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-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.

Esterificación quimioselectiva de fitosteroles / Chemoselective transesterification of wood steroles by lipases

La esterificacion selectiva de fitosteroles de madera representa una aplicación novedosa de las lipasas, que se inscribe dentro de una plataforma tecnológica de valoración del licor negro en el proceso Kraft. Los fitosteroles de madera son mezclas de esteroles y estanoles (esteroles saturados) donde más del 90 por ciento es beta-sitosterol y beta-sitostanol. Ambos productos tienen mercados potenciales diferentes por lo que el fraccionamiento de los fitosteroles que los contienen implica un valor agregado considerable. Ambas sustancias son muy similares, lo que impide su separación por métodos físicos, siendo la esterificación selectiva de esta mezcla con ésteres de ácidos grasos mediante lipasas quimioselectivas una interesante opción tecnológica para separarlas. Se evaluaron diversas lipasas comerciales en su capacidad de esterificar selectivamente los estanoles, seleccionándose una enzima inmovilizada y una no soportada. Se optimizó el proceso con la enzima inmovilizada obteniéndose grados de esterificación de estanoles por sobre el 90 por ciento y de esteroles en torno al 20 por ciento, lo que satisface el criterio de selectividad establecido. La enzima inmovilizada comercial tuvo baja estabilidad operacional debido a la desorción de la proteína por lo que se desarrollaron estrategias de inmovilización de la lipasa comercial no soportada, obteniéndose los mejores resultados con butil Sepabeads® como soporte. Con dicho biocatalizador se realizó la reacción de transesterificacion en modalidad de lotes repetidos demostrándose la elevada estabilidad de la enzima y comprobándose que es posible realizar cinco lotes productivos sin merma de la conversión ni de la productividad, lo que satisface los criterios de rentabilidad del proceso. La tecnología ha sido transferida al sector productivo y se ha presentado una patente de invención sobre la elaboración del biocatalizador.