RT Journal Article
T1 Designing tailor-made steric matters to improve the immobilized ficin specificity for small versus large proteins
A1 Siar, El Hocine
A1 Abellanas Pérez, Pedro
A1 Morellon Sterling, Roberto
A1 Bolívar Bolívar, Juan Manuel
A1 Rocha Martín, Javier
A1 Fernandez Lafuente, Roberto
AB The development of strategies that can permit to adjust the size specificity of immobilized proteases by the generation of steric hindrances may enlarge its applicability. Using as a model ficin immobilized on glyoxyl agarose, two strategies were assayed to generate tailor made steric hindrances. First, ficin has been coimmobilized on supports coated with large proteins (hemoglobin or bovine serum albumin (BSA)). While coimmobilization of ficin with BSA presented no effect on the activity versus any of the assayed substrates, coimmobilization with hemoglobin permitted to improve the immobilized ficin specificity for casein versus hemoglobin, but still significant activity versus hemoglobin remained. Second, aldehyde-dextran has been employed to modify the immobilized ficin, trying to generate steric hindrances to avoid the entry of large proteins (hemoglobin) while enabling the entry of small ones (casein). This also increased the size specificity of ficin, but still did not suppress the activity versus hemoglobin. The combination of both strategies and the use of 37ºC during the proteolysis enabled to almost fully nullify the hydrolytic activity versus hemoglobin while preserving a high percentage of the activity versus casein. The modifications improved enzyme stability and the biocatalyst could be reused for 5 cycles without alteration of its properties.
PB Elsevier
SN 0168-1656
YR 2024
FD 2024
LK https://hdl.handle.net/20.500.14352/111933
UL https://hdl.handle.net/20.500.14352/111933
LA eng
NO Siar, E. H., Abellanas-Perez, P., Morellon-Sterling, R., Bolivar, J. M., Rocha-Martin, J., & Fernandez-Lafuente, R. (2024). Designing tailor-made steric matters to improve the immobilized ficin specificity for small versus large proteins. Journal of Biotechnology, 395, 12-21. https://doi.org/10.1016/j.jbiotec.2024.09.005
NO This research was funded by Ministerio de Ciencia e Innovación and Agencia Estatal de Investigación (Spanish Government) (PID2022–136535OB-I00). We gratefully recognize Prof. Ángel Berenguer-Murcia for his suggestions and help during the writing of this paper.
NO Ministerio de Ciencia, Innovación y Universidades (España)
DS Docta Complutense
RD 29 ago 2025