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PublicationRSC Advances
Pages20639 - 20649

Characterization of supports activated with divinylsulfone as a tool to immobilize and stabilize enzymes via multipoint covalent attachment. Application to chymotrypsin

Authors:José Cleiton Sousa dos Santos, Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Jose C. S. dos Santosa,b,  Nazzoly Ruedaa,c,  Oveimar Barbosad, Jorge F. Fernández-Sáncheze, Antonio L. Medina-Castillof, Teresa Ramón-Márqueze,  María C. Arias-Martosf , Mª Carmen Millán-Linaresg, Justo Pedrocheg, María del Mar Yustg, 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: Department of Analytical Chemistry University of Granada Avd. Fuentenueva s/n, 18071 Granada, Spain.
f: NanoMyP, Nanomateriales y Polimeros S.L. Spin-Off company of the UGR, BIC building Avd. Innovacion 1, 18100, Granada, Spain.
g: Instituto de la Grasa-CSIC. Av Padre García Tejero 4. 41012 Sevilla. Spain.
Divinylsulfone (DVS) has been used to activate agarose beads. The DVS activated agarose resulted quite stable in the pH range 5-10 at 25ºC under wet conditions, and can react rapidly with α-amides of Cys and His, at pH 5-10, with Lys mainly at pH 10 and with Tyr in a much slower fashion. After blocking with different nucleophiles, the support lost all reactivity, confirming that this protocol could be useful as an enzyme-support reaction end point. Then, chymotrypsin was immobilized on this support at pH 5, 7 and 10. Even though the enzyme was immobilized at all pH values, the immobilization rate decreased with the pH value. The effect of the immobilization on the activity depended on the immobilization pH, at pH 7 the activity decreased (to 50%) more than at pH 10 (by a 25%), while at pH 5 the immobilization has not effect. Then, the effect of the blocking with different reagents was analyzed. It was found that the blocking with ethylenediamine improved the enzyme activity (by a 70%) and gave the best stability. The stability of all enzyme preparations improved when 24 h incubation was performed at pH 10, but the qualitative stabilization depended on the inactivation condition. The analysis of aminoacids of the preparation immobilized at pH 10 showed that Lys, Tyr and Cys residues were involved in the immobilization, involving a minimum of 10 residues (glyoxyl agarose gave 4 Lys involved in the immobilization). The new preparation was 4-5 fold more stable than glyoxyl agarose preparation, considered a very stable one, and in some instances was more active than the free enzyme (170% for the enzyme immobilized at pH 10). Thus, DVS activated supports are very promising to permit the multipoint covalent attachment of enzymes, and that way to improve their stability.

Keywords:Enzyme immobilization, versatile immobilization, blocking of the support, multipoint covalent attachment, divinyl sulfone, enzyme stabilization.
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