Solid-phase modification with succinic polyethyleneglycol of aminated lipase B from Candida antarctica: Effect of the immobilization protocol on enzyme catalytic properties |
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| Institution: | 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;1. Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;2. Tsinghua Innovation Center in Dongguan, Guangdong 523808, China;1. Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan;2. Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-2 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan;1. University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstraße 28/30, 48149 Münster, Germany;2. NRW Graduate School of Chemistry, Wilhelm-Klemm-Straße 10, 48149 Münster, Germany;3. European Virtual Institute for Speciation Analysis (EVISA), Mendelstraße 11, 48149 Münster, Germany;1. School of Engineering, Laurentian University, Sudbury, ON, Canada;2. SINTEF Materials and Chemistry, Department of Materials and Nanotechnology, Trondheim, Norway;1. Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 9 Heroon Polytechniou Str., Zographou Campus, 15780 Athens, Greece;2. Laboratory of Thermodynamics and Transport Phenomena, School of Chemical Engineering, National Technical University of Athens, 9 Heroon Polytechniou Str., Zographou Campus, 15780 Athens, Greece |
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| Abstract: | 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. |
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