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PublicationPROCESS BIOCHEMISTRY
Year2016
Volume51
Pages48-52
International

IMPROVED IMMOBILIZATION AND STABILIZATION OF LIPASE FROM Rhizomucor miehei ON OCTYL-GLYOXYL AGAROSE BEADS BY USING CaCl2.

Authors:José Cleiton Sousa dos Santos, Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Laura Fernandez-Lopeza,+, Nazzoly Ruedaa,b;+,  Rocio Bartolome-Cabreroa, Maria D. Rodrigueza,c, Tiago L. Albuquerquea,d, Jose C.S. dos Santosa,d, Oveimar Barbosae  and Roberto Fernandez-Lafuentea*
 
a Departamento de Biocatálisis. Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid. Spain.
b  Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
c Laboratorio de Biotecnología Molecular. Instituto de Biotecnología Misiones "María Ebe Reca". (FCEQyN – UnaM). Posadas, Argentina.
d Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
e Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia


A strategy to stabilize octyl (OC)-Rhizomucor miehei lipase (RML) and OC-Candida rugosa lipase (CRL) at pH 10 is necessary to take full advantage of the immobilization of these enzymes on OC-glyoxyl (OCGLX) supports. CaCl2 and MnCl2 have been reported to stabilize OC-RM and OC-CRL at pH 5.0 and 7.0. After screening different buffers, 5 mM CaCl2 was found to be fully soluble in Gly at pH 10. OC-RML was 15 folds stabilized at this pH value by CaCl2, while OC-CRL was not. This salt was used in the preparation of octyl-glyoxyl (OCGLX)-RML, permitting to maintain almost unaltered the OC-RML activity that was 3 fold higher than that of the free. This preparation was 30% and 3 folds more stable in thermal or acetonitrile inactivations respectively  than the standard  OCGLX one.

The stabilizing effect of CaCl2 and MnCl2 on the OCGLX preparations was studied. These salts stabilized both OCGLX-RML preparations, although the one prepared using Ca2+ during the covalent attachment was more stabilized than the standard one by the presence of Ca2+, even 7-8 folds in the presence of aceonitrile.  Thus, this additive permits to recover an OCGLX-RML preparation more stable and active than the standard protocol.
Keywords:covalent immobilization, Lipase stabilization, cations, lipase interfacial activation, octyl-glyoxyl, stabilizing additives
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