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PublicationRSC Advances
Pages11212 - 11222


Authors:José Cleiton Sousa dos Santos, Roberto Fernandez-Lafuente
Groups of research:Optimization of biocatalysts and bioprocesses
Nazzoly Ruedaa,b, Jose C. S. dos Santosa,c, Rodrigo Torresb,d,
Claudia Ortize, Oveimar Barbosaf*, 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 Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil
d Current address: Laboratorio de Biotecnología, Instituto Colombiano del Petróleo-Ecopetrol, Piedecuesta, Bucaramanga, Colombia.
eEscuela de Bacteriología y Laboratorio Clínico, Universidad Industrial de Santander, Bucaramanga, Colombia.
f Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia

A new heterofunctional support, octyl-glyoxyl agarose, is proposed in this study. The supports were prepared by simple periodate oxidation of the commercial octyl-agarose, introducing 25 µmol of glyoxyl groups per wet gram of support. This support was assayed with three different lipases (those from Candida antarctica (form B), Thermomyces lanuginosus (TLL) or Rhizomucor miehei) and the artificial phospholipase Lecitase Ultra. Used at pH 7, the new support maintained as first immobilization step the lipase interfacial activation. Thus,  it was possible to have the  purification and immobilization of the enzyme in one step. Moreover, stabilization of the open form of the lipase was achieved. The covalent enzyme/support bonds cannot be obtained if the immobilized enzyme was not incubated at alkaline pH value. This incubation at pH 10 of the previously immobilized enzymes produced a smaller decrease in enzyme activity when compared to the direct immobilization of the enzymes on glyoxyl-agarose at pH 10, because the immobilization via interfacial activation promoted a stabilization of the lipases. Except in the case of TLL (covalent attachment involved 70% of the enzyme molecules), covalent immobilization yield was over 80%. The non-covalent attached enzyme molecules were discarded by washings with detergent solutions and the new biocatalysts were compared to the octyl-agarose immobilized enzymes. While the stability in thermal and organic solvents inactivations was increased for Lecitase Ultra, CALB and RML, TLL improved its stability in organic media but its thermal stability decreased after covalent attachment of the interfacially activated enzyme. This stabilization resulted in octyl-glyoxyl-lipase preparations which presented higher activity in the presence of organic solvents. Finally, while octyl-agarose released enzyme molecules after incubation at high temperatures or in the presence of organic solvents and detergents, the covalently immobilized enzyme remained attached to the support even after boiling the enzyme in SDS, eliminating the risks of product contamination
Keywords:covalent immobilization, lipase, heterofunctional supports, interfacial activation, phospholipase, glyoxyl supports, octyl supports
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