International Journal of Universal Pharmacy and Bio Sciences 2(1): January-February 2013 INTERNATIONAL JOURNAL of UNIVERSAL PHARMACY and BIO SCIENCES
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International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 2(1): January-February 2013 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES Bio Sciences Research Article……!!! Received: 21-02-2013; Accepted: 23-02-2013 BENCH SCALE PRODUCTION OF PHENYLACETIC ACID USING ALCALIGENES SP. MTCC 10675 Bhatia S.K, Kumar D, Bhatia R.K and Bhalla T.C* Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla- 171005 (India). ABSTRACT KEYWORDS: The intracellular nitrilase of Alcaligenes sp. MTCC 10675 has Arylacetonitrilase, exhibited very high arylacetonitrilase activity and it has been Biotransformation, explored for the biotransformation of phenylacetonitrile in to a Phenylacetonitrile, pharmaceutical important compound phenylacetic acid. This Phenylacetic Acid. enzyme has maximum activity at pH 6.5 and 45 ºC. However the For Correspondence: enzyme was stable at 35 ºC for 16.9 h. Phenylacetonitrile Bhalla T.C* hydrolyzing activity of Alcaligenes sp. MTCC 10675 decrease up Address: Department of to 91.6 % at 55 ºC. Vmax and Km value for phenylacetonitrile as Biotechnology, Himachal - - substrate were respectively 0.34 µmol emg min and 28 mM. The Pradesh University, 0.47 U resting cell showed full conversion of 40 mM Summer Hill, Shimla- 171005 (India). phenylacetonitrile into phenylacetic acid in 80 min. Fed batch reaction at 1L scale having 200 mM phenylacetonitrile as substrate Tel: +91-177-2831948, added in five feeds resulted into 5.0 gg-dcw phenylacetic acid with Fax: +91-177-2831954. catalytic productivity of 0.75 gh-g-dcw. 16 Full Text Available On www.ijupbs.com International Standard Serial Number (ISSN): 2319-8141 1. INTRODUCTION : Nitriles are organic cyanides, containing –C≡N group and are widespread in nature and used by a large number of industries as organic solvents, herbicides, organic feed stocks, extractants, recrystallizing agents, precursors in the synthesis of plastics, synthetic fibers, resin, dye stuffs, emulsifiers and pharmaceuticals [1,2]. Nitrilases (nitrile aminohydrolases, EC 3.5.5.1) catalyses the hydrolysis of triple bond of cyano group (-C≡N) of nitriles to form corresponding carboxylic acids. This enzyme is distributed in microbes (bacteria, cyanobacteria and fungi), plants and animals. The nitrilases are capable of hydrolysing various aliphatic/aromatic nitriles, arylacetonitriles, aminonitriles and hydroxynitriles at physiological pH and temperature [3-5]. In recent years some of the fine chemicals of pharmaceutical importance are being commercially produced using nitrile hydrolysing enzymes e.g. α-hydroxyisobutyric acid, phenyl acetic acid (PAA), (R)-mandelic acid, (R)-4-cyano-3-hydroxybutyric acid and (S)- ibuprofen from their corresponding nitriles [3,6,7]. PAA is one of the important organic acid, which is widely used in the field of medicine, pesticide and aromatizer [8]. Its derivatives are valuable intermediates in the manufacture of pharmaceuticals and cosmetics [9]. It had also exhibits antimicrobial activity [10] and is used as important constituent of medium or reaction for the synthesis penicillin G salt [11] and for making phenobarbital, primidone, pesticide and fungicide (3-chlorophenylacetic acid and rodenticide). Phenylacetic acid esters are also used as flavouring agents in food industries and in cosmetics. In medicine, phenylacetic acid esters have been used for the synthesis of isochromanes which exhibit a wide variety of pharmacological activities [12]. PAA is the major metabolite of phenylethylamine, a "trace amine" which has become the focus of considerable clinical attention [13]. Chemically phenylacetic acid is synthesized by various methods i.e. cyanobenzyl hydrolysis, phenylacetamide hydrolysis, electrochemical process, carbonyl process [8]. These routes requires harsh conditions either alkali/acid treatment or requirement of very high temperature, pressure and expensive catalysts. In contrast to the chemical routes, the enzymatic processes for the synthesis of industrially important chemicals are gaining importance. Mild reaction conditions, ease of biocatalyst production and stereoselective transformation are the main advantages of the enzyme based processes. Keeping in view the wide applications of phenylacetic acid, a bench scale process for the conversion of phenylacetonitrile into phenylacetic acid using resting cell of Alcaligenes sp. MTCC 10675 has developed in the present study. Full Text Available On www.ijupbs.com 17 International Standard Serial Number (ISSN): 2319-8141 2. MATERIAL AND METHODS: 2.1 Chemicals: Isobutyronitrile and phenylacetonitrile were purchased from Alfa Aesar, Johnson Matthey Company (previously Lanchaster Synthesis). Media components were obtained from Hi- Media (Mumbai, India). All other chemicals used were of analytical grade and procured from standard companies. 2.2 Preparation of resting cells: Alcaligenes sp. MTCC 10675 has been used as source of nitrilase. The bacterium was routinely sub cultured on nutrient agar slants at 30°C and maintained at 4°C. A loopfull of bacterial cells of Alcaligenes sp. MTCC 10675 from the slant was inoculated in 50 ml of seed medium containing (per litre) 25 g nutrient broth L-, and incubated at 30°C for 18 h in an incubater shaker (160 rpm). One milliliter of 18 h preculture was added into 50 ml of nitrilase production medium containing 5 g peptone, 3 g yeast extract and 3 g malt extract L- supplemented with 0.3% (v/v) isobutyronitrile in 250 ml Erlenmeyer flask and incubated at 30°C for 24 h in an incubator shaker at 160 rpm. The cells from the culture were harvested by centrifugation at 10,000 g for 10 min at 4°C washed with 0.1 M sodium phosphate buffer (6.5 pH) and resuspended in the same buffer. 2.3 Nitrilase assay: The nitrilase assay was carried out in a reaction mixture (1.0 ml) containing 0.1 M NaH2PO4/Na2HPO4 buffer (pH 6.5), 50 mM nitrile and resting cells at 30˚C. After 60 min of incubation, reaction was stopped with equal volume of 0.1 N HCl. The amount of ammonia released in the reaction mixture was estimated calorimetrically through a phenate- hypochlorite method [14]. One unit of nitrilase activity was defined as that amount of enzyme which catalysed the release of one micromole of ammonia per min by the hydrolysis of phenylacetonitrile under assay conditions. 2.4 Optimization of reaction conditions: 2.4.1 Buffer pH and molarity To study the effect of buffer systems and pH on the nitrilase activity, various buffers were screened e.g. citrate buffer (pH 4.0-6.0), sodium phosphate buffer (pH 6.0-8.0), potassium phosphate buffer (pH 6.5-8.0), borate buffer (pH 7.5-9.0), sodium carbonate buffer (pH 9.5- 10.5). Molarity of selected buffer was further varied from 25 mM to 150 mM to select optimum strength of buffer. Full Text Available On www.ijupbs.com 18 International Standard Serial Number (ISSN): 2319-8141 2.4.2 Substrate concentration and incubation temperature Different concentrations of phenylacetonitrile were used (10 mM to 100 mM) in potassium phosphate buffer to study the effect of substrate concentration on nitrilase activity. To find out the optimum temperature of nitrilase the enzyme activity was estimated at different temperature from 25°C to 55°C. 2.5 Biocatalyst amount In order to work out the optimum amount of biocatalyst for hydrolysis of phenylacetonitrile the resting cells ranging from 3 mg to 28 mg dcw/ml were used in the reaction. 2.6 Time course of incubation The optimization of reaction time at 45°C was investigated by terminating the reaction after 5, 10, 15, 30, 60, 80, 100 and 120 min. 2.7 Stability of enzyme To evaluate the stability of enzyme, resting cell suspension were incubated at different temperature (25ºC, 35ºC, 45ºC and 55ºC) up to 10 h and the enzyme activity was assayed after an interval of one hour. 2.8 Effect of substrate on the activity of nitrilase Alcaligenes sp. MTCC 10675 The effect of substrate concentration on the activity of nitrilase was studied by adding different concentrations of phenylacetonitrile (20mM-80mM) in the reaction. 2.9 Bench scale production of phenylacetic acid The resting cells of Alcaligenes sp. MTCC 10675 were used for the development of bioprocess for the production of phenylacetic acid at bench scale. 2.10 Time course of phenylacetonitrile conversion To achieve 100% conversion of the substrate (40 mM phenylacetonitrile) to product (phenylacetic acid) the reaction mixture at 100 ml scale was incubated at 35°C for about 150 min by using resting cells and samples were withdrawn after every 10 min for HPLC analysis. 2.11 Fed batch reaction at 100 ml To achieve maximum product formation 40 mM of phenylacetonitrile was added at zero time and subsequent feedings were made at an interval of 80 min in 250 ml conical flask containing 100 ml of the reaction mixture. 2.12 Bench scale at 1L scale On the basis of results at 100 ml, the conversion of phenylacetonitrile to phenylacetic acid was scaled up to 1L and carried out in a bio-flow C-32 fermenter (New Brunswick Scientific, U.S.A.). The initial reaction contained potassium phosphate buffer (pH 6.5, 100 mM), 470 U Full Text Available On www.ijupbs.com 19 International Standard Serial Number (ISSN): 2319-8141 (1.8 g resting cell) and 40 mM phenylacetonitrile was added in each feed. The temperature of the vessel was maintained at 35°C and the impeller speed was set to 160 rpm. At the end of reaction, the cells were separated by centrifugation at 6000g and phenyl acetic acid was recovered from the supernatant by using diethyl ether as solvent. Lypholization (Flexi-Dry TMFTS systems, Stone Ridge, New York, USA) and vaccum drying of reaction supernatant was done to dry the phenyl acetic acid formed in the reaction. 3. RESULTS AND DISCUSSION: Optimization of reaction condition Buffer pH and molarity: Optimum phenylacetonitrile hydrolyzing activity (0.25 U/mg dcw) of Alcaligenes sp.