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Título: | Site-directing an intense multipoint covalent attachment (MCA) of mutants of the Geobacillus thermocatenulatus lipase 2 (BTL2): Genetic and chemical amination plus immobilization on a tailor-made support |
Autor: | Godoy, César A. CSIC ORCID; Rivas, Blanca de las CSIC ORCID; Guisán, José Manuel CSIC ORCID | Palabras clave: | Tailor-made support PUFAs Amination Lipase mutants Site-directed immobilization Covalent immobilization |
Fecha de publicación: | 2014 | Editor: | Elsevier | Citación: | Process Biochemistry 49: 1324- 1331 (2014) | Resumen: | Immobilized enzymes are preferred over their soluble counterparts due to their robustness in harsh industrial processes; the most stable enzyme derivatives are often produced through multipoint covalent attachment (MCA). However, most enzymes are unable to establish optimal MCA to electrophile-type supports given the heterogeneous distribution and/or low content of primary amino groups on their surfaces; this restricts both the diversity of areas prone to react and the number of attachments to the support. To overcome this we propose combining site-directed immobilization and protein engineering to increase the number of bonds between a specific enzyme surface and a tailor-made support. We applied this novel strategy to engineered mutants of the lipase 2 from Geobacillus thermocatenulatus with one Cys exposed residue, that after genetic amination and/or chemical amination, were immobilized on glyoxyl-disulfide support using a site-directed MCA protocol. Two highly stabilized derivatives of chemically aminated lipase variants, in which site-directed MCA implied the surrounding surface of residues Cys344 or Cys40, were produced: the first one was 2.4-fold more productive than the reference derivative (648 g of hydrolyzed ester); the second derivative was 40% more selective (EPA/DHA molar ratio) and as active (1 μmol g catalyst-1 min-1) as the reference in the production of PUFAs. © 2014 Elsevier Ltd. | URI: | http://hdl.handle.net/10261/101910 | DOI: | 10.1016/j.procbio.2014.04.020 | Identificadores: | doi: 10.1016/j.procbio.2014.04.020 issn: 1359-5113 |
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