Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing nitrogen-containing organic compound
Reexamination Certificate
2001-01-22
2003-04-22
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing nitrogen-containing organic compound
C435S136000
Reexamination Certificate
active
06551804
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an improved process for converting a nitrile to the corresponding carboxylic acid by using an enzyme catalyst having nitrilase activity. More particularly, the instant invention converts 2-methylglutaronitrile to the ammonium salt of 4-cyanopentanoic acid in aqueous solution using an enzyme catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity.
BACKGROUND OF THE INVENTION
A nitrilase enzyme directly converts a nitrile to the corresponding carboxylic acid ammonium salt in aqueous solution without the intermediate formation of an amide. Nitrilases have been identified in a variety of microorganisms, for example, Kobayashi et al. (
Tetrahedron
46:5587-5590 (1990);
J. Bacteriology,
172:4807-4815 (1990)) have described an aliphatic nitrilase isolated from
Rhodococcus rhodochrous
K22 that catalyzed the hydrolysis of aliphatic nitrites to their corresponding carboxylic acid ammonium salts. A stereospecific nitrilase of
Alcaligenes faecalis
1650 has been used to resolve racemic nitrites in the manufacture of chiral carboxylic acids, and the gene encoding the nitrilase has been cloned and expressed (WO 00/23577). A nitrilase has been isolated from the thermophilic bacterium
Bacillus pallidus
strain Dac521 that catalyzed the hydrolysis of aliphatic, aromatic and heterocyclic nitrites (Almatawah et al.,
Extremophiles
3:283-291 (1999)). A nitrilase from
Rhodococcus rhodochrous
NCIMB 40757 or NCIMB 40833 has been used to convert acrylonitrile to ammonium acrylate (U.S. Pat. No. 5,998,180). A nitrilase from
Comamonas testosteroni
has been isolated that can convert a range of aliphatic &agr;,&ohgr;-dinitriles to either the corresponding &ohgr;-cyanocarboxylic acid ammonium salt or dicarboxylic acid diammonium salt (CA 2,103,616; S. Lévy-Schil et al.,
Gene
161:15-20 (1995)). The regioselective hydrolysis of aliphatic &agr;,&ohgr;-dinitriles to the corresponding &ohgr;-cyanocarboxylic acid ammonium salts by the nitrilase activity of
Acidovorax facilis
72W has also been reported (Gavagan et al.,
J. Org. Chem.,
63:4792-4801 (1998)).
A combination of two enzymes, nitrile hydratase and amidase, can also be used to convert aliphatic nitrites to the corresponding carboxylic acid ammonium salts in aqueous solution. Here the aliphatic nitrile is initially converted to an amide by the nitrile hydratase and then the amide is subsequently converted by the amidase to the corresponding carboxylic acid ammonium salt. A wide variety of bacterial genera are known to possess a diverse spectrum of nitrile hydratase and amidase activities, including Rhodococcus, Pseudomonas, Alcaligenes, Arthrobacter, Bacillus, Bacteridium, Brevibacterium, Corynebacterium, and Micrococcus. Cowan et al. (
Extremophiles
2:207-216 (1998)) have recently reviewed both the nitrilase and nitrile hydratase/amidase enzyme systems of nitrile-degrading microorganism.
2-Methylglutaronitrile is one example of an aliphatic &agr;,&ohgr;-dinitrile that can be regioselectively converted to a &ohgr;-cyanocarboxylic acid ammonium salt (i.e., the ammonium salt of 4-cyanopentanoic acid) using a biocatalyst. The biocatalytic preparation of 4-cyanopentanoic acid has been described previously in U.S. Pat. No. 5,814,508 and its divisionals U.S. Pat. Nos. 5,858,736, 5,908,954, 5,922,589, 5,936,114, 6,077,955, and U.S. Pat. No. 6,066,490. These patents relate to a process in which an aliphatic &agr;,&ohgr;-dinitrile is converted to an ammonium salt of an &ohgr;-cyanocarboxylic acid in aqueous solution using a catalyst having an aliphatic nitrilase (EC 3.5.5.7) activity, or a combination of nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) activities. When the aliphatic &agr;,&ohgr;-dinitrile is also unsymmetrically substituted at the &ohgr;-carbon atom, the nitrilase produces the &ohgr;-cyanocarboxylic acid ammonium salt resulting from hydrolysis of the &ohgr;-nitrile group with greater than 98% regioselectivity. U.S. Pat. No. 5,814,508 specifically discloses a method for converting 2-methylglutaronitrile to 4-cyanopentanoic acid in aqueous solution, where
Acidovorax facilis
72W is subjected to a 10-120 minute heat treatment (35-70° C.) before use as an enzyme catalyst. This heat treatment was critical to select for the desirable regioselective aliphatic nitrilase (EC 3.5.5.7) activity while destroying an undesirable, non-regioselective nitrile hydratase activity. 4-Cyanopentanoic acid can then serve as substrate in a one-step chemical process for the commercial preparation of 1,5-dimethyl-2-piperidone. 1,5-Dimethyl-2-piperidone has many uses as an industrial solvent, including electronics cleaning, photoresist stripping, industrial degreasing and metal cleaning, resin cleanup, ink formulations, industrial adhesives, and as a reaction solvent for polymers and chemicals.
For commercial-scale applications using a biocatalyst, immobilizing microbial cells has many known economical advantages compared to the use of unimmobilized cells. Some advantages are the capacity to use them repeatedly, their ease of separation, and their use in continuous reactions. Cell inclusion into a polymer matrix permits entrapment of living or metabolically inactive cells while maintaining high diffusion of product and substrate. Examples of typical matrices for immobilization are sodium alginate (Bucke,
Methods in Enzymology
135:175-189 (1987)) or carrageenan (Chibata et al.,
Methods in Enzymology
135:189-198 (1987)). Methods of entrapment are relatively simple, and gel material is non-toxic and low priced.
“Operational” stability of immobilized cells can be further increased by subsequent treatment of the cell beads with crosslinking agents that covalently crosslink cells with multifunctional reagents, such as glutaraldehyde and polyethylenimine or hexamethylenediamine. In one example, stability was studied with respect to immobilized
E. coli
cells in kappa-carrageenan for the production of L-aspartic acid (Chibata, I. In
Immobilized Microbial Cells
; Venkatsubramanian, K., Ed.; ACS Symposium Series 106; American Chemical Society; Washington, D.C., 1979, pp 187-201). With optimized concentrations of hexamethylenediamine and glutaraldehyde used as a crosslinking treatment, the half-life of immobilized cells was remarkably extended to over five times that of untreated immobilized cells.
Furthermore, Birnbaum et al. (
Biotechnol. Lett.
3:393-400 (1981)) disclose methods of increasing the physical stability of calcium alginate immobilized cells. One stabilization method uses polyethylenimine treatment (24 hours), followed by glutaraldehyde cross-linking (1-5 minutes). Bead stability was examined by incubating the immobilized cells in 0.1 M sodium phosphate buffer for ten days. Little cell release was noted from the immobilized cells, thereby demonstrating improved bead integrity. At the same time, overall catalyst activity was detrimentally affected by this protocol. Birnbaum suggests this effect was likely due to glutaraldehyde toxicity.
Finally, the preferred order of adding polyethylenimine and glutaraldehyde for directly immobilizing whole microbial cells or microbial cell material has been understood to depend on the sensitivity of the immobilized enzyme activity to glutaraldehyde. U.S. Pat. No. 4,288,552 discloses that glutaraldehyde-sensitive enzymes (such as thiol-enzymes 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. For these types of enzyme catalysts, the invention requires that the microbial cell material be treated with polyethylenimine first, the glutaraldehyde being added simultaneously or subsequently, to negate potential loss of enzyme activity. In contrast, U.S. Pat. No. 4,355,105 teaches that it is desirable to introduce glutaraldehyde before polyethylenimine when immobilizing microorganisms whose enzymes are not sensitive to glutaraldehyde. In this instance the resulting immobilized cells are more readily recovered from the aqueous medium than cells immobilized with p
DiCosimo Robert
Fallon Robert D.
Gavagan John E.
E. I. Du Pont de Nemours and Company
Marx Irene
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