METHOD FOR PRODUCTION OF 4-CYANO BENZOIC ACID OR SALTS THEREOF

Abstract

Described herein are methods for the production of 4-cyano benzoic acid or salts thereof from terephthalonitrile using nitrilase as catalyst. Also described herein are compositions including 4-cyano benzoic acid.

Claims

1. An isolated nitrilase capable of catalysing a reaction from terephthalonitrile to ammonium 4-cyano benzoic acid in an aqueous medium comprising water, nitrilase and terephthalonitrile and/or ammonium 4-cyano benzoic acid, wherein the concentration of ammonium 4-cyano benzoic acid in the aqueous medium after incubation is at least 5% (w/w) and the concentration of terephthalonitrile is below 1.0% (w/w).

2. The isolated nitrilase of claim 1 wherein after incubation the aqueous medium comprises below 0.5% (w/w) terephthalic acid.

3. The isolated nitrilase of claim 1 comprising a sequence selected from the group consisting of a. An amino acid molecule of SEQ ID NO: 2, 4, 6 or 8, b. An amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: 2, 4, 6 or 8 or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 1, 3, 5 or 7 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5 or 7 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 1, 3, 5 or 7 or a functional fragment thereof, wherein the amino acid molecule as defined in b., d. and e. catalyzes the reaction from terephthalonitrile to ammonium 4-cyano benzoic acid in an aqueous medium.

4. An isolated nitrilase sequence selected from the group consisting of a. An amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22, and b. An amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22 or a functional fragment thereof, and c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, and d. An amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, wherein the amino acid molecule as defined in b., c., d. and e. catalyzes a reaction from terephthalonitrile to ammonium 4-cyano benzoic acid in an aqueous medium.

5. A process for producing 4-cyano benzoic acid or salt thereof comprising the steps of i. Providing an aqueous medium comprising water, one or more nitrilase and terephthalonitrile, ii. Incubating the aqueous medium and iii. Optionally isolating the 4-cyano benzoic acid or salt thereof from the reaction mixture, wherein the one or more nitrilase is capable of catalysing the reaction from terephthalonitrile to ammonium 4-cyano benzoic acid in an aqueous medium comprising water, nitrilase and terephthalonitrile and/or ammonium 4-cyano benzoic acid, wherein the concentration of ammonium 4-cyano benzoic acid in the aqueous medium after incubation is at least 5% (w/w) and the concentration of terephthalonitrile is below 1.0% (w/w).

6. The process of claim 5 wherein after incubation the aqueous medium comprises below 0.5% (w/w) terephthalic acid.

7. The process of claim 5 wherein the nitrilase comprises a sequence selected from the group consisting of a. An amino acid molecule of SEQ ID NO: 2, 4, 6 or 8, b. An amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: 2, 4, 6 or 8 or a functional fragment thereof, c. An amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO: 1, 3, 5 or 7 or a functional fragment thereof, d. An amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5 or 7 or a functional fragment thereof, and e. An amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO: 1, 3, 5 or 7 or a functional fragment thereof, wherein the amino acid molecule as defined in b., d. and e. catalyzes the reaction from terephthalonitrile to ammonium 4-cyano benzoic acid in an aqueous medium.

8. A process for producing 4-cyano benzoic acid or salt thereof comprising the steps of i. Providing an aqueous medium comprising water, one or more nitrilase and terephthalonitrile, ii. Incubating the aqueous medium and iii. Optionally isolating the 4-cyano benzoic acid or salt thereof from the reaction mixture, wherein the one or more nitrilase is selected from the group consisting of a. an amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22 or a functional fragment thereof, b. an amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22 or a functional fragment thereof, c. an amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, d. an amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, and e. an amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, wherein the amino acid molecule as defined in ii., iii., iv. and v. has an activity of converting terephthalonitrile to ammonium 4-cyano benzoic acid in an aqueous medium.

9. The process of claim 8 wherein the aqueous medium further comprises a divalent cation.

10. The process of claim 9 wherein the divalent cation is Mg.sup.2+, Mn.sup.2+, Ca.sup.2+, Fe.sup.2+, Zn.sup.2+ or Co.sup.2+.

11. The process of claim 8 wherein the terephthalonitrile is added to the aqueous medium before incubation in a concentration of between 1% and 30% w/w.

12. The process of claim 8 wherein a pH-value of the aqueous medium is adjusted to below 5 by adding acid to the aqueous medium during or after incubation.

13. The process of claim 8 wherein a product is isolated by filtration or centrifugation after incubation.

14. The process of claim 8 wherein the aqueous medium is incubated for at least 2 h.

15. The process of claim 8 wherein the aqueous medium is incubated between 15 and 50° C.

16. The process of claim 8 wherein the nitrilase is produced by fermentation.

17. A recombinant construct comprising a nitrilase wherein the nitrilase is selected from the group consisting of a. an amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22 or a functional fragment thereof, b. an amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22 or a functional fragment thereof, c. an amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, d. an amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, and e. an amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, wherein the amino acid molecule as defined in ii., iii., iv. and v. catalyzes a reaction from terephthalonitrile to 4-cyano benzoic acid in an aqueous medium.

18. The recombinant construct of claim 17, wherein the nitrilase is functionally linked to a heterologous promoter.

19. A recombinant vector comprising the recombinant construct of claim 17.

20. A recombinant microorganism comprising the recombinant construct of claim 17 or a recombinant vector comprising the recombinant construct of claim 17.

21. The recombinant microorganism of claim 20 wherein the microorganism is Rhodococcus rhodochrous, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Escherichia coli, Myceliophthora thermophila, Aspergillus sp., Saccharomyces cerevisiae, or Pichia pastoris.

22. A microorganism of the genus Comamonas testosteroni, Agrobacterium rubi, Candidatus Dadabacteria bacterium, Tepidicaulis marinus, Sphingomonas wittichii, Rhizobium spec., Synechococcus sp. CC9605, Flavihumibacter solisilvae or Salinisphaera shabanensis E1L3A expressing the nitrilase of claim 1.

23. A method for producing a nitrilase, comprising the steps of a. providing a recombinant microorganism according to claim 20, and b. cultivating the microorganism under conditions allowing for the expression of a nitrilase gene.

24. A composition comprising water, a nitrilase, terephthalonitrile and/or 4-cyano benzoic acid wherein the nitrilase is selected from the group consisting of a. an amino acid molecule of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22 or a functional fragment thereof, b. an amino acid molecule having at least 55% identity to the amino acid molecule of SEQ ID NO: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 20 or 22 or a functional fragment thereof, c. an amino acid molecule encoded by a nucleic acid molecule of SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, d. an amino acid molecule encoded by a nucleic acid molecule having at least 70% identity to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, and e. an amino acid molecule encoded by a nucleic acid molecule hybridizing under stringent conditions to a fragment of at least 250 bases complementary to SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 19 or 21 or a functional fragment thereof, wherein the amino acid molecule as defined in ii., iii., iv. and v. catalyzes a reaction from terephthalonitrile to ammonium 4-cyano benzoic acid in an aqueous medium.

25. A composition consisting of a) 95 wt % to 99.5 wt % 4-cyano benzoic acid, b) 0.0 wt % to 0.5 wt % terephthalic acid, c) 0.2 wt % to 1.5 wt % chloride, d) 0.05 wt % to 0.2 wt % water, and e) and optionally up to 4.75 wt % other components.

26. A composition consisting of a) 95 wt % to 97 wt % 4-cyano benzoic acid, b) 0.0 wt % to 0.5 wt % terephthalic acid, c) 0.3 wt % to 1.5 wt % ammonium, d) 2.0 wt % to 0.4 wt % sulfate, e) 0.4 wt % to 1.0 wt % natrium, and f) and optionally up to 2.3 wt % other components.

27. A method for making an aqueous solution containing at least 5% (w/w) ammonium 4-cyano benzoic acid, below 1.0% (w/w) terephthalonitrile, and below 0.5% (w/w) terephthalic acid, comprising the steps of I. Providing an aqueous medium comprising water, one or more nitrilase and terephthalonitrile and II. Incubating the aqueous medium, wherein the nitrilase is capable of catalysing a reaction from terephthalonitrile to 4-cyano benzoic acid in an aqueous medium.

28. The method of claim 27 wherein the aqueous medium further comprises a divalent cation.

29. The method of claim 28 wherein the divalent cation is Mg.sup.2+, Mn.sup.2+, Ca.sup.2+, Fe.sup.2+, Zn.sup.2+ or Co.sup.2+.

30. The method of claim 27 wherein the terephthalonitrile is added to the aqueous medium before incubation in a concentration of between 1% and 30% w/w.

31. The method of claim 27 wherein a pH-value of the aqueous medium is adjusted to below 5 by adding acid to the aqueous medium during or after incubation.

32. The method of claim 27 wherein the product is isolated by filtration or centrifugation after incubation.

33. The method of claim 27 wherein the aqueous medium is incubated for at least 2 h.

34. The method of claim 27 wherein the aqueous medium is incubated between 15 and 50° C.

35. The method of claim 27 wherein the nitrilase is produced by fermentation.

Description

FIGURES

[0156] FIG. 1 shows the reaction catalyzed by the nitrilases of the invention.

[0157] FIG. 2: Bioconversion of terephthalonitrile by heterologous E. coli cells expressing the nitrilase from Comamonas testosteroni (Seq. ID 2).

EXAMPLES

Example 1

[0158] 89 potential nitrilases were screened for activity of conversion terephthalonitril to 4-cyanobenzoic acid. Donor organism and SEQ ID of the amino acid sequence of 18 nitrilases active in screening and one non-functional nitrilase are listed in Table 1. The coding region of the nitrilases were optimized for expression in E. coli, these sequences synthesized and cloned in the expression vector pDHE (Stueckler et al. (2010) Tetrahedron 66(3-2)).

[0159] E. coli strains were transformed with the expression vectors, expression of the nitrilases induced and the culture harvested and tested for activity as described below.

TABLE-US-00007 TABLE 1 Donor Organism, SEQ ID, and 4-cyanobenzoic acid formation of 18 active nitrilases. Seq. 4-cyanobenzoic ID Donor Organism acid [mM] 10 Tepidicaulis marinus 88 101 Smithella sp. SDB 2 4 Unknown prokaryotic organism 328 103 Bradyrhizobium diazoefficiens 35 105 Aquimarina atlantica 2 107 Arthrobacter sp. Soil736 7 12 Sphingomonas wittichii RW1 126 111 Pseudomonas sp. RIT357 87 113 Nocardia brasiliensis NBRC 14402 22 109 Pseudomonas mandelii JR-1 14 8 Candidatus Dadabacteria bacterium CSP1-2 268 22 Salinisphaera shabanensis E1L3A 81 16 Synechococcus sp. CC9605 158 14 Rhizobium sp. YK2 192 6 Agrobacterium rubi 293 20 Flavihumibacter solisilvae 106 115 Defluviimonas alba 60 2 Comamonas testosteroni 704 24 Erythrobacter sp. JL475 0

[0160] 128 mg of terephthalonitrile were weighed to a 1.5 mL Eppendorf tube and mixed with 50 mM phosphate buffer solution at pH 7. To start the reaction, 50-100 μL of E. coli cell suspension containing different nitrilases were added and the mixture shaken at 37° C. The final terephthalonitrile concentration in the reaction tube was 1 M. After 48 hours, the entire reaction mixture was diluted in DMSO. A sample of this solution was withdrawn, diluted in water and subjected to HPLC analysis. The results are reported as concentration of 4-cyanobenzoic acid present in the 1 mL reaction mixture prior to dilution with DMSO.

Example 2

[0161] 1100 mL water and 100 g of terephthalonitrile were placed in a reactor.

[0162] The biocatalyst was used in the form of a concentrate cell suspension containing the nitrilase from Comamonas testosteroni (Seq. ID 2) and it was added to the reactor, whereby the bioconversion started. The temperature was kept at 37° C. and the reactor was mixed by an overhead-stirrer. The mixture was stirred for 21 h and samples for the analysis of 4-cyanobenzoic acid were taken from the reactor. The time course of terephthalonitrile conversion and 4-cyanobenzoic acid formation is given in FIG. 2.

[0163] After the bioconversion, the reaction mixture was removed from the reactor and filtered through Celite535 to remove the heterologous E. coli cells expressing the nitrilase. Acid, in this case sulfuric acid, was added to precipitate 4-cyanobenzoic acid, which was separated from the aqueous reaction mixture by filtration. The wet product was dried until a constant weight was reached. 111.5 g 4-cyanobenzoic acid were recovered.

Example 3

[0164] 128 mg of terephthalonitrile were weighed to a 1.5 mL Eppendorf tube and mixed with water or 50 mM phosphate buffer solution at pH 7. To start the reaction, 50-100 μL of E. coli cell suspension containing different nitrilases were added and the mixture shaken at 37° C. The final terephthalonitrile concentration in the reaction tube was 1 M. After 24 hours, the entire reaction mixture was diluted in DMSO. A sample of this solution was withdrawn, diluted in water and subjected to HPLC analysis. The results are reported as concentration of 4-cyanobenzoic acid present in the 1 mL reaction mixture prior to dilution with DMSO.

TABLE-US-00008 TABLE 2 4-cyanobenzoic acid formation from the nitrilases with the sequence IDs 4 (Unknown prokaryotic organism), 8 (Candidatus Dadabacteria bacterium CSP1-2), 6 (Agrobacterium rubi), and 2 (Comamonas testosteroni) and from the six nitrilases described in CN107641622A Ara Nit (Arabidopsis thaliana), Bras Nit (Brassica oleracea), Can Nit (Camelia sativa), Panto Nit (Pantoea sp. AS-PWVM4), Acid Nit (Acidovorax facilis 72W), Lepto Nit (Leptolyngbya sp.). Either water or an aqueous buffered solution (50 mM potassium phosphate buffer, pH 7) was used as reaction medium. 4-cyanobenzoic acid formation is given in mM as analysed after the incubation phase and also as mM/OD600 for normalization of the produced amount to the applied heterologous E. coli biomass in each reaction. 4-cyanobenzoic 4-cyanobenzoic acid acid [mM] [mM/OD600] Seq. ID Water Buffer Water Buffer 4 297 341 44 50 8 284 294 24 24 6 275 345 26 32 2 752 655 136 118 Ara Nit 46 56 5 6 Bras Nit 79 82 7 7 Can Nit 50 38 5 4 Panto Nit 198 229 22 26 Acid Nit 105 136 13 17 Lepto Nit 178 222 15 19

Example 4

[0165] The effect of the addition of Mg.sup.2+ ions to the reaction mixture was investigated. 128 mg of terephthalonitrile were weighed to a 1.5 mL Eppendorf tube and mixed with water. MgSO.sub.4 was added from a 1 M stock solution in water yielding different final concentrations of MgSO.sub.4 in the reaction. To start the reaction, 100 μL of an E. coli cell suspension containing the nitrilase from Comamonas testosteroni (Seq ID No. 2) were added and the mixture was shaken at 1000 rpm in an Eppendorf Thermomixer at 37° C. The final terephthalonitrile concentration in the reaction tube was 1 M. After 23 hours, the entire reaction mixture was diluted in DMSO. A sample of this solution was withdrawn, diluted in water and subjected to HPLC analysis. The results are reported as concentration of 4-cyanobenzoic acid present in the 1 mL reaction mixture prior to dilution with DMSO.

TABLE-US-00009 TABLE 3 4-cyanobenzoic acid formation from the nitrilase with the sequence ID 2 (Comamonas testosteroni) when different MgSO.sub.4 concentrations are used in the biocatalytic reaction. MgSO.sub.4 4-cyanobenzoic Residual Sum [mM] acid [mM] Terephthalonitrile [mM] [mM] 0 855 186 1041 10 931 162 1093 25 958 51 1009 40 1038 66 1104 50 955 19 973 100 1075 0 1075 125 1003 0 1003 150 1026 0 1026 175 1012 0 1012 200 1016 0 1016 250 1042 7 1049

[0166] The highest 4-cyanobenzoic acid concentration was achieved when 100 or more mM MgSO4 is added to the reaction. In these cases, complete conversion of the terephthalonitrile was observed.

Example 5

[0167] The effect of the addition of Mg.sup.2+ ions to the reaction mixture was investigated in combination with higher terephthalonitrile concentrations. 128 mg or 256 mg of terephthalonitrile were weighed to a 1.5 mL Eppendorf tube and mixed with water. MgSO.sub.4 was added from a 1 M stock solution in water yielding 100 or 200 mM MgSO.sub.4, respectively, in the reaction mixture. To start the reaction, 100 μL of an E. coli cell suspension containing the nitrilase from Comamonas testosteroni (Seq ID No. 2) were added and the mixture was shaken at 1000 rpm in an Eppendorf Thermomixer at 37° C. The final terephthalonitrile concentration in the reaction tube was 1 M or 2 M, respectively. After previously defined time points, the entire reaction mixture was diluted in DMSO. Samples of this solutions were withdrawn, diluted in water and subjected to HPLC analysis. The results are reported as concentration of 4-cyanobenzoic acid present in the 1 mL reaction mixture prior to dilution with DMSO.

TABLE-US-00010 TABLE 4 4-cyanobenzoic acid formation from the nitrilase with the sequence ID 2 (Comamonas testosteroni) when different MgSO.sub.4 concentrations and different terephthalonitrile concentrations are used in the biocatalytic reaction. Residual Terephthaloni- MgSO.sub.4 Reaction 4-cyanobenzoic Terephthaloni- trile [mM] [mM] Time [h] acid [mM] trile [mM] 1000 100 0.5 392 643 1 651 450 2 1028 99 4 1104 6 6 1054 11 23 1072 0 2000 100 0.5 381 1075 1 649 927 2 932 854 4 1015 867 6 1038 894 23 1083 860 1000 200 0.5 370 711 1 616 445 2 973 70 4 1059 3 6 1078 5 23 1080 0 2000 200 0.5 355 1072 1 569 902 2 1033 877 4 1307 783 6 1314 795 23 1355 805

[0168] The highest product concentration was achieved when the reaction mixture is supplemented with 200 mM MgSO.sub.4 and 2 M terephthalonitrile. Complete conversion of 2 M terephthalonitrile, however, was not achieved.

Example 6

[0169] The effect of the temperature on the reaction performance was investigated in the presence of absence of MgSO4. Approximately 128 mg of terephthalonitrile were weighed to a 1.5 mL Eppendorf tube and mixed with water. MgSO.sub.4 was added from a 1 M stock solution in water yielding 0 or 100 mM MgSO.sub.4, respectively, in the reaction mixture. To start the reaction, 100 μL of an E. coli cell suspension containing the nitrilase from Comamonas testosteroni (Seq ID No. 2) were added and the mixture was shaken at 1000 rpm in an Eppendorf Thermomixer at different temperatures (i.e., 20° C., 25° C., 30° C., 37° C.). The final terephthalonitrile concentration in the reaction tube was approximately 1 M. After previously defined time points, the entire reaction mixture was diluted in DMSO. Samples of this solutions were withdrawn, diluted in water and subjected to HPLC analysis. The results are reported as concentration of 4-cyanobenzoic acid present in the 1 mL reaction mixture prior to dilution with DMSO.

TABLE-US-00011 TABLE 5 4-cyanobenzoic acid formation from the nitrilase with the sequence ID 2 (Comamonas testosteroni) at different temperatures in the presence or absence of MgSO.sub.4 in the biocatalytic reaction. Temper- 4- Residual ature MgSO.sub.4 Reaction cyanobenzoic Terephthalonitrile [° C.] [mM] Time [h] acid [mM] [mM] 20 0 0.5 118 634 1 256 622 2 549 520 22 1079 0 20 100 0.5 76 680 1 176 752 2 407 650 22 1034 0 25 0 0.5 217 714 1 438 561 2 761 326 22 1072 0 25 100 0.5 182 675 1 364 656 2 706 350 22 1043 0 30 0 0.5 321 701 1 531 520 2 812 216 22 1045 0 30 100 0.5 246 744 1 497 568 2 897 181 22 1090 0 37 0 0.5 611 442 2 853 245 23 855 186 37 100 0.5 480 648 1 1049 58 2 1075 0

[0170] At a reaction temperature of 37° C., full conversion of 1 M terephthalonitrile was only achieved when the reaction mixture was supplemented with MgSO.sub.4. This implies that the beneficial effect of Mg.sup.2+ addition is more pronounced at higher reaction temperatures.

Example 7

[0171] The applied biocatalyst (E. coli cell suspension containing the nitrilase from Comamonas testosterone (Seq ID No. 2) principally catalyzes the conversion of terephthalonitrile to 4-cyanobenzoic acid as the main reaction.

[0172] The reaction conditions during the biocatalytic conversion can be adjusted in order to minimize excessive terephthalic acid formation. 8.14 g terephthalonitrile were added to 91.36 g deionized water in a 100 mL working volume EasyMax 102 reactor (Eppendorf, Germany). The temperature was adjusted to 33° C. and the stirrer speed was set to 400 rpm. Mixing was mediated by an impeller stirrer. 0.5 g of an E. coli cell suspension in potassium phosphate buffer containing the nitrilase from Comamonas testosteroni (Seq ID No. 2) were added to start the bioconversion. Samples were withdrawn for analysis of 4-cyanobenzoic acid and terephthalic acid at regular intervals. After 10.5 h the reaction was terminated, and cells were removed by filtration over Celite535. The final 4-cyanobenzoic acid content was 93 g/kg and the final terephthalic acid content was 0.2 g/kg. This corresponds to full conversion of the applied terephthalonitrile to these two products of the biocatalytic reaction. The fraction of 4-cyanobenzoic acid relative to the total product amount was 99.8%. 0.2% of the total product fraction was terephthalic acid. The terephthalic acid fraction is dependent on the mixing efficiency, the amount of biocatalyst added to the reaction and the temperature.

TABLE-US-00012 TABLE 6 Reaction conditions and reaction parameter for the bioconversion of terephthalonitrile. Parameter Unit Data Reaction temperature [° C.] 33 Reaction scale [kg] 0.1 TDN [g] 8.136 Biomass concentration [gBDW/kg] 0.183 Initial specific initial activity [kU/gBDW] 5.2 Full conversion YES/NO YES Total reaction duration [h] 10.5 Final 4-CBA concentration [g/kg] 93.02 Final TA concentration [g/kg] 0.20 Mass fraction 4-CBA of total product [%] 99.79 Specific yield Yp/x [g4-CBA/ 508 gBDW] Conversion [%] 100 TDN: terephthalonitrile, BDW: biomass dry weight, 4-CBA: 4-cyanobenzoic acid, TA: terephthalic acid. The initial specific activity of the catalyst is determined in the first hour of reaction and is given in kU. 1 kU corresponds to 1 mmol of 4-cyanobenzoic acid formed per minute.

Example 8

[0173] 3515 g water and 445 g terephthalonitrile were placed in a reactor. The biocatalyst was used in the form of a concentrated cell suspension containing the nitrilase from Comamonas testosteroni (Seq. ID 2) and added to the reactor, whereby the bioconversion started. The temperature was kept at 30° C. and the reactor was mixed by an overhead-stirrer. The mixture was stirred for 23 h. After the bioconversion, the pH was adjusted with NaOH to pH 9.4 and the reaction mixture was removed from the reactor. An ultrafiltration on a Sartoflow Advance (Sartorius) machine was performed using a membrane with a molecular weight cut-off of 10 kDa to remove the heterologous E. coli cells expressing the nitrilase. The resulting filtrate was split into two portions. 1748 g of the resulting filtrate were diluted with 1500 g water and the pH was adjusted to pH 2.2 by titration with 32 wt-% hydrochloric acid to precipitate the 4-cyanobenzic acid. Another 500 g of water were added to facilitate mixing during the addition of the hydrochloric acid solution. The suspension was filtered and washed with 1×1500 g water. The wet product was dried until a constant weight was reached. 193 g crystalline product were recovered and analyzed by HPLC and for chloride as well as water content (portion 1). 2029 g of the resulting filtrate were diluted with 1500 g water and the pH was adjusted to pH 1.89 by titration with 32 wt-% hydrochloric acid to precipitate the 4-cyanobenzic acid. Another 250 g of water were added to facilitate mixing during the addition of the hydrochloric acid solution. The suspension was filtered and washed with 2×1500 g water. The wet product was dried until a constant weight was reached. 223 g crystalline product were recovered and analyzed by HPLC and for chloride as well as water content (portion 2) The amount of water used for washing of the filter cake is decisive for the resulting product purity. Larger washing volume reduce the amount of residual chloride and other unwanted components in the final product. The fraction missing to give a sum of 100% is composed of ammonium and sodium and contaminants from the preceding biotransformation such as phosphate.

TABLE-US-00013 TABLE 7 Chemical composition of the product when hydrochloric acid is used for the precipitation of 4-cyanobenzoic acid. Compound Content in portion 1 Content in portion 2 4-cyanobenzoic acid 97.3 [wt-%] 99.0 [wt-%] Terephthalic acid  0.3 [wt-%]  0.3 [wt-%] Chloride (IC)  1.0 [wt-%]  0.3 [wt-%] Water  0.1 [wt-%]  0.1 [wt-%] IC: ion chromatography

Example 9

[0174] 881 g water and 108.9 g terephthalonitrile were placed in a reactor. The biocatalyst was used in the form of a concentrate cell suspension containing the nitrilase from Comamonas testosteroni (Seq. ID 2) and added to the reactor, whereby the bioconversion started. The temperature was kept at 30° C. and the reactor was mixed by an overhead-stirrer. The mixture was stirred for 24 h.

[0175] 612 g of the filtrate was diluted with 1000 g water and the pH was adjusted to pH 2.1 by titration with 98 wt-% sulfuric acid to precipitate the 4-cyanobenzic acid. The suspension was filtered and washed with 250 g water. The wet product was dried until a constant weight was reached. 56 g crystalline product were recovered and analyzed by HPLC for 4-cyanobenzoic acid and for ammonium, sulfate, and sodium content.

TABLE-US-00014 TABLE 8 Chemical composition of the product when sulfuric acid is used for the precipitation of 4-cyanobenzoic acid. Compound Content 4-cyanobenzoci acid (HPLC) 96.00 [wt-%] Terephthalic acid Not determined Ammonium (IC)  0.60 [wt-%] Sulfate (IC)  3.10 [wt-%] Na (Elementary analysis)  0.70 [wt-%] Water Not determined IC: ion chromatography.