Nitrile Hydratase Activity of Nocardia corallina B-276

Nitrile is a functional group that usually is transformed to amides or carboxylic acids under strong reaction conditions in acidic or basic media and high temperatures. Amides have also been prepared from nitriles at room temperature using strong oxidizing agents such as hydrogen peroxide or sodium superoxide in DMSO. On the other hand biocatalytic hydrolysis of nitriles mediated by nitrilase, nitrile hydratase (NHase) or amidase, is an important option for these processes. In general, these bioconversions, are fast under mild reaction conditions, there are no formation of secondary products, contamination due to chemical side reactions can be avoided or diminished, and the reaction can be regioand stereoselective, and thus it is an important alternative for the preparation of chiral compounds. These biocatalysts have important applications in industry for the enantioselective preparation of amides, for the transformation of waste nitrile to the less toxic amides or carboxylic acids. In Japan acrylamide is produced by the hydrolysis of acrylonitrile using resting cells of Rhodococcus rhodochrous J1; 5 and 2-arylpropionitriles have been enantioselectively hydrolyzed to the corresponding amides or carboxylic acids using the bacteria of the genera Rhodococcus, Agrobacterium, and Pseudomonas. Microorganisms of the genera Rhodococcus are some of the most common sources of NHases and amidases and, due to the fact that some authors have taxonomically classified Nocardia corallina B-276 (bacteria which we have used previously in reactions of oxidation) as Rhodococcus corallinus, we decided to study the possibility that Nocardia corallina B-276 could be used as a biocatalyst to hydrolyze nitriles.


Introduction
Nitrile is a functional group that usually is transformed to amides or carboxylic acids under strong reaction conditions in acidic or basic media and high temperatures.Amides have also been prepared from nitriles at room temperature using strong oxidizing agents such as hydrogen peroxide 1 or sodium superoxide in DMSO. 2 On the other hand biocatalytic hydrolysis of nitriles mediated by nitrilase, nitrile hydratase (NHase) or amidase, is an important option for these processes. 3In general, these bioconversions, are fast under mild reaction conditions, there are no formation of secondary products, contamination due to chemical side reactions can be avoided or diminished, and the reaction can be regioand stereoselective, and thus it is an important alternative for the preparation of chiral compounds.
These biocatalysts have important applications in industry for the enantioselective preparation of amides, for the transformation of waste nitrile to the less toxic amides or carboxylic acids.In Japan acrylamide is produced by the hydrolysis of acrylonitrile using resting cells of Rhodococcus rhodochrous J1; 4, 5 and 2-arylpropionitriles have been enantioselectively hydrolyzed to the corresponding amides or carboxylic acids using the bacteria of the genera Rhodococcus, [6][7][8][9] Agrobacterium, 9,10 and Pseudomonas. 11croorganisms of the genera Rhodococcus are some of the most common sources of NHases and amidases 12 and, due to the fact that some authors have taxonomically classified Nocardia corallina B-276 (bacteria which we have used previously in reactions of oxidation [13][14][15][16] ) as Rhodococcus corallinus, 17 we decided to study the possibility that Nocardia corallina B-276 could be used as a biocatalyst to hydrolyze nitriles.

Results and Discussion
The nitriles used to explore their possible hydrolysis using Nocardia corallina as biocatalyst were benzonitrile (1a), 4-chlorophenylacetonitrile (2a), phenylacetonitrile (3a), 4-methoxybenzonitrile (4a) and 3,4-dimethoxyphenylacetonitrile (5a), (Scheme 1).Nitriles are known as potential raw materials for fine chemistry. 3lso, the selection of the aromatic compounds that have substituents in the meta and para position of the ring is important, because NHases are metalloenzymes that can coordinate better with these compounds. 18][15][16] The biotransformation of nitriles by N. corallina was carried out using two methods.In method A we use the cells in the culture media and in method B we use the resting cells suspended in a phosphate buffer (pH= 7.0).
The hydrolysis of benzonitrile (1a, entry 1, Table 1) using method A gave the product with an isolated yield of 26%, after 30 h of reaction, and this method also has the disadvantage that the isolation of the corresponding benzamide was difficult due to its high solubility in water.To achieve better results, we decided to use resting cells of N. corallina and, as can be observed in Table 1 (entries 2 -5), an increase in the cell ratio improved substantially the conversion degree.For example, when the ratio changed from 1:40 to 1:100 the conversion was greatly improved, from 44 to 89%, meaning that the conversion degree was doubled.The corresponding benzoic acid was not detected during the biotransformation using any of the two methods.
To determine the possible hydrolysis of benzamide (1b) to benzoic acid under the conditions of method B, the reaction was performed with a ratio substrate:cells of 1:40, and after 48 h the corresponding carboxylic acid was not detected by HPLC, and the amide 1b remained unchanged.
In the case of 4-chlorophenylacetonitrile (2a) hydrolysis using method A, and after 118 h, the isolated yield of amide was only 11% (entry 1, Table 2).Also, in this case the results of the biocatalyzed hydrolysis under the reaction conditions of method B were much better.The biotransformation of 2a to the corresponding amide 2b, with a ratio of 1:400 gave a 90% conversion in 24 h, with an increase of only 6% after 48 h (entries 2 and 3, Table 2).Similar results were obtained using a ratio of 1:200 (entries 4 and 5, Table 2).In contrast with the hydrolysis of benzonitrile (1a) to benzamide (1b) that needed a substrate:cells ratio of 1:100 to get a conversion of 89% (entry 5, Table 1), for 4-chlorophenylacetamide (2a) using a lower ratio 1:15, the conversion was quantitative after 89 h (entry 6, Table 2).Once again the corresponding carboxylic acid was not detected by IR or TLC, under any of the reaction conditions using the two methods.
The hydrolysis of nitriles 3a, 4a and 5a to the corresponding amides resulted in very low conversions under method A, even after 192 h.Most of the starting material was recovered unchanged, and similar results were obtained for nitriles 1a and 2a.
Contrary to what was expected, and in spite of a high substrate: cells ratio 1:200, after 48 h the hydrolysis of phenylacetonitrile (3a) to the amide 3b was 31% (entry 2, Table 3).
To the best of our knowledge, this is the first report about the use of N. corallina B-276 for the hydrolysis of nitriles to amides, and it is worth mentioning that the hydrolysis continues until the amide, and is not further hydrolyzed to the carboxylic acid under the reaction conditions of methods A and B. This is important because amides are very useful intermediates and, in many cases are difficult to prepare, 19 since they are more easily hydrolyzed to the carboxylic acid than the nitrile itself.It has been reported that microorganisms of the genera Nocardia and Rhodococcus are able to hydrolyze nitriles to amides but frequently the reaction cannot be stopped in the amide stage and a mixture of amide and acid or only the acid is obtained. 20This is not the case of N. corallina B-276 which can produce only amides selectively, in high yields depending on the experimental conditions and substrate structure.Then the use of an amidase inhibitor is not necessary. 21
Method A: biotransformation using cells in culture medium 13 Pre-culture I.A 125 mL Erlenmeyer flask containing 50 mL of sterile culture medium was inoculated from an agar plate (three days old) and incubated at 28-30 °C on an orbital shaker (170 rpm) for 20-24 h.
Pre-culture II.The content of the pre-culture I flask was aseptically poured into a 250 mL Erlenmeyer flask containing 100 mL of fresh sterile culture medium.The flask was incubated at 28-30 °C on an orbital shaker (170 rpm) for 24 h.
Biotransformation.Under aseptic conditions the substrate (0.85-1.45 mmol), in 1 mL of N,N-dimethylformamide, was added to the flask containing pre-culture II, followed by the addition of n-octane (10 mL).The reaction mixture (161 mL final volume) was incubated at 28-30 °C on an orbital shaker (170 rpm).The progress of the biotransformation was then monitored by TLC, and stopped at the time indicated for each substrate, the reaction mixture was saturated with NaCl and filtered through Celite, the pH was adjusted to 10-10.5 with aqueous NaOH (2 mol L -1 ).Amides and unreacted nitrile were extracted with ethyl acetate (4 x 25 mL), and after drying over anhydrous sodium sulfate the solvent was evaporated under vacuum.The aqueous phase was then acidified to pH 2 with aqueous HCl (0.5 mol L -1 ), and the acids were extracted with CH 2 Cl 2 (4 x 25 mL) after drying over anhydrous sodium sulfate, and the solvent was then evaporated under vacuum.The products were purified by flash chromatography.
Method B: biotransformation using resting cells 24 Pre-culture.A 500 mL Erlenmeyer flask containing 200 mL of sterile culture medium was inoculated from an agar plate (three days old) and incubated at 28-30 °C on an orbital shaker (170 rpm) for 48 h.The cells were collected by centrifugation at 4500 rpm for15 min.Cells were washed twice with potassium phosphate buffer (0.1 mol L -1 , pH 7.0).
Biotransformation.The wet cells were incubated in 50 mL phosphate buffer, pH 7.0, for 30 min at 28-30 °C on an orbital shaker (170 rpm), the substrates were added in the substrate:cells ratio (m/m) as indicated in each case and were shaken under the same conditions for 20-48 h, depending on the substrate.The reaction mixture was then centrifuged at 4500 rpm for 15 min.Samples were analyzed by HPLC, using a C-18 column, and MeOH-H 2 O (80:20) as the eluent, to determine the conversion degree.

Conclusions
These results demonstrate that N. corallina B-276 has the ability to hydrolyze nitriles to amides, and because the corresponding carboxylic acid was not detected in any case,

Table 3 .
Biotransformation of nitriles 3a, 4a and 5a with resting cells of Nocardia corallina a Determined by HPLC.Reaction conditions were not optimized.

Table 2 .
Biotransformation of 4-chlorophenylacetonitrile 2a c Determined by HPLC; Reaction conditions were not optimized.