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.
Microbial catalysts having a combination of nitrile hydratase and amidase activities had a significantly-higher specific activity for hydrolysis of 3-hydroxyalkanenitriles than microbial nitrilase catalysts. Comamonas testosteroni 22 ± 1, Dietzia sp. ADL1 and Comamonas testosteroni 5-MGAM-4D nitrile hydratase/amidase biocatalysts each hydrolyzed 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid (as the ammonium salt) in 99 ± 100% yields, but in consecutive batch reactions with catalyst recycle, alginate-immobilized C. testosteroni 5-MGAM-4D had superior enzyme stability and volumetric productivity. In a series of 85 consecutive batch reactions with biocatalyst recycle for the production of 1.0 M 3-hydroxyvaleric acid, the recovered nitrile hydratase and amidase activities in the final reaction were 29% and 40%, respectively, of the initial activities. The catalyst productivity for this series of reactions was 670 g 3-hydroxyvaleric acid/g dry cell weight (50 g 3-hydroxyvaleric acid/g biocatalyst bead), and the volumetric productivity of the initial reaction in the series was 44 g 3-HVA/L/h. Similar results were obtained with alginate-immobilized C. testosteroni 5-MGAM-4D for the hydrolysis of 3-hydroxybutryonitrile and 3-hydroxypropionitrile to the corresponding 3-hydroxyalkanoic acid ammonium salts.
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