New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts

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-I...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Casablancas,Antoni, Cárdenas-Fernández,Max, Álvaro,Gregorio, Benaiges,Maria Dolors, Caminal,Glòria, Mas,Carles de, González,Glòria, López,Carmen, López-Santín,Josep
Lenguaje:English
Publicado: Pontificia Universidad Católica de Valparaíso 2013
Materias:
Acceso en línea:http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000300004
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:scielo:S0717-34582013000300004
record_format dspace
spelling oai:scielo:S0717-345820130003000042013-08-22New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalystsCasablancas,AntoniCárdenas-Fernández,MaxÁlvaro,GregorioBenaiges,Maria DolorsCaminal,GlòriaMas,Carles deGonzález,GlòriaLópez,CarmenLópez-Santín,Josep amine transaminases ammonia lyases E. coli fed-batch immobilized biocatalysts 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.16 n.3 20132013-05-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000300004en10.2225/vol16-issue3-fulltext-4
institution Scielo Chile
collection Scielo Chile
language English
topic amine transaminases
ammonia lyases
E. coli
fed-batch
immobilized biocatalysts
spellingShingle amine transaminases
ammonia lyases
E. coli
fed-batch
immobilized biocatalysts
Casablancas,Antoni
Cárdenas-Fernández,Max
Álvaro,Gregorio
Benaiges,Maria Dolors
Caminal,Glòria
Mas,Carles de
González,Glòria
López,Carmen
López-Santín,Josep
New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts
description 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.
author Casablancas,Antoni
Cárdenas-Fernández,Max
Álvaro,Gregorio
Benaiges,Maria Dolors
Caminal,Glòria
Mas,Carles de
González,Glòria
López,Carmen
López-Santín,Josep
author_facet Casablancas,Antoni
Cárdenas-Fernández,Max
Álvaro,Gregorio
Benaiges,Maria Dolors
Caminal,Glòria
Mas,Carles de
González,Glòria
López,Carmen
López-Santín,Josep
author_sort Casablancas,Antoni
title New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts
title_short New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts
title_full New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts
title_fullStr New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts
title_full_unstemmed New ammonia lyases and amine transaminases: Standardization of production process and preparation of immobilized biocatalysts
title_sort new ammonia lyases and amine transaminases: standardization of production process and preparation of immobilized biocatalysts
publisher Pontificia Universidad Católica de Valparaíso
publishDate 2013
url http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582013000300004
work_keys_str_mv AT casablancasantoni newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT cardenasfernandezmax newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT alvarogregorio newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT benaigesmariadolors newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT caminalgloria newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT mascarlesde newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT gonzalezgloria newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT lopezcarmen newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
AT lopezsantinjosep newammonialyasesandaminetransaminasesstandardizationofproductionprocessandpreparationofimmobilizedbiocatalysts
_version_ 1718441871971188736