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Thermostability improvement of sucrose isomerase PalI NX-5: a comprehensive strategy

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Abstract

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.

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Acknowledgements

AJSP thank the Consejo Nacional de Ciencia y Tecnología (CONACyT) for the Ph.D. studies grant.

Author information

Authors and Affiliations

  1. Laboratorio de Biotecnología I, Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito Universitarios s/n Nuevo Campus Universitario, C. P. 31125, Chihuahua, México

    A. J. Sardiña-Peña, T. S. Siqueiros-Cendón, E. A. Espinoza-Sánchez, B. F. Iglesias-Figueroa & Q. Rascón-Cruz

  2. Laboratorio de Química Analítica III, Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Circuito Universitarios s/n Nuevo Campus Universitario, C. P. 31125, Chihuahua, México

    L. Ballinas-Casarrubias

  3. Laboratorio Virtual NANOCOSMOS, Departamento de Medio Ambiente y Energía, Centro de Investigación en Materiales Avanzados, Chihuahua, México

    N. R. Flores-Holguín

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  1. A. J. Sardiña-Peña
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  2. L. Ballinas-Casarrubias
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  3. T. S. Siqueiros-Cendón
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  4. E. A. Espinoza-Sánchez
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  5. N. R. Flores-Holguín
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  6. B. F. Iglesias-Figueroa
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  7. Q. Rascón-Cruz
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Correspondence to Q. Rascón-Cruz.

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Sardiña-Peña, A.J., Ballinas-Casarrubias, L., Siqueiros-Cendón, T.S. et al. Thermostability improvement of sucrose isomerase PalI NX-5: a comprehensive strategy. Biotechnol Lett 45, 885–904 (2023). https://doi.org/10.1007/s10529-023-03388-6

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  • DOI: https://doi.org/10.1007/s10529-023-03388-6

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