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PublicationJournal of Molecular Catalysis B: Enzymatic
Year2015
Volume117
Pages38-44
International

BOVINE TRYPSIN IMMOBILIZATION ON AGAROSE ACTIVATED WITH DIVINYLSULFONE: IMPROVED ACTIVITY AND STABILITY VIA MULTIPOINT COVALENT ATTACHMENT

Authors:Roberto Fernandez-Lafuente, José Cleiton Sousa dos Santos
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Jose C. S. dos Santosa,b,  Nazzoly Ruedaa,c,  Oveimar Barbosad, Maria del Carmen Millán-Linarese, Justo Pedrochee, María del Mar Yustee, Luciana R.B. Gonçalvesb and Roberto Fernandez-Lafuentea*.
a: ICP-CSIC. Campus UAM-CSIC. Cantoblanco. 28049 Madrid. Spain.
b: Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
c: Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia
d: Facultad de Ciencias, departamento de Química, Universidad del Tolima, Ibagué, Colombia
e: Instituto de la Grasa-CSIC. Av Padre García Tejero 4. 41012 Sevilla. Spain.
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.


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