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PublicationJournal of Molecular catalysys B: Enzymatic

SOLID-PHASE MODIFICATION WITH SUCCINIC POLYETHYLENEGLYCOL OF AMINATED LIPASE B FROM Candida antarctica: Effect of the immobilization protocol on enzyme catalytic properties

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimization of biocatalysts and bioprocesses
Mónica Ruiz1, Magaly Galvis1, Oveimar Barbosa1, Claudia Ortiz2,
Rodrigo Torres1,* and Roberto Fernandez-Lafuente3,*
1       Escuela de Química, Grupo de Investigación en Bioquímica y Microbiología (GIBIM), Universidad Industrial de Santander, Bucaramanga, Colombia
2  Escuela de Bacteriología y Laboratorio Clínico, Universidad Industrial de Santander, Bucaramanga, Colombia
3 Departamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC, C/Marie Curie 2, Madrid, Cantoblanco 28049, Spain

Lipase B from Candida antarctica (CALB) has been modified using succinic polyethyleneglycol via the carbodiimide route.  Immobilized enzyme (on octyl Sepharose or Eupergit C) has been used, to take advantage of the solid phase. Modification of immobilized CALB´s native amino groups did not produce a significant alteration of CALB. However, if the enzyme was previously aminated, around 14-15 PEG molecules could be introduced per enzyme molecule. Also, it has been found that succinic groups are far more reactive than acetic acid following this strategy.
Even after this drastic double modification, the functional properties of the enzyme have not been impoverished to a large extent: stability decreased only to some extent (by a 5-6 fold factor), activity versus some substrates even increased (e.g., around 60% using p-nitrophenyl butyrate). It has been found that both modifications (amination and pegylation) have very different effects on enzyme properties when performed on CALB immobilized on Eupergit C or octyl Sepharose. For example, activity versus pNPP increased using CALB-octyl Sepharose while it decreased when using Eupergit C following amination and PEGylation. The effects also depend on the reaction and substrate, for example in hydrolysis of methyl mandelate, the activity decreased by 50% using CALB-octyl Sepharose after PEGylation of the aminated enzyme, while using CALB-Eupergit C had no effect. In this last case, enantioselecitvity in this hydrolysis significantly improved after both chemical modifications (from 7.5 to 20), while using CALB-octyl Sepharose almost had no effect.
Keywords:chemical amination, Chemical modification of proteins, Polyethyleneglycol, modulation of lipase properties, solid phase modification, ethylenediamine
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