Title of Invention	"A PROCESS FOR THE BIOTRANSFORMATION OF CYANOHYDROCARBONS TO CORRESPONDING AMIDES"
Abstract	The present invention relates to a process for the biotransformation of cyanohydrocarbons to corresponding amides. It relates to a process for the biotransformation of cyanohydrocarbos to corresponding amides using whole cells of a novel Rhodococcus sp. Strain RL4 having nitrile hydratase enzyme. The present invention particularly relates to conversion of aliphatic and aromatic cyanohydrocarbons such as acrylonitrile, propionitrile, isobutyronitrile, benzyl cyanide, 3-cyanopyridine and the like to the corresponding amides, and more particularly relates to an enzymatic process for the hydration of acrylonitrile to acrylamide catalyzed by nitrile hydratase present in the resting cells of Rhodococcus sp. RL4. The main usage of the invention is to provide a process fro the preparation of Rhodococcus sp. RL4 cells having nitrile hydratase enzyme which can be used under mild reaction conditions e.g. atmospheric pressure, low temperature, near neutral pH to hydrate acrylonitrile to acrylamide without any by-product formation.

Title of Invention

"A PROCESS FOR THE BIOTRANSFORMATION OF CYANOHYDROCARBONS TO CORRESPONDING AMIDES"

Abstract

The present invention relates to a process for the biotransformation of cyanohydrocarbons to corresponding amides. It relates to a process for the biotransformation of cyanohydrocarbos to corresponding amides using whole cells of a novel Rhodococcus sp. Strain RL4 having nitrile hydratase enzyme. The present invention particularly relates to conversion of aliphatic and aromatic cyanohydrocarbons such as acrylonitrile, propionitrile, isobutyronitrile, benzyl cyanide, 3-cyanopyridine and the like to the corresponding amides, and more particularly relates to an enzymatic process for the hydration of acrylonitrile to acrylamide catalyzed by nitrile hydratase present in the resting cells of Rhodococcus sp. RL4. The main usage of the invention is to provide a process fro the preparation of Rhodococcus sp. RL4 cells having nitrile hydratase enzyme which can be used under mild reaction conditions e.g. atmospheric pressure, low temperature, near neutral pH to hydrate acrylonitrile to acrylamide without any by-product formation.

Full Text	The present invention relates to a process for the biotransformation of cyanohydrocarbons to corresponding amides. The present invention particularly relates to a process for the biotransformation of cyanohydrocarbons to corresponding amides using whole cells of a novel Rhodococcus sp. strain RL4 having nitrile hydratase enzyme. The present invention particularly relates to conversion of aliphatic and aromatic cyanohydrocarbons such as acrylonitrile, propionitrile, isobutyronitrile, benzyl cyanide, 3-cyanopyridine and the like to the corresponding amides , and more particularly relates to an enzymatic process for the hydration of acrylonitrile to acrylamide catalyzed by nitrile hydratase present in the resting cells of Rhodococcus sp. RL4. The main usage of the invention is to provide a process for the preparation of Rhodococcus sp. RL4 cells having nitrile hydratase enzyme which can be used under mild reaction conditions e.g. atmospheric pressure, low temperature, near neutral pH to hydrate acrylonitrile to acrylamide without any by-product formation. Acrylamide is produced industrially as monomer for synthetic fibres, flocculant agents, etc. Conventional method for production of acrylamide involves the use of copper salts as catalysts. The method , however, has many disadvantages, e.g., the complexity of preparation of the catalyst, the difficulty in regenerating the catalyst used, and the complexity of purifying and isolating the acrylamide. In addition, acrylamide readily polymerized. Therefore, it is desirable that acrylamide is produced under mild reaction conditions. In recent years, acrylamide is being produced industrially using nitrile hydratase enzyme produced by bacteria.The reaction occurs at low temperatures (usually 5 to 10 °C) by incubation of acrylonitrile with bacterial cells that have high nitrile hydratase activity. In contrast with the conventional chemical process, the recovery of unreacted acrylonitrile is not necessary because the yield of the enzymatic conversion is almost 100%; the removal of copper ions as a catalyst from the reaction mixture is also no longer necessary. In fact, Nitto Chemical Company (Japan) started industrial production of acrylamide (3000 tons/yr) in 1985 using the nitrile hydratase of Rhodococcus sp. N-774, the first generation strain (Watanabe,!, Satoh, Y, and Enomoto, K (1987J Agric. Biol. Chem. 51, 3193-3199: Yamada, H and Kobayashi, M (1996) Biosci. Biotech. Biochem. 60, 1391-1400). In 1988 this strain was replaced by the second-generation strain, Pseudomonas chlororaphis B23 (Yamada, H and Kobayashi, M (1996) Biosci. Biotech. Biochem. 60, 1391-1400). Acrylamide (6000 tons/yr) had been produced using immobilized cells of Pseudomonas chlororaphis B23, entrapped on a cationic acrylamide -based polymer gel ( Watanabe, I (1987) In: Methods in Enzymology, vol. 136 ( K Mosbach, ed.) pp 523-530. New York. Academic Press; Nakai, K, Watanabe, I, Sato, Y and Enomoto, K. (1988) Nippon Nogeikagaku Kaishi 62, 1443-1450). In 1991, the same Company replaced P. chlororaphis B23 with the third generation strain Rhodococcus rhodochorous Jl whereby producing 30,000 tons of acrylamide/yr using a bioreactor containing immobilized cells ofR. rhodochorous Jl ( Kobayashi, M, Nagasawa, T, Yamada, H (1992) Trends in Biotechnol. 10, 402-408; Yamada, H and Kobayashi, M (1996) Biosci. Biotech. Biochem. 60, 1391-1400). The formation of nitrile hydratase in Rhodococcus sp. N-774 and P. chlororaphis B23 is highly enhanced by the addition of Fe+2 or Fe+3 ions to the medium ( Watanabe, I, Satoh, Y, Enomoto, K, Seiki, S and Sakashita, K ( 1987) Agric Biol chem 51, 3201-3206). The nitrile hydratase protein of R.rhodochorous Jl is not synthesized without the addition of Co+2 ions in the medium. ( Yamada, H and Kobayashi, M ( 1996) Biosci Biotech Biochem 60, 1391-1400). Fe+2 or Fe+3 ions can not replace Co+2 ions. In order to produce nitrile hydratase in the process of growing bacteria, it is necessary to add an inducer to the nutrient medium. Nitriles and amides of organic acids (. US Patent No. 4,555,487; European Patent No. 0109083; European Patent No. 0204555), urea or its derivatives (European Patent Application No. 0362829) may be used as inducers. Known in the prior art are strains of Corynebacterium N- 774 (US Patent No. 4,248,968) (now known as Rhodococcus sp.N-774) and Rhodococcus sp. S-6 (US Patent No. 5,179,014) for which no inducer is required. Disadvantages of strain N-774 reside in its low nitrile hydratase activity (its specific activity is 50-60 Units/mg, measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight), and narrow range of substrate nitriles (only aliphatic nitriles). A disadvantage of strain S-6 resides in its capacity to hydrate the produced amides to acids due to the presence of nitrilase enzyme, whereby the quality of amides is sharply affected and its low productivity (it is incapable of producing more than 20% acrylamide in the solution). In case of strain Pseudomonas chlororaphis B23, methacrylamide was essential to be present in the medium as an inducer of nitrile hydratase activity ( Watanabe, I, Satoh, Y, Enomoto, K, Seki, S and Sakashita, K (1987) Agric. Biol. Chem. 51, 3201-3206) . Glucose inhibited the formation of nitrile hydratase in P. chlororaphis B23 (Yamada, H, Ryuno, K, Nagasawa, T, Enomoto, K and Watanabe, I (1986) Agric Biol Chem. 50, 2859-2865). One of the important technical disadvantage of P. chlororaphis B23 was that when this organism was grown on a medium containing sucrose, mucilage polysaccharides,which resemble levan , were produced causing ineffective aeration and difficulty in harvesting of cells by brief centrifugation (at 8000 X g for 10 min) due to high viscosity of the culture medium ( Yamada, H and Kobayashi M (1996) Biosci Biotech Biochem 60, 1391-1400). The strain Rhodococcus rhodochrous Jl necessitates the use of urea, methacrylamide, or crotonamide in the medium to induce the formation of nitrile hydratase. When Rhodococcus rhodochrous Jl is cultured in the optimum culture medium consisting of 2g of glucose, 50 mg of KH2PO4, 50 mg of K2HPO4, 50 mg of MgSO4.7H2O, 0.1 g of yeast extract, 0.5 g of polypeptone, 0.75g of urea , 1 mg of CoCl2.6H2O per 100 ml tap water(pH 7.2), it produces a nitrile hydratase having a specific activity of 76 Units/mg ,( measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight) (Nagasawa, T, Takeuchi, K, Yamada, H (1991) Eur J Biochem 196, 581-589). Unlike P.chlororaphis B23, sucrose did not promote much of cell growth or nitrile hydratase activity of R. rhodochorous Jl (Nagasawa, T, Kobayashi, M and Yamada, H (1988) Arch Micrbiol 150, 89-94 ). The strain Rhodococcus sp. RL4 described in the present invention when cultured in the optimal culture medium containing 1g of sucrose, 2 g of peptone, 0.05 g of KH2P04, 0.05 g of K2HPO4 0.05 g of MgSO4.7H2O, 0.001 g of FeSO4.7H2O, 0.2 g of methacrylamide per 100 ml of water (pH 7.2), it produces a nitrile hydratase having a specific activity of 140 Units/mg (measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight). Unlike Rhodococcus rhodochrous Jl, glucose did not inhibit the formation of nitrile hydratase in Rhodococcus sp. RL4. The nitrile hydratase of strain Rhodococcus sp. RL4 has broad substrate specificity with respect to aliphatic and aromatic cyanohydrocarbons similar to the strain Rhodococcus rhodochrous Jl (European Patent Application No. 0362829). The resting cells of Rhodococcus sp. RL4 can be used to produce acrylamide at a concentration as high as 40%; the acrylamide solution obtained by separating the bacterial cells from the reaction system is colorless. On the other hand, although the resting cells of Brevibacterium R312 (US Patent 4,001,081) could also produce acrylamide at a high concentration (20%), the solution from which bacterial cells were separated was colored an extremely dark yellow and contains various impurities originating from the cells, and hence an onerous purifying step was necessary (cited from US Patent 4,248,968). The aforementioned strain RL4 can be maintained easily and is found stable with respect to enzyme yields and culture requirements. The main objective of the present invention is to provide a process for the biotransformation of cyan hydrocarbons to corresponding amides using novel Rhodococcus sp. RL4 cells having nitrile hydratase enzyme. Accordingly, the present invention provides a process for the biotransformation of cyan hydrocarbons to corresponding amides which comprises; a) growing strain Rhodococcus sp. RL4 at pH range of 6.5 to 8.5 and temperature 20 to 35°C in a conventional nutrient medium comprising a carbon source, a nitrogen source, salts, and an inducer such as herein described, recovering the Rhodococcus sp. RL4 cells having nitrile hydratase activity by known methods, b) contacting cyanohydrocarbons such as herein described in known medium at temperature of 0 to 30°c, at pH 6.0 to 9.0 with Rhodococcus sp. RL4 cells having nitrile hydratase enzyme on various supports, c) separating the cells from the reaction mixture by known method such as herein described, recovering the corresponding amide from the said supernatant by known method. The conventional nutrient medium used for growing Rhodococcus sp. RL4 having high nitrile hydratase activity consists or sucrose or glucose (carbon source). ( 0.5-3 g%), KH2P04 (0.05-2 g%), K2HPO4 (0.05-2 g%), MgSO4.7H2O (0.02-1 g%), FeSO4.7H20 (0.0002-0.002 g%) , peptone, yeast extract, beef extract, casaminoacid in combination or alone (complex nitrogen source) (0.5-2 g%), and an inducer (0.05-0.6 g%). The microorganism was grown under aerobic conditions for 48 to 72 h in shaking flasks or in a fermentor. The present invention provides a process for the preparation of Rhodococcus sp. RL4 cells having nitrile hydratase enzyme which can be used under mild reaction conditions e.g. atmospheric pressure, pH in the range of 7.0 to 8.5, temperature above freezing point to 30 °C to hydrate acrylonitrile to produce up to 400 g acrylamide per litre of reaction mixture without any by-product formation which obviates the drawbacks encountered in the copper salt-catalyzed chemical synthesis of acrylamide and the drawbacks encountered in enzymatic synthesis of acrylamide of formation of mucilage polysaccharide in P. chlororaphis B23 and of using expensive glucose instead of sucrose in the culture medium for the formation of nitrile hydratase in Rhodococcus rhodococcus Jl. In an embodiment of the present invention the cyanohydrocarbons used for the conversion to corresponding amides by Rhodococcus sp. RL4 cells may be aliphatic cyanohydrocarbons such as acrylonitile, propionitrile, isobutyronitrile and the aromatic cyanohydrocarbons such as benzylcyanide, 3-cyanopyridine. In another embodiment of the present invention the reaction conditions under which resting cells of Rhodococcus sp. RL4 may be used for hydrating acrylonitrile to acrylamide may be varied over a relatively wide range. The reaction can be carried out either in aqueous medium or in phosphate or Tris-buffer (pH 7.0 to 8.5) and the typical temperature range is 0 °C to 30 °C, but it is preferred that the hydration reaction be carried out at a temperature below 10 °C. Acrylonitrile as a substrate was added intermittently in small portions, since it is inhibitory for nitrile hydratase enzyme when added to the reaction mixture at a concentration exceeding 2%. In another embodiment of the invention the Rhodococcus sp RL4 may be used as free cells or in lyophilized form or in immobilized form on various known support materials such as polyacrylamide, agar, calcium alginate, K-carrageenan etc. In another embodiment of the present invention the conditions under which Rhodococcus sp. RL4 could be cultivated to produce cells having nitrile hydratase enzyme may be varied over a relatively wide range. A variety of carbon sources (0.5 to 3g%) such as sucrose, glucose, mannitol and sodium citrate may be suitable for growing the microorganism. Also, a variety of complex nitrogen sources (0.5 to 2g%) alone or in combination such as yeast extract, peptone, casein hydrolysate and beef extract may be used for growing the microorganism. A variety of inducers such as methacrylamide, isobutyronitrile and propionitrile may be used in the medium for preparation of Rhodococcus sp. RL4 cells having nitrile hydratase activity. However, the preferred carbon source is sucrose and glucose, preferred complex nitrogen source is any one of yeast extract, peptone and casein hydrolysate or a combination, and preferred inducer is methacrylamide. In another embodiment of the present invention the pH and temperature of the nutrient medium for growing Rhodococcus RL4 cells may also vary but, of course, should not be such that would result in inactivating or otherwise detrimentally affecting the microorganism to such an extent that production of nitrile hydratase is significantly decreased. The typical pH range at which Rhodococcus sp. RL4 may be grown is 6.5 to 8.5 and the typical temperature range is 20 to 35 °C, but is preferred that the microorganism be grown at pH 7.0 to 8.5 and at 28 to 30 °C. In yet another embodiment of the invention the inducer used may be such as propionitrile, isobutyronitrile or methacrylamide. In yet another embodiment of the invention the known method used for recovery of Rhodococcus RL4 cells may be such as centrifugation, decantation etc. In yet another embodiment of the present invention the organism used in the present invention was isolated from a soil samlpe of Assam , tentatively identified as Rhodococcus species and deposited at RRL Jorhat culture collection centre giving an accession number RL4. The morphological and biochemical characteristics of the bacterial strain RL4 is given in Table 1. Table 1. The morphological and biochemical characteristics of bacterial strain RL4. Characteristics Observation Cell shape At initial stage of cultivation, the cell is in a long rod-shaped form and irregular branches are observed. Later it breaks into a short rod-shaped or spherical form (pleomorphism) Spore none Motility none Pigment production negative Acid fastness Gram reaction Methyl red test Oxygen relationship State in nutrient agar plate culture Growth range Growth on sole carbon source: glucose maltose rhamnose sodium benzoate sodium citrate p-hydroxy benzoic acid cresol serine Growth in 5% NaCl Glucose fermentation Lactose fermentation Sucrose fermentation Indole production H2S production Nitrate reduction Denitrification Catalase Urease Oxidase Arginine dihydrolase Starch hydrolysis Gelatin hydrolysis negative positive negative aerobic smooth, convex colonies pH 5 to 10 ; temp. 10 °C to 37 °C positive positive positive negative positive positive negative negative positive acid formed, no gas produced no acid, no gas no acid, no gas negative negative negative negative positive positive negative negative negative negative To determine taxonomic position of the bacterial strain RL4 based on the description of bacteriological characteristics, according to Bergey's Manual of Determinative Bacteriology, 8th ed (1974) and Bergey's Manual of Systematic Bacteriology, vol.2 (1986) the strain RL4 falls under the aerobic, gram-positive, non acid fastness,and catalase-positive bacillary catagory having no endo-spore and no flagella. From the fact that the bacterium is in a long rod-shaped form at the initial stage of growth, showing hypha-like appearance, and grows with branching to break and split latter into short rod or spherical form (pleomorphism), it is considered to belong to the genus Rhodococcus. However, the biochemical characteristics of this organism clearly indicates that the strain RL4 differs from other high acrylamide- producing industrial Rhodococcus strains viz. Rhodococcus sp.N-774 ( US Patent 4,248,968 ) and Rhodococcus rhodochrous Jl( US Patent 5,334,519 ) with respect to nitrate reduction test ( nitrate reduction is negative for strain RL4, whereas in case of strains N-774 and Jl nitrate reduction is positive). In another embodiment of the present invention immobilized cells of Rhodococus sp. RL4 may be used for the conversion of cyanohydrocarbons to corresponding amides.The conventional methods of cell immobilization may be used for entrapping Rhodococcus sp. RL4 cells in polymers . In case of polyacrylamide gel entrapped cells, the immobilization can be conducted by suspending the cells in a suitable aqueous medium (e.g. a physiological saline, a buffer solution, etc) containing an acrylamide series monomer( e.g. acrylamide) and a cross linking agent (e.g. N,N' methylene bisacrylamide), adding a suitable polymerization initiator (e.g. ammonium persulfate) and a polymerization accelerator(e.g. N,N,N',N' tetramethyl ethylene diamine) to the suspension, and conducting polymerization and gellation at about 0 °C to 30 °C, preferably 0 °C to 15 °C, at a pH of about 5 to 9, preferably about 6 to 8. While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. The following examples are given by way of illustration of the present invention and therefore should not be construed to the limit of the present invention. Example 1 Rhodococcus sp. RL4 was cultivated at 30 °C for 48 h with shaking at 200 rev/min in an Erlenmayer flask containing 50 ml of a medium consisting of 1g of sucrose, 2g of peptone, 0.05 g of KH2PO4, 0.05 g of K2HP04, 0.05 g of MgSO4.7H2O, 0.001 g of FeSO4.7H2O, 0.2 g of methacrylamide per 100 ml of distilled water (pH 7.2). Cells were harvested by centrifugation at 10,000 X g at 10 °C for 12 min and then washed with 0.05 M potassium phosphate (pH 7.5), centrifuged again and suspended in 0.05 M potassium phosphate buffer (pH 7.5). A small portion of the suspension was sampled and used for measuring the dry weight of the bacterial cells. The yield of cells was 7.0 g dry cells per litre medium.The specific activity of the nitrile hydratase in strain RL4 reached 140 Units/ml (measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight), its total activity being 1000 Units/ml. The washed cells obtained as above were used for conversion of cyanohydrocarbons to corresponding amides. Example 2 The washed cells of Rhodococcus sp. RL4 (20 rng dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml of 0.05 M potassium phosphate buffer (pH 7.5) and acrylonitrile (5.4 ml) was added intermittently (0.2 ml every 30 min) over a period of 13.5 h under occasional stirring and the hydration reaction was allowed to proceed at 5 °C.Thereafter, reaction was allowed to proceed for another 1h to drive the reaction to completion. Removal of cells by centrifugation gave a colorless, transparent supernatant. This supernatant contained 30 % acrylamide, but contained no unreacted acrylonitrile and no by-products like acrylic acid. The yield of acrylamide was found to be 99.99%. Thus the reaction proceeded quantitatively to completion.The aforesaid supernatant was then vacuum-concentrated while bubbling air at a temperature of 45 °C by use of a flash evaporator to obtain more concentrated acrylamide solution or crystals thereof. The colorless crystals of acrylamide were recrystalized from methanol. This compound was identified as acrylamide based on m.p, elementary analysis, IR, NMR and Mass spectra. Example 3 The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and benzyl cyanide (0.2 ml) was added and the hydration reaction was allowed to proceed at 10 0C. Within two hours crystals of a -phenylacetamide separated out from the reaction mixture and was recovered by filtration. The conversion was 100%. This compound was identified as a -phenylacetamide based on m.p, elementary analysis, IR, NMR and Mass spectra. Example 4 The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and propionitrile (0.2 ml) was added and the hydration reaction was allowed to proceed at 10 °C for two hours. After the completion of the reaction, the clear solution obtained by removal of the cells by centrifugation was dried at 70 °C.The crystals of propionamide thus obtained were recrystalized from methanol. The conversion was 100%. This compound was identified as propionamide based on m.p, elementary analysis, IR, NMR and Mass spectra. Example 5 The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and isobutyronitrile (0.2 ml) was added and the hydration reaction was allowed to proceed at 10 °C for two hours. After the completion of the reaction, the clear solution obtained by removal of the cells by centrifugation was dried at 70 "C.The crystals of isobutyramide thus obtained were recrystalized from methanol. The conversion was 100%. This compound was identified as isobutyramide based on m.p, elementary analysis, IR, NMR and Mass spectra. Example 6 The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and 3-cyanopyridine (0.2g) was added. The reaction was allowed to proceed at 10 °C for 4 h with stirring. After the completion of the reaction, the clear solution obtained by removal of the cells by centrifugation was dried at 70 °C.The crystals of nicotinamide thus obtained were recrystalized from methanol. The conversion was 100%. This compound was identified as nicotinamide based on m.p, elementary analysis, IR, NMR and Mass spectra. Example 7 The washed cells of Rhodococcus sp. RL4 (0.3 g dry weight equivalent) prepared as given in Example 1 were suspended in 12 ml of 0.05 M potassium phosphate buffer (pH 7.5) and added to 12 ml of a solution of monomer (3.25 g acrylamide) and crosslinker (0.38 g N,N'-methylene bisacrylamide) in 0.05 M potassium phosphate buffer (pH 7.5) to prepare a uniform suspension. The polymerization was initiated by adding 0.5 ml of N,N,N',N'- tetramethyl ethylene diamine solution (0.07 g dissolved in water) and 0.5 ml of ammonium persulfate solution (0.025 g dissolved in water). Polymerization was performed in ice-water bath (10 °C) for 1 h. The gel block was then cut into cubes, and these were then thoroughly washed with 0.05 M potassium phosphate buffer (pH 7.5) in order to remove nonpolymerized monomers and residues. (FigureRemoved) Figure 1. Schematic diagram of perlmental setup for reactor operation PI Acrylonitrile reservoir, (2) substrate and product reservoir, (3) Water bath, (4) peristaltic pump, (5) fluidized-bed with immobilized cells, (6) water jacket By using the immobilized strain as obtained above, conversion of acrylonitrile to acrylamide was conducted in a fluidized-bed reactor. A schematic diagram of the experimental equipment for fluidized- bed reactor operation is shown in Figure 1. A jacketed glass column (2.8 cm bed diameter and 13.5 cm height) equipped with perforated glass bottom to support immobilized cells ( 20 g gel cubes) was used. The substrate product reservoir was placed in water bath at 5 °C. The fluidized- bed was maintained at the same temperature as that of the reservoir by recirculating water from the water bath through plastic tube. The fluidized-bed reactor operation was carried out in a fed-batch manner by recirculating the substrate solution at the rate of 30 L per hour into the substrate-product reservoir containing 150 ml of 0.05 M potassium phosphate buffer( pH 7.5) and the fluidized-bed (2.8 cm bed diameter and 13.5 cm height). At the same time acrylonitrile was continuously fed to the substrate-product reservoir at the rate of 3.5 ml/h by a peristaltic pump. Samples were withdrawn from substrate-product reservoir periodically and acrylamide concentration was measured by HPLC. After the completion of the addition of acrylonitrile (35 ml), 21% acrylamide was obtained and the conversion of substrate was 100%. After the completion of one batch which took llh, the reaction mixture was drained out from the reactor, the gel cubes in the reactor was washed with 0.05 M phosphate buffer (pH 7.5) and the reactor was operated for another 12 batches under similar conditions as above. The productivity of the reactor system was found to remain constant (100% conversion) upto 6th batch and then the conversion falls from 90% in the 7 th batch to 50.5% in the 13th batch.The main advantage of the present invention is that the resting cells of Rhodococcus sp. RL4 can be used for the biotransformation of acrylonitrile to acrylamide at concentrations of 20 to 40% without any by-product formation under very mild reaction conditions e.g. low temperature(above freezing point to 10 ° C) and pH range of 7.0 to 8.5; the acrylamide solution obtained by separating the bacterial cells from the reaction system is colourless. This obviates the drawback encountered in biotransformation of acrylonitrile to acrylamide using the resting cells of Brevibacterium R312 (cited from US Patent 4,248,968) which produced dark coloured acrylamide solution, and hence an onerous purifying step was necessary. Furthermore, both sucrose and glucose act as a carbon source to promote much of cell growth and nitrile hydratase activity of Rhodococcus sp. RL4. Also,the nitrile hydratase of Rhodococcus species RL4 has broad substrate specificity thereby converting aliphatic and aromatic cyanohydrocarbons to corresponding amides. The aforementioned strain RL4 can be maintained easily and is found stable with respect to enzyme yields and culture requirements. We Claim: 1. A process for the biotransformation of cyanohydrocarbons to corresponding amides which comprises; a) growing strain Rhodococcus sp. RL4 at pH range of 6.5 to 8.5 and temperature 20 to 35 °C in a conventional nutrient medium comprising a carbon source, a nitrogen source, salts, and an inducer such as herein described, recovering the Rhodococcus sp. RL4 cells having nitrile hydratase activity by known methods, b) contacting cyanohydrocarbons such as herein described in known medium at temperature of 0 to 30°c, at pH 6.0 to 9.0 with Rhodococcus sp. RL4 cells having nitrile hydratase enzyme bn various supports, c) separating the cells from the reaction mixture by known method such as herein described, recovering the corresponding amide from the said supernatant by known method. 2. A process as claimed in claim 1 wherein the strain Rhodococcus sp RL4 is used as free cells or in lyophilized form or in immobilized form on various supports such as polyacrylamide, agar, calcium alginate, K-carrageen an. 3. A process as claimed in claim 1 wherein the carbon sources (0.5 to 3g %) used for cultivating the strain Rhodococcus sp. RL4 are selected from a member of the group consisting of sucrose, glucose, mannitol and sodium citrate. 4. A process as claimed in claim 1 wherein the nitrogen sources (0.5 to 2g%) used for cultivating the strain Rhodococcus sp. RL4 are selected from a member of the group consisting of peptone, yeast extract, beef extract and casein hydrolystate. 5. A process as claimed in claim 1 for producing Rhodococcus sp. RL4 cells having high nitrile hydratase activity wherein, the inducer capable of inducing nitrile hydratase is selected from a member of the group consisting of propionitrile, isobutyronitrile and methacrylamide. 6. A process as claimed in claim 1 wherein for growing the microorganism the preferred range of pH is 7.0 to 8.5 and temperature 28 to 30°C. 7. A process as claimed in claim wherein the known method used for recovery of Rhodococcus RL4 cells is centrifugation, decantation. 8. A process as claimed in claims 1 wherein the cyanohydrocarbons used for the conversion to corresponding amides by Rhodococcus sp. RL4 cells are aliphatic cyanohydrocarbons selected from acrylonitrile, propionitrile and isobutyronitrile and the aromatic cyanohydrocarbons selected from benzyl cyanide and 3-cyano pyridine. 9. A process as claimed in claim 1 wherein the medium used is aqueous medium or phosphate buffer or Tris buffer 10. A process for the biotransformation of cyanohydrocarbons to corresponding amides substantially as herein described with reference to the examples.

Full Text

The present invention relates to a process for the biotransformation of cyanohydrocarbons to corresponding amides. The present invention particularly relates to a process for the biotransformation of cyanohydrocarbons to corresponding amides using whole cells of a novel Rhodococcus sp. strain RL4 having nitrile hydratase enzyme. The present invention particularly relates to conversion of aliphatic and aromatic cyanohydrocarbons such as acrylonitrile, propionitrile, isobutyronitrile, benzyl cyanide, 3-cyanopyridine and the like to the corresponding amides , and more particularly relates to an enzymatic process for the hydration of acrylonitrile to acrylamide catalyzed by nitrile hydratase present in the resting cells of Rhodococcus sp. RL4.
The main usage of the invention is to provide a process for the preparation of Rhodococcus sp. RL4 cells having nitrile hydratase enzyme which can be used under mild reaction conditions e.g. atmospheric pressure, low temperature, near neutral pH to hydrate acrylonitrile to acrylamide without any by-product formation.
Acrylamide is produced industrially as monomer for synthetic fibres, flocculant agents, etc. Conventional method for production of acrylamide involves the use of copper salts as catalysts. The method , however, has many disadvantages, e.g., the complexity of preparation of the catalyst, the difficulty in regenerating the catalyst used, and the complexity of purifying and isolating the acrylamide. In addition, acrylamide readily polymerized. Therefore, it is desirable that acrylamide is produced under mild reaction conditions. In recent years, acrylamide is being produced industrially using nitrile hydratase enzyme produced by bacteria.The reaction occurs at low temperatures (usually 5 to 10 °C) by incubation of acrylonitrile with bacterial cells that have high nitrile hydratase activity. In contrast with the

conventional chemical process, the recovery of unreacted acrylonitrile is not necessary because the yield of the enzymatic conversion is almost 100%; the removal of copper ions as a catalyst from the reaction mixture is also no longer necessary. In fact, Nitto Chemical Company (Japan) started industrial production of acrylamide (3000 tons/yr) in 1985 using the nitrile hydratase of Rhodococcus sp. N-774, the first generation strain (Watanabe,!, Satoh, Y, and Enomoto, K (1987J Agric. Biol. Chem. 51, 3193-3199: Yamada, H and Kobayashi, M (1996) Biosci. Biotech. Biochem. 60, 1391-1400). In 1988 this strain was replaced by the second-generation strain, Pseudomonas chlororaphis B23 (Yamada, H and Kobayashi, M (1996) Biosci. Biotech. Biochem. 60, 1391-1400). Acrylamide (6000 tons/yr) had been produced using immobilized cells of Pseudomonas chlororaphis B23, entrapped on a cationic acrylamide -based polymer gel ( Watanabe, I (1987) In: Methods in Enzymology, vol. 136 ( K Mosbach, ed.) pp 523-530. New York. Academic Press; Nakai, K, Watanabe, I, Sato, Y and Enomoto, K. (1988) Nippon Nogeikagaku Kaishi 62, 1443-1450). In 1991, the same Company replaced P. chlororaphis B23 with the third generation strain Rhodococcus rhodochorous Jl whereby producing 30,000 tons of acrylamide/yr using a bioreactor containing immobilized cells ofR. rhodochorous Jl ( Kobayashi, M, Nagasawa, T, Yamada, H (1992) Trends in Biotechnol. 10, 402-408; Yamada, H and Kobayashi, M (1996) Biosci. Biotech. Biochem. 60, 1391-1400). The formation of nitrile hydratase in Rhodococcus sp. N-774 and P. chlororaphis B23 is highly enhanced by the addition of Fe+2 or Fe+3 ions to the medium ( Watanabe, I, Satoh, Y, Enomoto, K, Seiki, S and Sakashita, K ( 1987) Agric Biol chem 51, 3201-3206). The nitrile hydratase protein of R.rhodochorous Jl is not synthesized without the addition of Co+2 ions in the medium. ( Yamada, H and Kobayashi, M (

1996) Biosci Biotech Biochem 60, 1391-1400). Fe+2 or Fe+3 ions can not replace Co+2 ions.
In order to produce nitrile hydratase in the process of growing bacteria, it is necessary to add an inducer to the nutrient medium. Nitriles and amides of organic
acids (. US Patent No. 4,555,487; European Patent No. 0109083; European Patent

No. 0204555), urea or its derivatives (European Patent Application No. 0362829)
may be used as inducers. Known in the prior art are strains of Corynebacterium N-
774 (US Patent No. 4,248,968) (now known as Rhodococcus sp.N-774) and
Rhodococcus sp. S-6 (US Patent No. 5,179,014) for which no inducer is required.
Disadvantages of strain N-774 reside in its low nitrile hydratase activity (its specific activity is 50-60 Units/mg, measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight), and narrow range of substrate nitriles (only aliphatic nitriles). A disadvantage of strain S-6 resides in its capacity to hydrate the produced amides to acids due to the presence of nitrilase enzyme, whereby the quality of amides is sharply affected and its low productivity (it is incapable of producing more than 20% acrylamide in the solution). In case of strain Pseudomonas chlororaphis B23, methacrylamide was essential to be present in the medium as an inducer of nitrile hydratase activity ( Watanabe, I, Satoh, Y, Enomoto, K, Seki, S and Sakashita, K (1987) Agric. Biol. Chem. 51, 3201-3206) . Glucose inhibited the formation of nitrile hydratase in P. chlororaphis B23 (Yamada, H, Ryuno, K, Nagasawa, T, Enomoto, K and Watanabe, I (1986) Agric Biol Chem. 50, 2859-2865). One of the important technical disadvantage of P. chlororaphis B23 was that when this organism was grown on a medium containing sucrose, mucilage polysaccharides,which resemble levan , were produced causing ineffective aeration
and difficulty in harvesting of cells by brief centrifugation (at 8000 X g for 10 min) due to high viscosity of the culture medium ( Yamada, H and Kobayashi M (1996) Biosci Biotech Biochem 60, 1391-1400). The strain Rhodococcus rhodochrous Jl necessitates the use of urea, methacrylamide, or crotonamide in the medium to induce the formation of nitrile hydratase. When Rhodococcus rhodochrous Jl is cultured in the optimum culture medium consisting of 2g of glucose, 50 mg of KH2PO4, 50 mg of K2HPO4, 50 mg of MgSO4.7H2O, 0.1 g of yeast extract, 0.5 g of polypeptone, 0.75g of urea , 1 mg of CoCl2.6H2O per 100 ml tap water(pH 7.2), it produces a nitrile hydratase having a specific activity of 76 Units/mg ,( measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight) (Nagasawa, T, Takeuchi, K, Yamada, H (1991) Eur J Biochem 196, 581-589). Unlike P.chlororaphis B23, sucrose did not promote much of cell growth or nitrile hydratase activity of R. rhodochorous Jl (Nagasawa, T, Kobayashi, M and Yamada, H (1988) Arch Micrbiol 150, 89-94 ). The strain Rhodococcus sp. RL4 described in the present invention when cultured in the optimal culture medium containing 1g of sucrose, 2 g of peptone, 0.05 g of KH2P04, 0.05 g of K2HPO4 0.05 g of MgSO4.7H2O, 0.001 g of FeSO4.7H2O, 0.2 g of methacrylamide per 100 ml of water (pH 7.2), it produces a nitrile hydratase having a specific activity of 140 Units/mg (measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight). Unlike Rhodococcus rhodochrous Jl, glucose did not inhibit the formation of nitrile hydratase in Rhodococcus sp. RL4. The nitrile hydratase of strain Rhodococcus sp. RL4 has broad substrate specificity with respect to aliphatic and aromatic cyanohydrocarbons similar to the strain Rhodococcus rhodochrous Jl (European Patent Application No. 0362829). The resting cells of Rhodococcus sp.
RL4 can be used to produce acrylamide at a concentration as high as 40%; the acrylamide solution obtained by separating the bacterial cells from the reaction system is colorless. On the other hand, although the resting cells of Brevibacterium R312 (US Patent 4,001,081) could also produce acrylamide at a high concentration (20%), the solution from which bacterial cells were separated was colored an extremely dark yellow and contains various impurities originating from the cells, and hence an onerous purifying step was necessary (cited from US Patent 4,248,968). The aforementioned strain RL4 can be maintained easily and is found stable with respect to enzyme yields and culture requirements.
The main objective of the present invention is to provide a process for the biotransformation of cyan hydrocarbons to corresponding amides using novel Rhodococcus sp. RL4 cells having nitrile hydratase enzyme.
Accordingly, the present invention provides a process for the biotransformation of cyan hydrocarbons to corresponding amides which comprises;
a) growing strain Rhodococcus sp. RL4 at pH range of 6.5 to 8.5 and temperature 20
to 35°C in a conventional nutrient medium comprising a carbon source, a nitrogen source, salts, and an inducer such as herein described, recovering the Rhodococcus sp. RL4 cells having nitrile hydratase activity by known methods,
b) contacting cyanohydrocarbons such as herein described in known medium at
temperature of 0 to 30°c, at pH 6.0 to 9.0 with Rhodococcus sp. RL4 cells having
nitrile hydratase enzyme on various supports,
c) separating the cells from the reaction mixture by known method such as herein
described, recovering the corresponding amide from the said supernatant by
known method.
The conventional nutrient medium used for growing Rhodococcus sp. RL4 having high nitrile hydratase activity consists or sucrose or glucose (carbon source).
( 0.5-3 g%), KH2P04 (0.05-2 g%), K2HPO4 (0.05-2 g%), MgSO4.7H2O (0.02-1 g%), FeSO4.7H20 (0.0002-0.002 g%) , peptone, yeast extract, beef extract, casaminoacid in combination or alone (complex nitrogen source) (0.5-2 g%), and an inducer (0.05-0.6 g%). The microorganism was grown under aerobic conditions for 48 to 72 h in shaking flasks or in a fermentor.
The present invention provides a process for the preparation of Rhodococcus sp. RL4 cells having nitrile hydratase enzyme which can be used under mild reaction conditions e.g. atmospheric pressure, pH in the range of 7.0 to 8.5, temperature above freezing point to 30 °C to hydrate acrylonitrile to produce up to 400 g acrylamide per litre of reaction mixture without any by-product formation which obviates the drawbacks encountered in the copper salt-catalyzed chemical synthesis of acrylamide and the drawbacks encountered in enzymatic synthesis of acrylamide of formation of mucilage polysaccharide in P. chlororaphis B23 and of using expensive glucose instead of sucrose in the culture medium for the formation of nitrile hydratase in Rhodococcus rhodococcus Jl.
In an embodiment of the present invention the cyanohydrocarbons used for the conversion to corresponding amides by Rhodococcus sp. RL4 cells may be aliphatic cyanohydrocarbons such as acrylonitile, propionitrile, isobutyronitrile and the aromatic cyanohydrocarbons such as benzylcyanide, 3-cyanopyridine.
In another embodiment of the present invention the reaction conditions under which resting cells of Rhodococcus sp. RL4 may be used for hydrating acrylonitrile to acrylamide may be varied over a relatively wide range. The reaction can be carried out either in aqueous medium or in phosphate or Tris-buffer (pH 7.0 to 8.5)
and the typical temperature range is 0 °C to 30 °C, but it is preferred that the hydration reaction be carried out at a temperature below 10 °C. Acrylonitrile as a substrate was added intermittently in small portions, since it is inhibitory for nitrile hydratase enzyme when added to the reaction mixture at a concentration exceeding
2%.
In another embodiment of the invention the Rhodococcus sp RL4 may be used as free cells or in lyophilized form or in immobilized form on various known support materials such as polyacrylamide, agar, calcium alginate, K-carrageenan etc.
In another embodiment of the present invention the conditions under which Rhodococcus sp. RL4 could be cultivated to produce cells having nitrile hydratase enzyme may be varied over a relatively wide range. A variety of carbon sources (0.5 to 3g%) such as sucrose, glucose, mannitol and sodium citrate may be suitable for growing the microorganism. Also, a variety of complex nitrogen sources (0.5 to 2g%) alone or in combination such as yeast extract, peptone, casein hydrolysate and beef extract may be used for growing the microorganism. A variety of inducers such as methacrylamide, isobutyronitrile and propionitrile may be used in the medium for preparation of Rhodococcus sp. RL4 cells having nitrile hydratase activity. However, the preferred carbon source is sucrose and glucose, preferred complex nitrogen source is any one of yeast extract, peptone and casein hydrolysate or a combination, and preferred inducer is methacrylamide.
In another embodiment of the present invention the pH and temperature of the nutrient medium for growing Rhodococcus RL4 cells may also vary but, of course, should not be such that would result in inactivating or otherwise detrimentally
affecting the microorganism to such an extent that production of nitrile hydratase is significantly decreased. The typical pH range at which Rhodococcus sp. RL4 may be grown is 6.5 to 8.5 and the typical temperature range is 20 to 35 °C, but is preferred that the microorganism be grown at pH 7.0 to 8.5 and at 28 to 30 °C.
In yet another embodiment of the invention the inducer used may be such as propionitrile, isobutyronitrile or methacrylamide.
In yet another embodiment of the invention the known method used for recovery of Rhodococcus RL4 cells may be such as centrifugation, decantation etc.
In yet another embodiment of the present invention the organism used in the present invention was isolated from a soil samlpe of Assam , tentatively identified as Rhodococcus species and deposited at RRL Jorhat culture collection centre giving an accession number RL4. The morphological and biochemical characteristics of the bacterial strain RL4 is given in Table 1.
Table 1. The morphological and biochemical characteristics of bacterial strain RL4.
Characteristics Observation
Cell shape At initial stage of cultivation, the cell is in a
long rod-shaped form and irregular branches are observed. Later it breaks into a short rod-shaped or spherical form (pleomorphism)
Spore none
Motility none
Pigment production negative
Acid fastness
Gram reaction
Methyl red test
Oxygen relationship
State in nutrient agar plate culture
Growth range
Growth on sole carbon source:
glucose
maltose
rhamnose
sodium benzoate
sodium citrate
p-hydroxy benzoic acid
cresol
serine
Growth in 5% NaCl Glucose fermentation Lactose fermentation Sucrose fermentation Indole production H2S production Nitrate reduction Denitrification Catalase Urease Oxidase
Arginine dihydrolase Starch hydrolysis Gelatin hydrolysis
negative
positive
negative
aerobic
smooth, convex colonies
pH 5 to 10 ; temp. 10 °C to 37 °C
positive
positive
positive
negative
positive
positive
negative
negative
positive
acid formed, no gas produced
no acid, no gas
no acid, no gas
negative
negative
negative
negative
positive
positive
negative
negative
negative
negative
To determine taxonomic position of the bacterial strain RL4 based on the description of bacteriological characteristics, according to Bergey's Manual of Determinative Bacteriology, 8th ed (1974) and Bergey's Manual of Systematic Bacteriology, vol.2 (1986) the strain RL4 falls under the aerobic, gram-positive, non acid fastness,and catalase-positive bacillary catagory having no endo-spore and no flagella. From the fact that the bacterium is in a long rod-shaped form at the initial stage of growth, showing hypha-like appearance, and grows with branching to break and split latter into short rod or spherical form (pleomorphism), it is considered to belong to the genus Rhodococcus. However, the biochemical characteristics of this organism clearly indicates that the strain RL4 differs from other high acrylamide- producing industrial Rhodococcus strains viz. Rhodococcus sp.N-774 ( US Patent 4,248,968 ) and Rhodococcus rhodochrous Jl( US Patent 5,334,519 ) with respect to nitrate reduction test ( nitrate reduction is negative for strain RL4, whereas in case of strains N-774 and Jl nitrate reduction is positive).
In another embodiment of the present invention immobilized cells of Rhodococus sp. RL4 may be used for the conversion of cyanohydrocarbons to corresponding amides.The conventional methods of cell immobilization may be used for entrapping Rhodococcus sp. RL4 cells in polymers . In case of polyacrylamide gel entrapped cells, the immobilization can be conducted by suspending the cells in a suitable aqueous medium (e.g. a physiological saline, a buffer solution, etc) containing an acrylamide series monomer( e.g. acrylamide) and a cross linking agent (e.g. N,N' methylene bisacrylamide), adding a suitable polymerization initiator (e.g. ammonium persulfate) and a polymerization accelerator(e.g. N,N,N',N' tetramethyl ethylene diamine) to the suspension, and
conducting polymerization and gellation at about 0 °C to 30 °C, preferably 0 °C to 15 °C, at a pH of about 5 to 9, preferably about 6 to 8.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
The following examples are given by way of illustration of the present invention and therefore should not be construed to the limit of the present invention.
Example 1
Rhodococcus sp. RL4 was cultivated at 30 °C for 48 h with shaking at 200 rev/min in an Erlenmayer flask containing 50 ml of a medium consisting of 1g of sucrose, 2g of peptone, 0.05 g of KH2PO4, 0.05 g of K2HP04, 0.05 g of MgSO4.7H2O, 0.001 g of FeSO4.7H2O, 0.2 g of methacrylamide per 100 ml of distilled water (pH 7.2). Cells were harvested by centrifugation at 10,000 X g at 10 °C for 12 min and then washed with 0.05 M potassium phosphate (pH 7.5), centrifuged again and suspended in 0.05 M potassium phosphate buffer (pH 7.5). A small portion of the suspension was sampled and used for measuring the dry weight of the bacterial cells. The yield of cells was 7.0 g dry cells per litre medium.The specific activity of the nitrile hydratase in strain RL4 reached 140 Units/ml (measured in micro mole of acrylamide/min/mg of cells, based on dry matter weight), its total activity being 1000 Units/ml. The washed cells obtained as above were used for conversion of cyanohydrocarbons to corresponding amides.
Example 2
The washed cells of Rhodococcus sp. RL4 (20 rng dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml of 0.05 M potassium phosphate buffer (pH 7.5) and acrylonitrile (5.4 ml) was added intermittently (0.2 ml every 30 min) over a period of 13.5 h under occasional stirring and the hydration reaction was allowed to proceed at 5 °C.Thereafter, reaction was allowed to proceed for another 1h to drive the reaction to completion. Removal of cells by centrifugation gave a colorless, transparent supernatant. This supernatant contained 30 % acrylamide, but contained no unreacted acrylonitrile and no by-products like acrylic acid. The yield of acrylamide was found to be 99.99%. Thus the reaction proceeded quantitatively to completion.The aforesaid supernatant was then vacuum-concentrated while bubbling air at a temperature of 45 °C by use of a flash evaporator to obtain more concentrated acrylamide solution or crystals thereof. The colorless crystals of acrylamide were recrystalized from methanol.
This compound was identified as acrylamide based on m.p, elementary analysis, IR, NMR and Mass spectra.
Example 3
The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and benzyl cyanide (0.2 ml) was added and the hydration reaction was allowed to proceed at 10 0C. Within two hours crystals of a -phenylacetamide separated out from the reaction mixture and was recovered by filtration. The conversion was 100%.
This compound was identified as a -phenylacetamide based on m.p, elementary analysis, IR, NMR and Mass spectra.
Example 4
The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and propionitrile (0.2 ml) was added and the hydration reaction was allowed to proceed at 10 °C for two hours. After the completion of the reaction, the clear solution obtained by removal of the cells by centrifugation was dried at 70 °C.The crystals of propionamide thus obtained were recrystalized from methanol. The conversion was 100%.
This compound was identified as propionamide based on m.p, elementary analysis, IR, NMR and Mass spectra.
Example 5
The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and isobutyronitrile (0.2 ml) was added and the hydration reaction was allowed to proceed at 10 °C for two hours. After the completion of the reaction, the clear solution obtained by removal of the cells by centrifugation was dried at 70 "C.The crystals of isobutyramide thus obtained were recrystalized from methanol. The conversion was 100%.
This compound was identified as isobutyramide based on m.p, elementary analysis, IR, NMR and Mass spectra.
Example 6
The washed cells of Rhodococcus sp. RL4 (10 mg dry weight equivalent) prepared as given in Example 1 were suspended in 15 ml 0.05M potassium phosphate buffer (pH 7.5) and 3-cyanopyridine (0.2g) was added. The reaction was allowed to proceed at 10 °C for 4 h with stirring. After the completion of the reaction, the clear solution obtained by removal of the cells by centrifugation was dried at 70 °C.The crystals of nicotinamide thus obtained were recrystalized from methanol. The conversion was 100%.
This compound was identified as nicotinamide based on m.p, elementary analysis, IR, NMR and Mass spectra.
Example 7
The washed cells of Rhodococcus sp. RL4 (0.3 g dry weight equivalent) prepared as given in Example 1 were suspended in 12 ml of 0.05 M potassium phosphate buffer (pH 7.5) and added to 12 ml of a solution of monomer (3.25 g acrylamide) and crosslinker (0.38 g N,N'-methylene bisacrylamide) in 0.05 M potassium phosphate buffer (pH 7.5) to prepare a uniform suspension. The polymerization was initiated by adding 0.5 ml of N,N,N',N'- tetramethyl ethylene diamine solution (0.07 g dissolved in water) and 0.5 ml of ammonium persulfate solution (0.025 g dissolved in water). Polymerization was performed in ice-water bath (10 °C) for 1 h. The gel block was then cut into cubes, and these were then thoroughly washed with 0.05 M potassium phosphate buffer (pH 7.5) in order to remove nonpolymerized monomers and residues.
(FigureRemoved)

Figure 1. Schematic diagram of perlmental setup for reactor operation
PI Acrylonitrile reservoir, (2) substrate and product reservoir, (3) Water bath, (4) peristaltic pump, (5) fluidized-bed with immobilized cells, (6) water jacket
By using the immobilized strain as obtained above, conversion of acrylonitrile to acrylamide was conducted in a fluidized-bed reactor. A schematic diagram of the experimental equipment for fluidized- bed reactor operation is shown in Figure 1. A jacketed glass column (2.8 cm bed diameter and 13.5 cm height) equipped with perforated glass bottom to support immobilized cells ( 20 g gel cubes) was used. The substrate product reservoir was placed in water bath at 5 °C. The fluidized- bed was maintained at the same temperature as that of the reservoir by recirculating water from the water bath through plastic tube. The fluidized-bed reactor operation was carried out in a fed-batch manner by recirculating the substrate solution at the rate of 30 L per hour into the substrate-product reservoir containing 150 ml of 0.05 M potassium phosphate buffer( pH 7.5) and the fluidized-bed (2.8 cm bed diameter and 13.5 cm height). At the same time acrylonitrile was continuously fed to the substrate-product reservoir at the rate of 3.5 ml/h by a peristaltic pump. Samples were withdrawn from substrate-product reservoir periodically and acrylamide concentration was measured by HPLC. After the completion of the addition of acrylonitrile (35 ml), 21% acrylamide was obtained and the conversion of substrate was 100%. After the completion of one batch which took llh, the reaction mixture was drained out from the reactor, the gel cubes in the reactor was washed with 0.05 M phosphate buffer (pH 7.5) and the reactor was operated for another 12 batches under similar conditions as above. The productivity of the reactor system was found to remain constant (100% conversion) upto 6th batch and then the conversion falls from 90% in the 7 th batch to 50.5% in the 13th batch.The main advantage of the present invention is that the resting cells of Rhodococcus sp. RL4 can be used for the biotransformation of acrylonitrile to
acrylamide at concentrations of 20 to 40% without any by-product formation under very mild reaction conditions e.g. low temperature(above freezing point to 10 ° C) and pH range of 7.0 to 8.5; the acrylamide solution obtained by separating the bacterial cells from the reaction system is colourless. This obviates the drawback encountered in biotransformation of acrylonitrile to acrylamide using the resting cells of Brevibacterium R312 (cited from US Patent 4,248,968) which produced dark coloured acrylamide solution, and hence an onerous purifying step was necessary. Furthermore, both sucrose and glucose act as a carbon source to promote much of cell growth and nitrile hydratase activity of Rhodococcus sp. RL4. Also,the nitrile hydratase of Rhodococcus species RL4 has broad substrate specificity thereby converting aliphatic and aromatic cyanohydrocarbons to corresponding amides. The aforementioned strain RL4 can be maintained easily and is found stable with respect to enzyme yields and culture requirements.

We Claim:
1. A process for the biotransformation of cyanohydrocarbons to corresponding
amides which comprises;
a) growing strain Rhodococcus sp. RL4 at pH range of 6.5 to 8.5 and temperature 20
to 35 °C in a conventional nutrient medium comprising a carbon source, a nitrogen
source, salts, and an inducer such as herein described, recovering the
Rhodococcus sp. RL4 cells having nitrile hydratase activity by known methods,
b) contacting cyanohydrocarbons such as herein described in known medium at
temperature of 0 to 30°c, at pH 6.0 to 9.0 with Rhodococcus sp. RL4 cells having
nitrile hydratase enzyme bn various supports,
c) separating the cells from the reaction mixture by known method such as herein
described, recovering the corresponding amide from the said supernatant by
known method.

2. A process as claimed in claim 1 wherein the strain Rhodococcus sp RL4 is used as
free cells or in lyophilized form or in immobilized form on various supports such
as polyacrylamide, agar, calcium alginate, K-carrageen an.
3. A process as claimed in claim 1 wherein the carbon sources (0.5 to 3g %) used
for cultivating the strain Rhodococcus sp. RL4 are selected from a member of the
group consisting of sucrose, glucose, mannitol and sodium citrate.
4. A process as claimed in claim 1 wherein the nitrogen sources (0.5 to 2g%) used
for cultivating the strain Rhodococcus sp. RL4 are selected from a member of the
group consisting of peptone, yeast extract, beef extract and casein hydrolystate.
5. A process as claimed in claim 1 for producing Rhodococcus sp. RL4 cells having
high nitrile hydratase activity wherein, the inducer capable of inducing nitrile
hydratase is selected from a member of the group consisting of propionitrile,
isobutyronitrile and methacrylamide.
6. A process as claimed in claim 1 wherein for growing the microorganism the
preferred range of pH is 7.0 to 8.5 and temperature 28 to 30°C.
7. A process as claimed in claim wherein the known method used for recovery of
Rhodococcus RL4 cells is centrifugation, decantation.
8. A process as claimed in claims 1 wherein the cyanohydrocarbons used for the
conversion to corresponding amides by Rhodococcus sp. RL4 cells are aliphatic
cyanohydrocarbons selected from acrylonitrile, propionitrile and isobutyronitrile
and the aromatic cyanohydrocarbons selected from benzyl cyanide and 3-cyano
pyridine.
9. A process as claimed in claim 1 wherein the medium used is aqueous medium or
phosphate buffer or Tris buffer
10. A process for the biotransformation of cyanohydrocarbons to corresponding
amides substantially as herein described with reference to the examples.

Documents:

27-del-2000-abstract.pdf

27-del-2000-claims.pdf

27-del-2000-correspondence-others.pdf

27-del-2000-correspondence-po.pdf

27-del-2000-description (complete).pdf

27-del-2000-form-1.pdf

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Patent Number

216951

Indian Patent Application Number

27/DEL/2000

PG Journal Number

13/2008

Publication Date

31-Mar-2008

Grant Date

20-Mar-2008

Date of Filing

18-Jan-2000

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	BINOD KUMAR GOGOI	REGIONAL RESEARCH LABORATORY, JORHAT -785006, ASSAM, INDIA
2	RITU ARAMBAM	REGIONAL RESEARCH LABORATORY, JORHAT -785006, ASSAM, INDIA
3	TANKESHWAR NATH	REGIONAL RESEARCH LABORATORY, JORHAT -785006, ASSAM, INDIA
4	AJIT KUMAR HAZARIKA	REGIONAL RESEARCH LABORATORY, JORHAT -785006, ASSAM, INDIA
5	JAGDISH NARAYAN NIGAM	REGIONAL RESEARCH LABORATORY, JORHAT -785006, ASSAM, INDIA
6	NARENDRA NATH DUTTA	REGIONAL RESEARCH LABORATORY, JORHAT -785006, ASSAM, INDIA

PCT International Classification Number

C12P 013/02

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA