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 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. 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 - - 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]. (nitrile , 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 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 (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. MTCC 10675 was observed at pH 7.5 in 0.1 mM potassium phosphate buffer (Fig. 1). The nitrilase is active within a narrow pH range like other nitrilases [4]. In case of A. faecalis MTCC 10757, the maximum activity was observed at the pH 8.0 [15]. Substrate concentration, temperature and biocatalytic amount: Maximum nitrilase activity (0.3 U/mg dcw) for free cells of Alcaligenes sp. MTCC 10675 was recorded at 45ºC with 40 mM phenylacetonitrile. Nitrilase activity decreased up to 48 % on increasing the phenylacetonitrile concentration from 40 to 80 mM. Enzyme activity increased gradually up to 45ºC and beyond this temperature loss in activity was recorded like the arylacetonitrilase of Alcaligenes faecalis JM3 [16], Alcaligenes faecalis ATCC 8750 [17] 1992), Bradyrhizobium japonicum USDA110 [18], Aspergillus niger K10 [19], and Alcaligenes faecalis MTCC 10757 (Nageshwar et al. 2011). In order to work out optimum amount of free cells for the conversion of phenylacetonitrile to phenylacetic acid, amount of free cells was varied from 3 to 28 mg dcw/ml in the reaction and maximum specific activity 0.47 U/mg dcw was observed at 18 mg dcw/ml. The arylacetonitrilase of Alcaligenes sp. MTCC 10675 had - - Vmax and Km value respectively 0.34 µmolemg min and 28 mM respectively Thermal stability: The resting cells of Alcaligenes sp. MTCC 10675 were incubated at 25ºC, 35ºC, 45ºC and 55ºC for 6 h and nitrilase activity was assayed to assess half life of enzyme. Half life of nitrilase activity of free cell at these temperature 25ºC, 35ºC, 45ºC and 55ºC were respectively 17.9, 16.9 h, 5.31 h and 1.5 h (Fig. 2). Optimization of process parameters Time course of conversion of phenylacetonitrile: To achieve 100% conversion of substrate (40 mM phenylacetonitrile) to product (phenyl acetic acid) the reaction at 100 ml scale was carried out at 35ºC for about 140 min. The HPLC analysis of product formed after an interval of 20 min revealed that 40 mM phenylacetonitrile was added in the reaction completely converted into phenylacetic acid in 80 min (Fig. 3). Full Text Available On www.ijupbs.com 20

International Standard Serial Number (ISSN): 2319-8141 The nitrilase of the Alcaligenes sp. MTCC 10675 suffered substrate inhibition effect at high phenylacetonitrile concentrations (>40 mM), therefore fed batch mode of reaction was used for the conversion of phenylacetonitrile into phenylacetic acid. Fed batch reaction at 100 ml scale: Analysis of the product formed in 100 ml fed batch reaction exhibit that the complete conversion of substrate fed up to five feeding, further addition of substrates resulted into accumulation of phenylacetonitrile with decrease in conversion rate (Fig. 4). Phenylacetic acid production at 1L scale: Fed batch reaction at 1L scale resulted into full conversion of phenylacetonitrile and lypholization and vaccum drying of reaction supernatant resulted in the formation of white precipitates of phenylacetic acid. The filtration and drying of the precipitate finally led to recovery of 5.0 gg-dcw phenylacetic acid with 0.75 gh-g-dcw (catalytic productivity). For phenylacetonitrile biotransformation, there was no report of bench scale synthesis. However, at test tube scale various research group have reported nitrilase mediated conversion of phenylacetonitrile to phenylacetic acid i.e. Bradyrhizobium japonicum USDA 110 [18], Pseudomonas putida [20] and Exophiala oligosperma R1 [21]. CONCLUSION: Phenylacetic acid is a important organic acid having immense potential in medicinal and pharma industry. There are different chemical methods are available for the synthesis of PAA but there was no method reported till date which is ecofriendly. In this report a very efficient bioprocess for the biotransformation of phenylacetonitrile into phenylacetic acid using resting cells of Alcaligenes sp. MTCC 10674 has been reported first time. ACKNOWLEDGMENT: The authors acknowledge the Department of Biotechnology and University Grant Commission, India for financial support in the form of Senior Research Fellowship to Mr. Shashi Kant Bhatia, Devender Kumar and Ravi Kant Bhatia. The computational facility availed at Bioinformatics Centre, H P University is also duly acknowledged. REFERENCES: 1. Hoyale AJ, Bunch AW and Knowes C, (1998), The nitrilase of Rhodococcus rhodochrous NCIMB 11216. Enzyme. Microbial. technol, 23, 475-482. 2. Martínková L and Kren V, (2010), Biotransformations with nitrilases. Curr. Opin. Chem. Biol, 14(2), 130-137. 3. Bhatia SK, Mehta PK, Bhatia RK and Bhalla TC, (2012), An isobutyronitrile induced bienzymatic system of Alcaligenes sp. MTCC 10674 for the production of alpha- hydroxyisobutyric acid. Bioprocess. Biosyst. Eng, DOI 10.1007/s00449-012-0817-y Full Text Available On www.ijupbs.com 21

International Standard Serial Number (ISSN): 2319-8141 4. O'Reilly C and Turner PD, (2003), The nitrilase family of CN hydrolysing enzymes – a comparative study. J. Appl. Biol. 95, 1161-1174 5. Thuku RN, Brady D, Benedik MJ and Sewell BT, (2009), Microbial nitrilases: versatile, spiral forming, industrial enzymes. Appl. Microbiol. 106, 703-727. 6. Wang MX, Liu J, Wang DX and Zheng QY, (2005), Synthesis of optically active a- methylamino acids and amides through biocatalytic kinetic resolution of amides. Tetrahedron Asymmetry, 16, 2409-2416. 7. Sheldon RA, Arends I and Hanefeld U, (2007), Green Chemistry and Catalysis. WILEY-VCH Verlag GmbH & C, KgA, Weinheim. 8. Duan J, Jiang J, Gong J, Fan Q and Jiang D, (2000), Synthesis of phenylacetic acid by carbonylation. J. Mole. Catal. A: Chem. 159, 89–96. 9. Qiu. Z., Y. He, D. Zheng and Liu. F, (2005), Study on the Synthesis of Phenylacetic Acid by Carbonylation of Benzyl Chloride under Normal Pressure. J. Nat. Gas. Chem, 14, 40-46. 10. Kim CJY, Cho JY, Kuk JH, Moon JH, Cho JL, Kim YC and Park KH, (2004), Identification and Antimicrobial Activity of Phenylacetic Acid Produced by Bacillus licheniformis Isolated from Fermented Soybean. Curr. Microbiol, 48, 312-317. 11. Dirk J. Hillenga, Hanneke J.M. Versantvoort, Siep van der Molen, Arnold J.M. Driessen and Wil N. Konings, (1995), Uptake of the Penicillin G Precursor Phenylacetic Acid. Appl. Environm. Microbiol, 61, 2589-2595. 12. Ivanov I, Nikolova S, Kochovska E and Stela SA, (2007), Application of ortho- acylated phenylacetic acid esters to the synthesis of 1-substituted isochromanes. ARKIVOC, 15, 31-44. 13. Sandler M, Ruthven CRJ, Goodwin BL, Lees A, Stern GM, (1982), Phenylacetic acid in human body fluids: high correlation between plasma and cerebrospinal fluid concentration values. J. Neuro. Neuro. Psy, 45, 366-368. 14. Fawcett JK and Scott JE, (1960), A rapid and precise method for the determination of urea. J. Clin. Pathol, 13, 156-159. 15. Nageshwar YVD, Sheelu G, Shambhu RR, Muluka H, Mehdi N, Shaheer M, Malik MS, and Kamal A, (2011), Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity. Bioprocess. Biosystem. Eng, 34, 515-523. 16. Nagasawa T, Mauger J and Yamada Y, (2010), A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3-purification and characterization. Eur. J. Bio, 194, 765-72. Full Text Available On www.ijupbs.com 22

International Standard Serial Number (ISSN): 2319-8141 17. Yamamoto K, Fujimatsu I and Komatsu K, (1992), Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC8750 responsible for enantio- selective hydrolysis of mandelonitrile. J. Ferment. Bioeng. 73, 425-430. 18. Zhu D, Mukherjee C, Yang Y, Rios BE, Gallagher DT, Smith NN, Biehl ER and Hua L, (2008), A new nitrilase from Bradyrhizobium japonicum USDA 110 Gene cloning, biochemical characterization and substrate specificity. J. Biotechnol. 133, 327-333. 19. Kaplan O, Vejvoda V, Plihal O, Pompach P, Kavan D, Bojarova P, Bezouska K, Mackova M, Cantarella M, Jirku V, Kren V and Martinkova L, (2006), Purification and characterization of a nitrilase from Aspergillus niger K10. Appl. Microbiol. Biotechnol, 73, 567-575. 20. Rustler S and Stolz A, (2007), Isolation and characterization of a nitrile hydrolyzing acidotolerant black yeast-Exophiala oligosperma R1. Appl. Microbiol. Biotechnol, 75, 899-908. 21. Banerjee A, Kaul P and Banerjee UC, (2006), Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida. Arch. Microbiol. 184, 407-418.

Fig. 1 Effect of pH on phenylacetonitrile hydrolyzing activity of Alcaligenes sp. MTCC 10675.

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International Standard Serial Number (ISSN): 2319-8141 Fig. 2 Effect of temperature on arylacetonitrilase activity of Alcaligenes sp. MTCC 10675.

Fig. 3 Time course of phenylacetonitrile conversion

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International Standard Serial Number (ISSN): 2319-8141 Fig. 4 Fed batch reaction for the production of phenylacetic acid at 100 ml scale

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