Isolation and Characterization of Rhodococcus Ruber CGMCC3090 That Hydrolyzes Aliphatic, Aromatic and Heterocyclic Nitriles
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African Journal of Biotechnology Vol. 8 (20), pp. 5467-5486, 19 October, 2009 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2009 Academic Journals Full Length Research Paper Isolation and characterization of Rhodococcus ruber CGMCC3090 that hydrolyzes aliphatic, aromatic and heterocyclic nitriles JinLi Zhang1,2, Min Wang1*, Hua Sun1, XiaoDan Li1 and LiPing Zhong1 1Key Laboratory of Industrial Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China. 2Department of Food Science and Engineering, Shandong Agricultural University, Taian 271018 Shandong, P. R. China. Accepted 29 September, 2009 A bacterial strain was isolated from soil samples that had been polluted by nitrile compounds. This strain converts acrylonitrile to acrylamide with high activity. The nitrile hydrolysis activity was tested using eight substrates, including aliphatic, aromatic and heterocyclic (di)nitriles. All of the nitrile compounds were hydrolyzed by the resting cells. The main (cyano-)amide products demonstrated that nitrile hydratase was abundantly produced in this strain and that it mediated monohydrolysis. The specific conversion rate decreased in the following order: acrylonitrile > 3-cyanopyridine > valeronitrile > adiponitrile > 2,3,4,5,6-pentafluorobenzonitrile > 4-hydroxyphenylacetonitrile > 3-indoleaceto-nitrile > phthalonitrile, suggesting a higher conversion capability towards aliphatic nitriles. The strain that had broad substrate spectra was identified and named Rhodococcus ruber CGMCC3090 based on the 16S rDNA sequence. Key words: Rhodococcus ruber, nitrile hydratase, 16S rDNA, substrate spectra, isolation, identification. INTRODUCTION Nitrile compounds are intermediates, products, by- agents. Rhodococcus spp. have been reported to be the products and waste products of agriculture, the chemical most robust, useful and versatile organisms (Bunch, and pharmaceutical industries and the processing of 1998; Martínková et al., 2009). The most notable fossil fuels (Martínková et al., 2009). The enzymatic example of this use is the commercial production of hydrolysis of nitriles to carboxylic acids or amides plays acrylamide and nicotinamide using Rhodococcus an increasingly important role in organic synthesis and rhodocrous J1 (Yamada and Kobayashi, 1996; Nagasawa environment remediation, due to the mild reaction et al., 1988). In addition, the immobilization of conditions, high activities and the high selectivities of the Rhodococcus sp. strains Novo ‘SP361’ and ‘SP409’ have enzymes (Banerjee et al., 2002; Mylerová and been achieved comercially by NOVO Industri (DK). R. Martínková 2003). erythropolis AJ270, which has an amidase that has a A wide range of prokaryotic and eukaryotic micro- greater rate of amide hydrolysis than of nitrile hydration, organisms has been isolated for use as nitrile-hydrolyzing is being exploited for its enantioselectivity in organic synthesis (Wang et al., 2000; Wang et al., 2003; Ma et al., 2006; Wang JY et al., 2007; Ma et al., 2008). Microorganisms of other different taxonomic groups *Corresponding author. E-mail: [email protected] or have also been studied and used. Nocardia sp. 163, [email protected]. Tel: +86-22-60601256. Fax: +86-22- isolated from Mount Tai in the 1980’s (which had the 60602298. highest nitirile hydratase activity at that time), is being used to produce acrylamide at the scale of several tens Abbreviations: DCW, Dry cell weight; DMF, N,N- of thousand tons per year at facilities in China (Zhang et dimethylformamide; DMSO-d6, dimethyl sulfoxide D6; TMS, tetramethylsilane; M. P., melting point; NJ, neighbor-joining; MP, al., 1998; Liu et al., 2001). Zheng and co-workers have maximum parsimony; TBR, tree bisection and reconnection; obtained several novel, versatile nitrile-amide-converting OTUs, operational taxonomic units. bacteria, such as Bacillus subtilis ZJB-063, Nocardia sp. 5468 Afr. J. Biotechnol. 108 and Arthrobacter nitroguajacolicus ZJUTB06-99 which point more acrylonitrile (3.0%, v/v) was added. Thereafter, (Zheng et al., 2008; Wang YJ et al., 2007; Shen et al., samples were removed at 6 h intervals and acidified to pH 4.0 using 2009). 6 M HCl. The supernatant obtained by centrifugation was analyzed using GC to determine the concentrations of acrylonitrile and acry- However, commercial sources of nitrilases and nitrile lamide. The strain with the highest conversion ratio from acry- hydratases and the appropriate nitrile-amide converting lonitrile to acrylamide was selected. biocatalysts are almost unavailable (Chen et al., 2009). Therefore, the isolation and screening of biocatalysts that have particular characteristics in the hydrolysis of nitriles Culture of the bacteria are still of great interest. In this work, we aimed at ob- The cells were precultured aerobically for 28 h at 30°C in 250-ml taining an acrylonitrile-hydrolyzing strain with broad sub- Erlenmeyer flasks filled with 30 ml of medium, consisting of (g/l) strate spectra. The characteristics of this strain and its glycerol 10.0, peptone 5.0, malt extract 3.0 and yeast extract 3.0. capabilities in the hydrolysis of eight nitrile compounds The cultivation was conducted at 28°C for 72 h in 250-ml Erlen- were examined. These include: 1) the hydrolysis of meyer flasks on a rotary shaker at 180 r/min. The medium was as aromatic nitriles; 2) the hydrolysis of heterocyclic nitriles; follows (g/l): glucose 15.0, yeast extract 5.0, urea 7.0, KH2PO4 0.5, K2HPO4 0.5, MgSO4·7H2O 0.5, monosodium glutamate 1.0 and 3) the hydrolysis of aliphatic nitriles; and 4) the selective -2 CoCl2·6H2O 2.4 × 10 . The pH of both media was adjusted to 7.2 mono-hydrolysis of symmetric dinitriles. Furthermore, the using aqueous diluted NaOH. hydrolytic profile of a perfluoronitrile, 2, 3, 4, 5, 6- penta- The resting cells were harvested from the fermentation broth by fluorobenzonitrile, was tested using a biocatalyst for the centrifugation and were washed with physiological saline buffer. first time. This strain, which has a broad substrate spectra Afterwards, the cells were resuspended in an appropriate volume of was then identified based on its 16S rDNA sequence. R. phosphate buffer (50 mmol/l, pH 7.5) to obtain a final concentration of 5.1 mg DCW/ml. ruber with its broad substrate spectra has demonstrated wide application potential in organic synthesis and environment remediation. Hydrolysis of nitrile compounds Due to their poor aqueous solubilities, the nitriles were first dis- MATERIALS AND METHODS solved in an appropriate volume of DMF. The mixture for biotrans- formation consisted of 1.0 ml of resting cells, 9.0 ml of phosphate Chemicals buffer (25 mmol/l, pH 7.5) and 0.5 ml (5% (v/v)) of substrate solution, giving a final concentration of 15 - 2,000 mmol/l in 100-ml Acrylonitrile and acrylamide were purchased from Sigma-Aldrich Erlenmeyer flasks. The reaction was carried out at 30°C on a rotary (USA). 3-cyanopyridine and 3-pyridinecarboxamide were pur- shaker (150r/min) for different times and terminated by addition of 6 chased from Lonza Chemical Industry Co. Ltd (China). Phthalo- M HCl to pH 2.5. The cells were removed by centrifugation. One nitrile, valeronitrile, adiponitrile and 2,3,4,5,6-pentafluorobenzonitrile part of the resulting supernatant was analyzed using HPLC. The were obtained from Alfa Aesar (USA). Methanol, acetonitrile and remaining part was saturated with sodium chloride and extracted tetrahydrofuran were of gradient grade for liquid chromatography three times with ethyl acetate (at pH 8.5-9 for extraction of the (Merck KGaA, Germany). All other chemicals were of analysis amides and at pH 2-2.5 for extraction of the carboxylic acids, res- grade. The TLC and preparative TLC were performed on silica gel pectively). The combined organic layers were filtered through anhydrous Na SO . The solvent (ethyl acetate) was removed by GF254 plates that were purchased from Qingdao Marine Chemical 2 4 Group Co. (China). evaporation under vacuum and the crude products were purified using preparative TLC. All of the experiments mentioned above were carried out in triplicate. Sample collection and screening Extraction of DNA from pure cultures Soil samples were taken from an area polluted by nitrile compounds in Tianjin, China. Nitrile-utilizing microorganisms were isolated from Genomic DNA samples from pure cultures were extracted as the soil samples by an enrichment culture method in basal medium described previously (Gliesche et al., 1997). The purified DNA was containing 0.5 g/l glucose, 0.5 g/l KH2PO4 0.5 g/l K2HPO4, 0.2 g/l dissolved in 10 mM Tris-EDTA (TE) buffer, pH 8.0 (Sigma, St. Louis, MgSO4·7H2O, 1.0 g/l NaCl, 13.9 mg/l FeSO4·7H2O and 11.9 mg/l Mo.). The concentration was determined spectrophotometrically CoCl2·6H2O at pH 7.2. The flasks were incubated on a rotary and adjusted to 10g/ml. shaker (180r/min) at 30°C at 3~4 d intervals, with increasing amounts of acrylonitrile (final concentrations of 0.3, 0.6 and 1.0% (v/v)) added to the sterilized medium. Then, the cultures were Amplification of the 16S rDNA diluted and 100 L aliquots were plated onto LB agar containing acrylonitrile (3.0% (v/v)) for the selection of acrylonitrile-utilizing The extracted DNA (50 ng) was amplified using a pair of universal cells. The incubation was carried out at 30°C for 5 d, and the bacterial primers (Wang et al., 2002). The amplification reactions colonies that grew more strongly were preserved in a slant were carried out in a 50 l reaction, containing 1×PCR Buffer containing (g/l) beef extract 5.0, peptone 10.0, NaCl 5.0 and agar (Qiagen, Inc., Valencia, CA), 20 pmol of each primer, 200 mol/l of 20.0. dNTPs, 2.5 U of Taq DNA polymerase (Qiagen, Inc., Valencia, CA) The cells from the slant were inoculated into 250-ml Erlenmeyer and sufficient ddH2O to bring the final volume to 50 l.