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Reuse of lipase from Pseudomonas fluorescens via its step by step coimmobilization on glyoxyl-octyl agarose beads with less stable lipases

Autores:Roberto Fernandez-Lafuente
Grupos de investigación:Optimización de biocatalizadores y bioprocesos enzimáticos
Nathalia S. Rios1,2 +, Sara Arana-Peña1,+, Carmen Mendez- Sanchez1, Claudia Ortiz3,
Luciana R. B. Gonçalves2 and Roberto Fernandez-Lafuente1,*
1   Departamento de Biocatálisis. ICP-CSIC, Campus UAM-CSIC, Madrid, Spain. NSR, nathaliarios25@yahoo.com.br, SAP, s.arana@csic.es, CMS, cmende02@ucm.es, RFL, rfl@icp.csic.es
2   Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Bloco 709, CEP 60455-760, Fortaleza, CE, Brazil, LRBG, lrg@ufc.br
2   3Escuela de Microbiología, Universidad Industrial de Santander, Bucaramanga, Colombia, CO, ortizc@uis.edu.co

Abstract: Coimmobilization of lipases may be interesting in many instances, but this means that the stability of the less stable enzyme determines the stability of the full combilipase. Here, we propose a strategy that permits the reuse of the most stable enzyme. Lecitase Ultra (LU) (a phospholipase) and the lipases from Rhizomucor miehei (RML) and from Pseudomonas fluorescens (PFL) were immobilized on octyl agarose and their stabilities were studied under a broad range of conditions. Immobilized PFL was found to be the most stable enzyme under all condition ranges studied. Furthermore, in many cases it keeps full activity while the other enzymes lose more than 50% of the initial activity. To coimmobilize these enzymes without discarding fully active PFL when LU or RML had been inactivated, PFL was covalently immobilized on glyoxyl-agarose beads. After biocatalysts reduction, the other enzyme was coimmobilized just by interfacial activation. After checking that glyoxyl-octyl-PFL was stable in 4% Triton X-100, the biocatalysts of PFL coimmobilized with LU or RML were submitted to inactivation under different conditions. Then, the inactivated least stable coimmobilized enzyme was desorbed (using 4% detergent) and a new enzyme reloading (in some instances using RML and in some others employing LU) was performed. The initial activity of immobilized PFL was maintained intact for several of these cycles. This shows the great potential of this lipase coimmobilization strategy.

Palabras clave:enzyme coimmobilization; glyoxyl-octyl; covalent immobilization; interfacial activation; reuse of coimmobilized enzymes
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