Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2001-02-23
2002-07-09
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S128000, C435S227000, C435S252100, C435S252300, C435S252330
Reexamination Certificate
active
06416980
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a process for the production of &agr;-hydroxy acids using an enzyme catalyst having nitrilase activity. More specifically, the invention pertains to production of glycolic acid from glycolonitrile using a catalyst having
Acidovorax facilis
72W nitrilase activity.
BACKGROUND OF THE INVENTION
Glycolic acid (HOCH
2
COOH; CAS Registry Number is 79-14-1) is the simplest member of the &agr;-hydroxy acid family of carboxylic acids. Its unique 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, and as a component in personal care products like skin creams. 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]). New technology to commercially produce glycolic acid would be eagerly received by industry.
Various methods for preparing &agr;-hydroxy acids are known, using the corresponding &agr;-hydroxy nitrile as the starting material and a microorganism as the catalyst. Examples of &agr;-hydroxy acids produced include: glycolic acid, lactic acid, 2-hydroxyisobutyric acid, 2-hydroxy-2-hydroxyphenyl propionic acid, mandelic acid, 2-hydroxy-3,3-dimethyl-4-butyrolactone, and 4-methylthiobutyric acid. These products are synthesized using microorganisms, such as those belonging to the genera Nocardia, Bacillus, Brevibacterium, Aureobacterium, Pseudomonas, Caseobacter, Alcaligenes, Acinetobacter, Enterobacter, Arthrobacter, Escherichia, Micrococcus, Streptomyces, Flavobacterium, Aeromonas, Mycoplana, Cellulomonas, Erwinia, Candida, Bacteridium, Aspergillus, Penicillium, Cochliobolus, Fusarium, Rhodopseudomonas, Rhodococcus, Corynebacterium, Microbacterium, Obsumbacterium and Gordona. (JP-A-4-99495, JP-A-4-99496 and JP-A-4-218385 corresponding to U.S. Pat. No. 5,223,416; JP-A-4-99497 corresponding to U.S. Pat. No. 5,234,826; JP-A-5-95795 corresponding to U.S. Pat. No. 5,296,373; JP-A-5-21987; JP-A-5-192189 corresponding to U.S. Pat. No. 5,326,702; JP-A-6-237789 corresponding to EP-A-0610048; JP-A-6-284899 corresponding to EP-A-0610049; JP-A-7-213296 corresponding to U.S. Pat. No. 5,508,181.)
However, most known methods for preparing &agr;-hydroxy acids from the corresponding &agr;-hydroxy nitrites as mentioned above do not produce and accumulate a product at a sufficiently high concentration to meet commercial needs. This is frequently a result of enzyme inactivation early in the reaction period. U.S. Pat. No. 5,756,306 teaches that “When an &agr;-hydroxy nitrile is enzymatically hydrolyzed or hydrated using nitrilase or nitrile hydratase to produce an &agr;-hydroxy acid or &agr;-hydroxy amide, a problem occurs in that the enzyme is inactivated within a short period of time. It is therefore difficult to obtain the &agr;-hydroxy acid or &agr;-hydroxy amide in high concentration and high yield.” (col. 1, lines 49-54). Maintaining the aldehyde concentration (formed by the disassociation of &agr;-hydroxy nitrile to aldehyde and hydrogen cyanide) and/or the &agr;-hydroxy nitrile concentration in the reaction mixture within a specified range is one method to avoid this problem.
U.S. Pat. No. 5,508,181 addresses further difficulties relating to rapid enzyme inactivation. Specifically, U.S. Pat. No. 5,508,181 mentions that &agr;-hydroxy nitrile compounds partially disassociate into the corresponding aldehydes, according to the disassociation equilibrium. These aldehydes inactivate the enzyme within a short period of time by binding to the protein, thus making it difficult to obtain &agr;-hydroxy acid or &agr;-hydroxy amide in a high concentration with high productivity from &agr;-hydroxy nitrites (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. U.S. Pat. No. 5,326,702 is similar to U.S. Pat. No. 5,508,181, except sulfite, disulfite, or dithionite ions are used to sequester aldehyde and prevent enzyme inactivation. However, the concentration of &agr;-hydroxy acid produced and accumulated even by using such additives as described above is not great.
And finally, U.S. Pat. No. 6,037,155 also teaches that low accumulation of &agr;-hydroxy acid products 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 (
Agricultural Biological Chemistry
, Vol. 46, page 1165 (1982)) generated in the partial disassociation of &agr;-hydroxy nitrile in water together with the corresponding aldehyde or ketone (
Chemical Reviews
, Vol. 42, page 189 (1948)). The inventors solved 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 &agr;-hydroxy nitrile to aldehyde and hydrogen cyanide.
With specific respect to the production of glycolic acid, glycolonitrile is known to reversibly disassociate to hydrogen cyanide and formaldehyde, either of which can inactivate enzyme activity. U.S. Pat. No. 3,940,316 describes a process for preparing an organic acid from the corresponding nitrile using a bacteria with “nitrilasic” activity, and lists glycolonitrile as a substrate. In particular, this patent describes the use of Bacillus, Bacteridium, Micrococcus, and Brevibacterium for this purpose. Though described as having nitrilasic activity, Brevibacterium R312 is the only strain used in all of the U.S. Pat. No. 3,940,316 examples. Brevibacterium R312 is known to have nitrile hydratase and amidase activities, but no nitrilase activity (Toumeix et al., Antonie van Leeuwenhoek, 1986, 52:173-182).
A method for preparing lactic acid, glycolic acid, and 2-hydroxyisobutyric acid by using a microorganism belonging to Corynebacterium spp. is disclosed in Japanese Patent Laid-open No. Sho 61-56086. JP 09028390 discloses a method for manufacturing high-purity glycolic acid from glycolonitrile by the action of Rhodococcus or Gordona hydrolase. Selectivity for glycolic acid is reported as almost 100%, without formation of glycolic acid amide. U.S. Pat. No. 6,037,155 also provides examples of methods for producing &agr;-hydroxy acids from &agr;-hydroxy nitrites, including glycolic acid. This disclosure acknowledges that not all microbial catalysts can produce high concentrations of glycolic acid due to the aforementioned problems and instructs that screening studies must be conducted in order to find industrially advantageous microorganisms. U.S. Pat. No. 6,037,155 specifically identifies microorganisms belonging to Variovorax spp. and Arthrobacter spp., which are resistant to the suppressing effect of &agr;-hydroxy nitrile or &agr;-hydroxy acid, have durable activity, and can produce the desired product at high concentration.
Acidovorax facilis
72W (ATCC 55746) is characterized by aliphatic nitrilase (EC 3.5.5.7) activity, as well as a combination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. U.S. Pat. No. 5,858,736 describes the use of the nitrilase activity of this microbe as a catalyst for the hydrolysis of aliphatic &agr;,&ohgr;-dinitriles to the corresponding &ohgr;-cyanocarboxylic acids and ammonia in an aqueous reaction mixture. The nitrilase was found to be highly regioselective, where hydrolysis of an &agr;-alkyl-&agr;,&ohgr;-dinitrile produced only the &ohgr;-cyanocarboxylic acid resulting from hydrolysis of the &ohgr;-nitrile group. U.S. Pat. No. 5,814,508 discloses
Chauhan Sarita
DiCosimo Robert
Fallon Robert D.
Gavagan John E.
Payne Mark S.
Achutamurthy Ponnathapu
E. I. du Pont de Nemours & Company
Fronda Christian L.
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