110⟩. When the He normal energy decreases below 100 meV, i.e., He-surface distances Z>2 Å, the corrugation unexpectedly increases up to an impressive â³85%. This is not due to van der Waals interactions but to the combination of soft potential effects and the evolution of He-cation and He-anion interactions with Z. This feature, not previously analyzed on alkali-halide surfaces, may favor the alignment properties of weakly interacting overlayers.