A chemoenzymatic process for the production of high-purity glycolic acid has been demonstrated, starting with the reaction of formaldehyde and hydrogen cyanide to produce glycolonitrile in > 99 % yield and purity. The resulting aqueous glycolonitrile was used without further purification in a subsequent biocatalytic conversion of glycolonitrile to ammonium glycolate. A high-activity biocatalyst based on an Acidovorax facilis 72W nitrilase was developed, where protein engineering and optimized protein expression in an E. coli transformant host were used to improve microbial nitrilase specific activity by 33-fold compared to the wild-type strain. A biocatalyst productivity of > 1000 g glycolic acid/g dry cell weight was achieved using a glutaraldehyde/ polyethylenimine cross-linked carrageenan-immobilized E. coli MG1655 transformant expressing the A. facilis 72W nitrilase mutant, where 3.2 M ammonium glycolate was produced in consecutive batch reactions with biocatalyst recycle, or in a continuous stirred-tank reactor. Direct conversion of the unpurified ammonium glycolate product solution to highpurity aqueous glycolic acid was accomplished by fixed-bed ion exchange over a strong acid cation resin.
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.
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.
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