Web of Science: 7 citas, Scopus: 7 citas, Google Scholar: citas
New ammonia lyases and amine transaminases : dtandardization of production process and preparation of immobilized biocatalysts
Casablancas Mira, Antoni (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
Cárdenas Fernández, Anthony Max (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
Álvaro Campos, Gregorio (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
Benaiges Massa, M Dolors (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
Caminal i Saperas, Glòria (Institut de Química Avançada de Catalunya)
Mas i Rocabayera, Carles de (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
González, Glòria (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
López, Carmen (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)
López Santín, Josep (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química)

Fecha: 2013
Resumen: Background: New enzymes for biotransformations can be obtained by different approaches including directed mutagenesis and in vitro evolution. These mutants have to be efficiently produced for laboratory research on bioreactions as well as for process development. In the framework of a European ERA-IB project, two different types of enzymes (ammonia lyases and aminotransferases) have been selected as biocatalysts for the synthesis of industrially relevant amines. New mutant enzymes have been obtained: a) aspartases able to recognize β-amino acids; b) ω-transaminases with improved activity. The objectives are to find out a common operational strategy applicable to different mutants expressed in E. coli with the same initial genetic background, the development of an integrated process for production and the preparation of stable useful biocatalysts. Results: Mutant enzymes were expressed in E. coli BL21 under the control of isopropylthiogalactoside (IPTG) inducible promoter. The microorganisms were grown in a formulated defined medium and a high-cell density culture process was set up. Fed-batch operation at constant specific growth rate, employing an exponential addition profile allowed high biomass concentrations. The same operational strategy was applied for different mutants of both aspartase and transaminase enzymes, and the results have shown a common area of satisfactory operation for maximum production at low inducer concentration, around 2 μmol IPTG/g DCW. The operational strategy was validated with new mutants and high-cell density cultures were performed for efficient production. Suitable biocatalysts were prepared after recovery of the enzymes. The obtained aspartase was immobilized by covalent attachment on MANA-agarose, while ω-transaminase biocatalysts were prepared by entrapping whole cells and partially purified enzyme onto Lentikats (polyvinyl alcohol gel lens-shaped particles). Conclusions: The possibility of expressing different mutant enzymes under similar operation conditions has been demonstrated. The process was standardized for production of new aspartases with β-amino acid selectivity and new ω-transaminases with improved substrate acceptance. A whole process including production, cell disruption and partial purification was set up. The partially purified enzymes were immobilized and employed as stable biocatalysts in the synthesis of chiral amines.
Nota: Número d'acord de subvenció MICINN/EUI2008-03615
Nota: Número d'acord de subvenció AGAUR/2009/SGR-281
Derechos: Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades. Creative Commons
Lengua: Anglès.
Documento: article ; recerca ; publishedVersion
Materia: Amine transaminases ; Ammonia lyases ; E. coli ; Fed-batch ; Immobilized biocatalysts
Publicado en: Electronic journal of biotechnology, Vol. 16, issue 3 (2013) , ISSN 0717-3458

DOI: 10.2225/vol16-issue3-fulltext-4

13 p, 530.3 KB

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