Bioconversion of Acrylonitrile to Acrylic Acid by Rhodococcus Ruber Strain AKSH-84

J. Microbiol. Biotechnol. (2011), 21(1), 37–42 doi: 10.4014/jmb.1006.06044 First published online 12 November 2010 Bioconversion of Acrylonitrile to Acrylic Acid by Rhodococcus ruber Strain AKSH-84 Kamal, Ahmed*, M. Shiva Kumar, C. Ganesh Kumar, and Thokhir Basha Shaik Chemical Biology Laboratory, Indian Institute of Chemical Technology, Hyderabad 500607, India Received: June 26, 2010 / Revised: September 20, 2010 / Accepted: September 23, 2010 A new versatile acrylonitrile-bioconverting strain isolated million tons in 2005, with an expectation to reach around from a petroleum-contaminated sludge sample and identified 3.7% by the year 2011 [4, 5]. The current industrial as Rhodococcus ruber AKSH-84 was used for optimization demand for acrylic acid is catered by the chemical of medium and biotransformation conditions for nitrilase production process, which is based on gas-phase catalytic activity to produce acrylic acid. A simple and rapid HPLC oxidation of propylene via acrolein in a single or two-step protocol was optimized for quantification of acrylic acid, process. The drawbacks of these methods include side acrylamide, and acrylonitrile. The optimal medium conditions reactions, very high temperatures, and risk of the radical- for nitrilase activity were pH of 7.0, temperature of 30oC, initiated exothermic polymerization with possibility of agitation of 150 rpm, and inoculum level of 2%. Glycerol dimerization of acrylic acid [14]. The environmental safety as a carbon source and sodium nitrate as the nitrogen and cost efficacy are other serious concerns. Biotechnological source provided good nutritional sources for achieving good and biocatalytic processes have been found to be more biotransformation. Nitrilase activity was constitutive in economical, green, and safe [1]. We isolated novel nature and was in the exponential growth phase after 24 h microorganisms from petroleum-contaminated sludge samples of incubation under optimal conditions without addition capable of performing biotransformation of acrylonitrile to of any inducer. The substrate preference was acrylonitrile acrylic acid. Furthermore, a cost-effective and reliable and acetonitrile. The present work demonstrates the reverse-phase HPLC protocol for the detection and biotransformation of acrylonitrile to acrylic acid with the quantification of acrylic acid, acrylamide, and acrylonitrile, new strain, R. ruber AKSH-84, which can be used in green using a C18 column with water-acetonitrile as a mobile biosynthesis of acrylic acid for biotechnological processes. phase, was optimized. The results were confirmed using The nitrilase produced by the isolate was purified and LC-MS. The environmental and nutritional conditions for characterized. nitrilase activity were optimized for a newly isolated strain Keywords: Rhodococcus ruber, acrylic acid, acrylonitrile, of Rhodococcus ruber AKSH-84 using whole cells. This is nitrilase, biotransformation the first report on the isolation and identification and the medium optimization for nitrilase activity of a Rhodococcus ruber strain that is able to convert acrylonitrile to acrylic acid. The biotransformation conditions from acrylonitrile Acrylic acid that is used traditionally for production of to acrylic acid by whole resting cells and purified nitrilase diverse acrylic esters, namely methyl acrylate, ethyl from the strain AKSH-84 were also optimized. acrylate, butyl acrylate, and 2-ethylhexyl acrylate [20], which find major application in diverse industries related to industrial coatings, adhesives, decoratives, masonry, MATERIALS AND METHODS paper, textile products, resins, flocculants, targeted drug delivery systems, etc. [8, 16, 22]; poly(acrylic acid) (PAA) Samples and Chemicals gels used in the fabrication of BioMEMS devices as Petroleum-contaminated sludge samples obtained from the Petroleum sensors and actuators [11]; and superabsorbent polymers Refinery Unit, Essar Oil Limited, Vadinar, Jamnagar, Gujarat, India [23] and detergent polymers [21]. There is a demand for were screened for isolating nitrile-degrading microorganisms. Acrylic crude acrylic acid, and the market reached around 3.2 acid, acrylamide, acrylonitrile, and all other chemicals and solvents (analytical grade) were purchased from Sigma, St. Louis, MO, USA. *Corresponding author o Phone: +91-40-27193157; Fax: +91-40-27193189; Acrylic acid was used after distillation and stored at 13 C. Solvents for E-mail: [email protected] HPLC analysis such as acetonitrile, methanol, and water of HPLC 38 Kamal et al. grade were obtained from Rankem Fine Chemicals, New Delhi, Purification of Nitrilase India. The HiPrep 16/10 DEAE FF and Sephadex G-200 superfine The Rhodococcus ruber strain AKSH-84 was cultured in 1 l of columns, marker protein for molecular mass determination, and production medium (pH 7) with 3% inoculum in a 5-l Erlenmeyer Coomassie Brilliant Blue R-250 were obtained from GE Biosciences, flask and incubated at 30oC with shaking at 150 rpm on an orbital USA. All the solutions prepared for the purification were filter- shaker (Innova 4230, New Brunswick Scientific, USA). The cells at sterilized by passing though 0.2-µm filters and stored at 4oC. log phase were harvested by centrifugation (10,000 rpm/4oC) for 10 min. The cell pellet was washed with 0.1 M phosphate buffer Isolation and Screening for Acrylonitrile-Converting containing 10 mM dithiothreitol and 10 mM β-mercaptoethanol to Microorganisms remove traces of medium constituents, and the cell pellet was later Enrichment of the sludge samples was carried out by suspending mixed with the same buffer above. The washed cells were further 1.0 g of each sludge sample in 100 ml of enrichment medium with suspended in 0.1 M potassium phosphate buffer and then disrupted the following composition: glucose 10 g, yeast extract 0.2 g, with a Vibram Cell ultrasonic oscillator (Model VC505, Sonics and KH2PO4 0.5 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O Materials, Inc., USA) at 19 kHz frequency for 10 min with a burst 0.01 g (per liter) spiked with 2% (v/v) acetonitrile as an inducer at interval of 30 s each. The mixture was centrifuged at 10,000 rpm for o pH 7.0. The flasks were incubated at 37 C with agitation at 150 rpm 10 min and the resultant supernatant was referred to as the cell-free for about 30 days. The second and third enrichments were followed extract. The cell-free crude extract was precipitated with solid by transferring culture suspension (1.0 ml) to the same fresh ammonium sulfate (45-70% saturation), and the precipitate was medium and incubating for 15 days each. Serial 10-fold dilutions collected after centrifugation at 12,000 rpm/4oC for 15 min, resuspended were performed on enrichment agar plates containing acrylonitrile in phosphate buffer (0.1 M), and dialyzed against the same buffer. (200 mM concentration) as substrate and bromothymol blue The dialysate was desalted using PD10 columns (GE Biosciences, o (0.01%); these indicator plates were incubated at 37 C for 48 h. USA) and loaded on a HiPrep 16/10 DEAE FF ion-exchange Acrylonitrile-converting microorganisms produced yellow-colored column (GE Biosciences, USA) equilibrated with the loading buffer halos around the colonies against a dark blue background, and these containing 50 mM HEPES (pH 7.5) and 1 mM EDTA and was isolates were purified for 2-3 times on nutrient agar plates and interfaced to a Biologic Duoflow Fast Precision Liquid Chromatography o o stored as nutrient agar slants (4 C) and glycerol stocks (-70 C). (FPLC) System (Bio-Rad, USA). The enzyme was loaded after equilibration of the column with loading buffer and washed with Identification of the Nitrilase-Producing Strain 100 ml of the loading buffer to remove the unbound protein. The Morphological characteristics of the strain AKSH-84 were observed enzyme was eluted with a step-wise gradient of 0-400 mM KCl in via a light microscope (Model BX51, Olympus Corporation, Tokyo, 50 mM HEPES buffer (pH 7.5) containing 1 mM EDTA. The eluted Japan). The culture was cultivated in nutrient broth and the cells enzyme fractions were assayed for nitrilase activity and protein o were separated by centrifugation at 10,000 rpm for 10 min at 4 C content using the Bradford method [7]. The active fractions were and washed with distilled water. The cells were fixed for 1 h in 4% pooled and concentrated. The concentrated enzyme solution glutaraldehyde in 0.2 M phosphate buffer, pH 6.9, for scanning obtained was passed through a Sephadex G-200 superfine column electron microscopy (SEM). The gold-coated stubs were scanned (1.5×23 cm) equilibrated with 0.1 M potassium phosphate buffer and micrographs taken on a SEM Model S-3000N (Hitachi, Japan) containing 0.1 M KCl and 1 mM EDTA, and the eluted active at the accelerating voltage of 10 kV. Routine physiological and fractions were pooled and concentrated using Centriplus concentrators biochemical characterizations were carried out following the methods (Amicon Inc.) and stored at -10oC until further use. All the purification listed in Bergey’s Manual of Determinative Bacteriology [10]. The steps were performed at 4oC. 16S rDNA gene sequencing was carried out using universal primers: forward primer 27f (5'-AGA GTT TGA TCM TGG CTC AG-3') Biotransformation of Acrylonitrile to Acrylic Acid by Whole and reverse primer 1492r (5'-ACG GTT ACC TTG TTA CGA CTT- Resting Cells Exhibiting Nitrilase Activity 3'). The phylogenetic tree was constructed using MEGA4 (version The nitrilase activity was assayed using acrylonitrile as substrate. 4.0) software [19]. The resting cell suspensions were prepared by suspending the cell pellet in 10 mM phosphate buffer (pH 7.2), so as to get a concentration Optimization of Medium and Reaction Conditions for Acrylic of 200 mg/ml. The reaction mixture was prepared by using 875 µl Acid Production by Rhodococcus ruber Strain AKSH-84 of 10 mM phosphate buffer, 25 µl of 500 mM acrylonitrile substrate, The APY medium [adjusted to pH 7.0 with following composition and 100 µl of cell suspension so as to get a final concentration of (per liter): ammonium acetate 10 g, peptone 5 g, yeast extract 5 g, 20 mg/ml cell pellet and 100 mM of substrate.

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