Nitrile-metabolizing potential of Amycolatopsis sp. IITR215 Vikash Babu, Shilpi, Bijan Choudhury * Department of Biotechnology, Indian Institute of Technology, IIT Roorkee, Roorkee-247667, Uttarakhand, India 1. Introduction Nitrile compounds are widely used by chemical industries for the synthesis of various chemicals. These compounds are highly neurotoxic and carcinogenic in nature and considered as environ- mental pollutants [1]. Therefore, remediation of such toxic compounds is necessary for a pollution-free environment. Nitrile compounds can be detoxified by transformation of the –CN group to –CONH 2 or –COOH. These types of transformations can be achieved chemically as well as enzymatically. Enzymatic biotrans- formation has many advantages over chemical transformation, namely: (1) it is effective at very low concentrations; (2) it acts under mild conditions; (3) it displays chemo-, regio- and enantio- selectivity; and (4) it is not restricted to a natural substrate and can work outside an aqueous environment. Nitrile metabolism is common in bacteria and plants but has also been reported in fungi. Nitrile-metabolizing enzymes are generally inducible in nature. Nitrile-hydrolyzing biocatalysis is due to the activity of two enzyme systems: nitrilase (EC 3.5.5.1) and nitrile hydratase (EC 4.2.1.84) plus amidase (EC 3.5.1.4). Multiple nitrile-metabolizing enzyme systems have been reported in some strains. Rhodococcus rhodochrous J1 was found to contain two nitrile hydratases, one of which was induced by urea and the other by cyclohexane–caboxamide–crotonamide [2,3]. A constitutive nitrilase was found in Bacillus subtilis ZJB- 063 whereas nitrile hydratase and amidase were induced by the addition of e-caprolactam [4]. Zhu et al. (2008) reported two nitrilase genes (blr 3397 and bll 6402) in Bradyrhizobium japonicum USDA 110. Both nitrilases were active against phenylacetonitrile. Nitrilase blr 3397 also showed highest activity with hydrocinnamonitrile while nitrilase bll 6402 showed comparable activity towards aliphatic nitriles [5]. Multiple nitrile-metabolizing enzymes have also been reported in Nocardia rhodochrous LL100–21 [6], R. rhodochrous NCIMB11216 [7,8], Bacillus pallidus DAC521 [9,10] and Nocardia globerula NHB-2 [11]. In the present study, we isolate a novel microbial strain from sewage that is capable of growth on agar plates containing polyacrylonitrile as the sole nitrogen source. The strain was identified by 16S rDNA sequence homology. This strain produced multiple nitrile-metabolizing enzymes having wide range of substrate specificities. To our knowledge this is the first report showing constitutive multiple nitrile-metabolizing enzymes from the Amycolatopsis genus. Process Biochemistry 45 (2010) 866–873 ARTICLE INFO Article history: Received 23 August 2009 Received in revised form 3 February 2010 Accepted 4 February 2010 Keywords: Nitrilase Nitrile hydratase/amidase Polyacrylonitrile Hexanenitrile Diethylphosphoramidate (DEPA) Amycolatopsis sp. IITR215 ABSTRACT Strain Amycolatopsis sp. IITR215 was isolated from a sewage sample using polyacrylonitrile powder as the sole nitrogen source. Identification was performed by 16S rDNA analysis. The isolated strain harbored multiple nitrile-metabolizing enzymes having a wide range of substrate specificities. It metabolized nitrile and amide compounds with constitutive enzymes. Studies using an amidase inhibitor showed that hydrolysis of acrylonitrile and acrylamide occurred due to nitrile hydratase and amidase, respectively, while hydrolysis of hexanenitrile was due to the action of either nitrilase or a second nitrile hydratase/amidase system. The inhibitory effects of N-bromosuccinimide and N- ethylmaleimide on enzymes of this culture were also studied and this further indicated the involvement of either a nitrilase or a second nitrile hydratase/amidase system for hydrolysis of hexanenitrile. Interestingly, hexanenitrile hydrolysis exhibited an optimum temperature of 55 8C, whereas acrylonitrile and acrylamide hydrolysis showed an optimum temperature of 45 8C. The optimum pH was 5.8 for hexanenitrile hydrolysis and 7.0 for acrylonitrile and acrylamide hydrolysis. Hexanenitrile hydrolysis by enzymes of this strain showed better organic solvent tolerance in the presence of alcohols. The maximum enzyme activity of nitrile-metabolizing enzymes was found using media containing isobutyramide as the nitrogen source. This is the first report on constitutive multiple enzymes from the Amycolatopsis genus. ß 2010 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +91 1332 285297; fax: +91 1332 273560. E-mail address: bijanfbs@iitr.ernet.in (B. Choudhury). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2010.02.008