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Immobilization of Lipases And Assay in Continuous Fixed Bed Reactor

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Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type II). Two immobilization methods were also used, namely: 1) adsorption, using as support the following silica derivatives (150-300μm e 450μ): phenyl, epoxy, amino and without derivation, and 2) covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U / gsupport for CRL and 0.97U / gsupport for PPL, and of transesterification, 2,8U / gsupport for CRL and 1,9U / gsupport for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40°C and 50°C and for PPL at 32°C and 40°C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continuos reactor. Lipases immobilized by covalent binding were used in the assays of operacional stability in continuos reactors. For PPL in aqueous medium, at 32°C, and CRL in organic medium at 40°C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40°C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40°C the loss was 33% after 20 days. Compairing both sources with each other, very different results were obtained. Higher activitiy was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.
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Keywords: continuous reactor; immobilization; lipases; silica

Document Type: Review Article

Affiliations: Departamento de Bioquimica e Tecnologia Quimica, Instituto de Quimica, Universidade Estadual Paulista, UNESP, Araraquara - SP, Departamento de Biotecnologia, Universidade de Ribeirao Preto, UNAERP, 14096- 380 Ribeirao Preto - SP, Brazil.

Publication date: 2003-12-01

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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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