Glycolic acid (HOCH2COOH; CAS Registry Number is 79-14-1) is the simplest member of the α-hydroxy acid family of carboxylic acids. Its properties make it ideal for a broad spectrum of consumer and industrial applications, including use in water well rehabilitation, the leather industry, the oil and gas industry, the laundry and textile industry, as a monomer in the preparation of polyglycolic acid (PGA), and as a component in personal care products. Glycolic acid also is a principle ingredient for cleaners in a variety of industries (dairy and food processing equipment cleaners, household and institutional cleaners, industrial cleaners [for transportation equipment, masonry, printed circuit boards, stainless steel boiler and process equipment, cooling tower/heat exchangers], and metals processing [for metal pickling, copper brightening, etching, electroplating, electropolishing]). It has also been reported that polyglycolic acid is useful as a gas barrier material (i.e., exhibits high oxygen barrier characteristics) for packing foods and carbonated drinks (WO 2005/106005 A1). However, traditional chemical synthesis of glycolic acid produces a significant amount of impurities that must be removed prior to use. New technology to commercially produce glycolic acid, especially one that produces glycolic acid in high purity and at low cost, would be eagerly received by industry.
Microbial enzyme catalysts can hydrolyze a nitrile (e.g., glycolonitrile) directly to the corresponding carboxylic acids (e.g., glycolic acid) using a nitrilase (EC 3.5.5.7), where there is no intermediate production of the corresponding amide (Equation 1), or by a combination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) enzymes, where a nitrile hydratase (NHase) initially converts a nitrile to an amide, and then the amide is subsequently converted by the amidase to the corresponding carboxylic acid (Equation 2):

It has been demonstrated that the enzyme catalyst specific activity measured as micromoles glycolic acid produced per minute per g dry cell weight of catalyst, decreases by from 35% to 50% of initial activity after a single use in consecutive batch reactions with biocatalyst recycle. This represents a significant loss in specific activity of the enzyme catalyst in batch reactions, and, in turn, overall enzyme activity and productivity. For a commercially feasible enzymatic process for producing glycolic acid, this loss in enzyme activity needs to be addressed.
One aspect of the loss of enzyme activity may be attributable to impurities and other components present when reacting the enzyme catalyst with glycolonitrile for glycolic acid production. Methods to synthesize glycolonitrile by reacting aqueous solutions of formaldehyde and hydrogen cyanide have previously been reported (U.S. Pat. No. 2,175,805; U.S. Pat. No. 2,890,238; and U.S. Pat. No. 5,187,301; Equation 3).

However, these methods typically result in an aqueous glycolonitrile reaction product that requires significant purification (e.g., distillative purification) as many of the impurities and/or byproducts of the reaction (including excess reactive formaldehyde) may interfere with the enzymatic conversion of glycolonitrile to glycolic acid, including suppression of catalyst activity (i.e., decreased specific activity). In particular, it is well known that formaldehyde can create undesirable modifications in proteins by reacting with amino groups from N-terminal amino acid residues and the side chains of arginine, cysteine, histidine, and lysine residues (Metz et al., J. Biol. Chem., 279 (8): 6235-6243 (2004)). Suppression of catalyst activity decreases the overall productivity of the catalyst (i.e., total grams of glycolic acid formed per gram of catalyst), adding a significant cost to the overall process that may make enzymatic production economically non-viable when compared to chemical synthesis. As such, reaction conditions are needed that can help to protect the enzymatic activity against undesirable impurities that decrease the activity of the catalyst.
A method of producing high purity glycolonitrile has been reported by subjecting the formaldehyde to a heat treatment prior to the glycolonitrile synthesis reaction (U.S. application Ser. No. 11/3143865 and U.S. application Ser. No. 11/314905; Equation 3). However, glycolonitrile can reversibly disassociate into formaldehyde and hydrogen cyanide. As such, there remains a need to protect nitrilase activity against the undesirable effects of both formaldehyde and hydrogen cyanide produced by dissociation of glycolonitrile.
U.S. Pat. No. 5,508,181 also describes similar difficulties related to rapid enzyme catalyst inactivation when converting nitrile compounds to α-hydroxy acids. Specifically, U.S. Pat. No. 5,508,181 provides that α-hydroxy nitrile compounds partially disassociate into the corresponding aldehydes, according to the disassociation equilibrium. These aldehydes were reported to inactivate the enzyme within a short period of time by binding to the protein, thus making it difficult to obtain α-hydroxy acid or α-hydroxy amide in a high concentration with high productivity from α-hydroxy nitriles (col. 2, lines 16-29). As a solution to prevent enzyme inactivation due to accumulation of aldehydes, phosphate or hypophosphite ions were added to the reaction mixture. Similarly, U.S. Pat. No. 5,326,702 describes the use of sulfite, disulfite, or dithionite ions to sequester aldehyde and prevent enzyme inactivation, but concludes that the concentration of α-hydroxy acid produced and accumulated even by using such additives is not sufficient for most commercial purposes.
Moreover, U.S. Pat. No. 6,037,155 teaches that low accumulation of α-hydroxy acid product is related to enzyme inactivation within a short time due to the disassociated-aldehyde accumulation. These inventors suggest that enzymatic activity is inhibited in the presence of hydrogen cyanide (Asano et al., Agricultural Biological Chemistry, Vol. 46, pages 1165-1174 (1982)) generated in the partial disassociation of the α-hydroxy nitrile in water together with the corresponding aldehyde or ketone (Mowry, David T., Chemical Reviews, Vol. 42, pages 189-283 (1948)). The inventors address the problem of aldehyde-induced enzyme inactivation by using microorganisms whose enzyme activity could be improved by adding a cyanide substance to the reaction mixture. The addition of a cyanide substance limited the disassociation of α-hydroxy nitrile to aldehyde and hydrogen cyanide. While this tactic provides a benefit to the system, it only addresses one aspect associated with enzyme inactivation in conversion of glycolonitrile to glycolic acid, in that, as stated above, glycolonitrile is known to reversibly disassociate to hydrogen cyanide and formaldehyde, and both are known to negatively effect enzyme catalyst activity.
A separate process has been developed to protect the specific activity of an enzyme catalyst having nitrilase activity when converting glycolonitrile to glycolic acid in the presence of formaldehyde (see copending U.S. application Ser. No. 11/931,069 incorporated herein by reference), where significant improvements in catalyst activity and stability were achieved by adding an amine protectant to the reaction mixture, or by immobilization of the nitrilase catalyst in or on a matrix that is comprised of an amine protectant, e.g. PEI, polyallylamine, PVOH/polyvinylamine, etc. In that system, the specific activity of the catalyst in the presence of formaldehyde is improved, but does not address, altogether, issues related to the loss in specific activity of recovered catalyst activity in consecutive batch reactions with catalyst recycle.
U.S. Pat. No. 4,288,552 discloses (column 1, lines 46-49, and column 2, lines 50-55) that glutaraldehyde-sensitive enzymes (such as thiol-enzymes (e.g, nitrilase) and others with an SH group in or very near the active site of the enzyme molecule) are inactivated by thiol-reactive agents such as glutaraldehyde. Therefore, use of glutaraldehyde to improve the retention of initial catalyst activity during hydrolysis of glycolonitrile to glycolic acid was heretofore unpredictable. Said unpredictable benefit is demonstrated herein.
Therefore there is a need for a process that provides improved retention of initial nitrilase activity during hydrolysis of glycolonitrile to glycolic acid, thereby improving overall catalyst activity, catalyst productivity, and volumetric productivity for the conversion of glycolonitrile to glycolic acid.