Abstract
Nitriles are important chemical building blocks for the synthesis of intermediates in fine chemical and pharmaceutical industries. Here, we report a new highly thermostable nitrilase from an Antarctic Pyrococcus sp. MC-FB, a hyperthermophilic archaeon. A gene that encoded a nitrilase was identified and subsequently cloned and overexpressed in Escherichia coli. The recombinant nitrilase, named NitMC-FB, is active as a homodimer (60 kDa) with an optimal temperature and pH of 90 °C and 7.0, respectively. NitMC-FB hydrolyzes preferentially aromatic nitriles, being the first aromatic nitrilase from an archaeon described so far. The K M and V max parameters were determined to be 13.9 mM and 3.7 μmol/min*mg, respectively, with 2-cyanopyridine as the substrate. Additionally, the recombinant nitrilase is highly thermostable with a half-life of 8 h at 90 °C.
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Abbreviations
- DTT:
-
Dithiothreitol
- EDTA:
-
Ethylenediaminetetraacetic acid
- IPTG:
-
Isopropyl-β-D-thiogalactopyranosid
References
Almatawah QA, Cramp R, Cowan DA (1999) Characterization of an inducible nitrilase from a thermophilic Bacillus. Extremophiles 3:283–291. doi:10.1007/s007920050129
Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi:10.1016/S0022-2836(05)80360-2
Banerjee A, Kaul P, Banerjee UC (2006) Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida. Arch Microbiol 184:407–418. doi:10.1007/s00203-005-0061-9
Barbier G, Godfroy A, Meunier J et al (1999) Pyrococcus glycovorans sp. nov., a hyperthermophilic archaeon isolated from the East Pacific Rise. Int J Syst Bacteriol 49(4):1829–1837. doi:10.1099/00207713-49-4-1829
Bhalla TC, Prashant Kumari N et al (2016) Synthesis of vanillic acid using whole cell nitrilase of wild and mutant Gordonia terrae. Bioprocess Biosyst Eng 39:67–73. doi:10.1007/s00449-015-1490-8
Bhatia SK, Mehta PK, Bhatia RK, Bhalla TC (2014) Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis. Appl Microbiol Biotechnol 98:83–94. doi:10.1007/s00253-013-5288-9
Birrien JL, Zeng X, Jebbar M et al (2011) Pyrococcus yayanosii sp. nov., an obligate piezophilic hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 61:2827–2831. doi:10.1099/ijs.0.024653-0
Brady D (2010) Biocatalytic hydrolysis of nitriles. In: Anastas P, Crabtree R (eds) Handbook of green chemistry, vol 3. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, pp 27–49
Cai W, Su E, Zhu S et al (2014) Characterization of a nitrilase from Arthrobacter aurescens CYC705 for synthesis of iminodiacetic acid. J Gen Appl Microbiol 60:207–214. doi:10.2323/jgam.60.207
Chen Z, Chen H, Ni Z et al (2015) Expression and characterization of a novel nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. J Microbiol Biotechnol 25:1660–1669. doi:10.4014/jmb.1502.02032
Dennett GV, Blamey JM (2016) A new thermophilic nitrilase from an Antarctic hyperthermophilic microorganism. Front Bioeng Biotechnol 4:5. doi:10.3389/fbioe.2016.00005
DeSantis G, DiCosimo R (2009) Applications of nitrile hydratases and nitrilases. In: Tao J, Lin G, Liese A (eds) Biocatalysis for the pharmaceutical industry: discovery, development, and manufacturing. Wiley, Singapore, pp 153–181
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. doi:10.1093/nar/gkh340
Fawcett JK, Scott JE (1960) A rapid and precise method for the determination of urea. J Clin Pathol 13:156–159. doi:10.1136/jcp.13.2.156
Flores PA, Amenábar MJ, Blamey JM (2013) Hot environments from Antarctica: source of thermophiles and hyperthermophiles, with potential biotechnological applications. In: Satyanarayana T, Littlechild J, Kawarabayasi Y (eds) Thermophilic microbes in environmental and industrial biotechnology: biotechnology of thermophiles, 2nd edn. Springer Netherlands, Dordrecht, pp 99–118
Gasteiger E, Hoogland C, Gattiker A et al (2005) Protein Identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics protocols handbook. Humana Press, Totowa, pp 571–607
Gong JS, Li H, Zhu XY et al (2012a) Fungal His-tagged nitrilase from Gibberella intermedia: gene cloning, heterologous expression and biochemical properties. PLoS ONE 7:e50622. doi:10.1371/journal.pone.0050622
Gong JS, Lu ZM, Li H et al (2012b) Nitrilases in nitrile biocatalysis: recent progress and forthcoming research. Microb Cell Fact 11:142. doi:10.1186/1475-2859-11-142
Harper DB (1985) Characterization of a nitrilase from Nocardia sp. (Rhodochrous group) N.C.I.B. 11215, using p-hydroxybenzonitrile as sole carbon source. Int J Biochem 17:677–683. doi:10.1016/0020-711X(85)90364-7
Hoyle AJ, Bunch AW, Knowles CJ (1998) The nitrilases of Rhodococcus rhodochrous NCIMB 11216. Enzyme Microb Technol 23:475–482. doi:10.1016/S0141-0229(98)00076-3
Kaplan O, Bezouska K, Plíhal O et al (2011) Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10. BMC Biotechnol 11:2. doi:10.1186/1472-6750-11-2
Kaur G, Soni P, Tewari R, Sharma R (2014) Isolation and characterization of a nitrile-hydrolysing bacterium Isoptericola variabilis RGT01. Indian J Microbiol 54:232–238. doi:10.1007/s12088-014-0453-0
Kobayashi M, Nagasawa T, Yamada H (1989) Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization. Eur J Biochem 182:349–356. doi:10.1111/j.1432-1033.1989.tb14837.x
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0
Legras JL, Chuzel G, Arnaud A, Galzy P (1990) Natural nitriles and their metabolism. World J Microbiol Biotechnol 6:83–108. doi:10.1007/BF01200927
Liese A, Seelbach K, Buchholz A, Haberland J (2007) Processes. In: Liese A, Seelbach K, Wandrey C (eds) Industrial biotransformations, 2nd edn. Wiley-VCH Verlag GmbH, Weinheim, pp 147–513
Littlechild J, Novak H, James P, Sayer C (2013) Mechanisms of thermal stability adopted by thermophilic proteins and their use in white biotechnology. In: Satyanarayana T, Littlechild J, Kawarabayasi Y (eds) Thermophilic microbes in environmental and industrial biotechnology: biotechnology of thermophiles, 2nd edn. Springer, Netherlands, Dordrecht, pp 481–507
Martínková L (2016) Nitrile-converting enzymes and their synthetic applications. In: Patel R (ed) Green biocatalysis, 1st edn. Wiley, Hoboken, pp 331–349
Martínková L, Rucká L, Nešvera J, Pátek M (2016) Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications. World J Microbiol Biotechnol 33:8. doi:10.1007/s11274-016-2173-6
Mueller P, Egorova K, Vorgias CE et al (2006) Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi. Protein Expr Purif 47:672–681. doi:10.1016/j.pep.2006.01.006
Muluka H, Sheelu G, Nageshwar YVD (2016) Bioconversion of iminodiacetonitrile to iminodiacetic acid with whole cells of Lysinibacillus boronitolerans MTCC 107614 (IICT-akl252). Bioprocess Biosyst Eng 39:413–420. doi:10.1007/s00449-015-1524-2
O’Reilly C, Turner PD (2003) The nitrilase family of CN hydrolysing enzymes—a comparative study. J Appl Microbiol 95:1161–1174. doi:10.1046/j.1365-2672.2003.02123.x
Pace HC, Brenner C (2001) The nitrilase superfamily: classification, structure and function. Genome Biol 2:1–9. doi:10.1186/gb-2001-2-1-reviews0001
Park WJ, Kriechbaumer V, Möller A et al (2003) The nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid. Plant Physiol 133:794–802. doi:10.1104/pp.103.026609
Qiu J, Su EZ, Wang HL et al (2014) Cloning, overexpression, and characterization of a high enantioselective nitrilase from Sphingomonas wittichii RW1 for asymmetric synthesis of (R)-phenylglycine. Appl Biochem Biotechnol 173:365–377. doi:10.1007/s12010-014-0845-y
Sharma NN, Sharma M, Bhalla TC (2011) An improved nitrilase-mediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced Nocardia globerula NHB-2. J Ind Microbiol Biotechnol 38:1235–1243. doi:10.1007/s10295-010-0902-7
Stalker DM, Malyj LD, McBride KE (1988) Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene. J Biol Chem 263:6310–6314
Thuku RN, Brady D, Benedik MJ, Sewell BT (2009) Microbial nitrilases: versatile, spiral forming, industrial enzymes. J Appl Microbiol 106:703–727. doi:10.1111/j.1365-2672.2008.03941.x
Wang H, Li G, Li M et al (2014) A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization. World J Microbiol Biotechnol 30:245–252. doi:10.1007/s11274-013-1445-7
Yamamoto K, Fujimatsu I, Komatsu KI (1992) Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile. J Ferment Bioeng 73:425–430. doi:10.1016/0922-338X(92)90131-D
Zhang CS, Zhang ZJ, Li CX et al (2012) Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata. Appl Microbiol Biotechnol 95:91–99. doi:10.1007/s00253-012-3993-4
Zhu D, Mukherjee C, Yang Y et al (2008) A new nitrilase from Bradyrhizobium japonicum USDA 110. Gene cloning, biochemical characterization and substrate specificity. J Biotechnol 133:327–333. doi:10.1016/j.jbiotec.2007.10.001
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We acknowledge the financial support of Becas CONICYT (Comisión Nacional de Investigación Científica y Tecnológica) and INACH (Instituto Antártico Chileno).
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Communicated by S. Albers.
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Cabrera, M.Á., Blamey, J.M. Cloning, overexpression, and characterization of a thermostable nitrilase from an Antarctic Pyrococcus sp.. Extremophiles 21, 861–869 (2017). https://doi.org/10.1007/s00792-017-0948-9
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DOI: https://doi.org/10.1007/s00792-017-0948-9