English   español  
Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/122931
Share/Impact:
Statistics
logo share SHARE logo core CORE   Add this article to your Mendeley library MendeleyBASE

Visualizar otros formatos: MARC | Dublin Core | RDF | ORE | MODS | METS | DIDL
Exportar a otros formatos:

Title

Bovine trypsin immobilization on agarose activated with divinylsulfone: Improved activity and stability via multipoint covalent attachment

AuthorsDos Santos, José C. S.; Rueda, Nazzoly; Barbosa, Oveimar ; Millán-Linares, María del Carmen ; Pedroche, Justo ; Yuste, María del Mar; Gonçalves, Luciana R. B.; Fernández-Lafuente, Roberto
Issue Date2015
PublisherElsevier
CitationJournal of Molecular Catalysis B: Enzymatic 117: 38- 44 (2015)
Abstract© 2015 Elsevier B.V. All rights reserved. Trypsin has been immobilized on divinyl sulfone (DVS) activated agarose at pH 5, 7 and 10. While at pH 5 and 7 immobilization was slow and presented a negative effect on enzyme activity, the immobilization at pH 10 produced a significant increment of activity (by a 24 fold factor). Using this preparation, the effect on enzyme activity/stability of different blocking reagents (used as an enzyme-support reaction end point) were evaluated, selecting ethylenediamine (EDA) because it produced an increase in enzyme activity (a 4 fold factor) and the best results in terms of stability. Next, the effect of alkaline incubation on enzyme activity/stability before the blocking step was analyzed. Activity decreased by 40% after 72 h (but it should be considered that previously it had increased by a 24 fold factor), but the stability significantly improved after this incubation. Thus, after immobilization at different pH values, the immobilized trypsin was submitted to 72 h of alkaline incubation and blocked with EDA. The most active and stable preparation was that immobilized at pH 10. This preparation was less stable than the glyoxyl preparation in thermal inactivations (by less than a twofold factor), but was more stable in organic solvent inactivation (also by less than a twofold factor). The number of groups involved in the enzyme support attachment was 6 Lys using glyoxyl and became a minimum of 13 (including Lys, Tyr and His) using the DVS-activated support (the precision of the method did not permit to analyze the implication of some of the 3 terminal amino groups). Thus, this DVS-agarose support seems to be a very promising support to permit a very intense enzyme-support multipoint covalent attachment.
URIhttp://hdl.handle.net/10261/122931
DOI10.1016/j.molcatb.2015.04.008
Identifiersdoi: 10.1016/j.molcatb.2015.04.008
issn: 1873-3158
Appears in Collections:(IG) Artículos
Files in This Item:
File Description SizeFormat 
Postprint_2015_JMolCatB_V117_P38.pdf617,51 kBAdobe PDFThumbnail
View/Open
Show full item record
Review this work
 

Related articles:


WARNING: Items in Digital.CSIC are protected by copyright, with all rights reserved, unless otherwise indicated.