2024-03-29T14:00:30Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/1690032020-01-15T09:26:27Zcom_10261_101com_10261_5com_10261_68com_10261_2col_10261_354col_10261_321
Orrego, Alejandro H.
Romero-Fernández, Maria
Millán-Linares, María del Carmen
Yust, María del Mar
Guisán, José Manuel
Rocha-Martín, Javier
2018-08-22T08:31:48Z
2018-08-22T08:31:48Z
2018-08-15
Catalysts 8(8): 333 (2018)
2073-4344
http://hdl.handle.net/10261/169003
10.3390/catal8080333
http://dx.doi.org/10.13039/501100000780
http://dx.doi.org/10.13039/501100010198
Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 ◦C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts.
http://creativecommons.org/licenses/by/4.0/
openAccess
Enzyme immobilization
2-picoline borane
Biocatalysis
Glyoxyl agarose
Schiff base
Enzyme stabilization
Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent
artículo