RESUMO
A key step in a chemoenzymatic process for the production of high-purity glycolic acid (GLA) is the enzymatic conversion of glycolonitrile (GLN) to ammonium glycolate using a nitrilase derived from Acidovorax facilis 72W. Protein engineering and over-expression of this nitrilase, combined with optimized fermentation of an E. coli transformant were used to increase the enzyme-specific activity up to 15-fold and the biocatalyst-specific activity up to 125-fold. These improvements enabled achievement of the desired volumetric productivity and biocatalyst productivity for the conversion of GLN to ammonium glycolate.
Assuntos
Acetonitrilas/química , Aminoidrolases/química , Aminoidrolases/metabolismo , Betaproteobacteria/enzimologia , Escherichia coli/enzimologia , Glicolatos/síntese química , Engenharia de Proteínas/métodos , Aminoidrolases/genética , Betaproteobacteria/genética , Escherichia coli/genética , Proteínas Recombinantes/metabolismoRESUMO
Hydroxycarboxylic acid monomers can be used to prepare industrially important polymers. Enzymatic production of such hydroxycarboxylic acids is often preferred to chemical production since the reactions are run at ambient temperature, do not require strongly acidic or basic reaction conditions, and produce the desired product with high selectivity at high conversion. However, native enzymes often do not perform desired reactions with the efficiency required for commercial applications. Protein engineering was used to significantly increase the specific activity of nitrilase from Acidovorax facilis 72W for the conversion of 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid. Overexpression of engineered nitrilase enzymes in Escherichia coli, combined with immobilization of whole cells in alginate beads that can be recycled many times has facilitated the development of a commercially viable bioprocess for production of 3-hydroxyvaleric acid.
Assuntos
Aminoidrolases/genética , Comamonadaceae/enzimologia , Escherichia coli/enzimologia , Microbiologia Industrial , Engenharia de Proteínas , Alginatos/química , Aminoidrolases/isolamento & purificação , Aminoidrolases/metabolismo , Reatores Biológicos/microbiologia , Células Imobilizadas , Escherichia coli/genética , Fermentação , Microesferas , Mutagênese Sítio-Dirigida , Transformação GenéticaRESUMO
The genes encoding a thermally stable and regio-selective nitrile hydratase (NHase) and an amidase from Comamonas testosteroni 5-MGAM-4D have been cloned and sequenced, and active NHase has been over-produced in Escherichia coli. Maximal activity requires co-expression of a small open reading frame immediately downstream from the NHase beta subunit gene. Compared to the native organism, the E. coli biocatalyst has nearly threefold more NHase activity on a dry cell weight basis, and this activity is significantly more thermally stable. In addition, this biocatalyst converts a wide spectrum of nitrile substrates to the corresponding amides. Such versatility and robustness are desirable attributes of a biocatalyst intended for use in commercial applications.