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PublicationPROCESS BIOCHEMISTRY
Year2014
Volume49
Pages1511-1515
International

STABILIZING HYPERACTIVATED LECITASE STRUCTURES THROUGH PHYSICAL TREATMENT WITH IONIC POLYMERS

Authors:Roberto Fernandez-Lafuente, Cristina Garcia-Galan, José Cleiton Sousa dos Santos
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Jose C. S. dos Santosa,b, Cristina Garcia-Galana, Rafael C. Rodriguesc, Hosiberto Batista de Sant'Anab, 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: Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil.



Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800 % when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1 % concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1 % SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01 % (v/v) CTAB, the Lecitase activity decreased to 10 %. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase. 


Keywords:enzyme hyperactivation, Lecitase, detergents, PEI, solid-phase physical modification, bioimprinting.
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