Abstract
In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V max and K m toward 3-cyanopyridine were 77.5 μmol min−1 mg−1 and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.
Similar content being viewed by others
References
Almatawah QA, Cowan DA (1999) Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine. Enzyme Microb Technol 25:718–724
Banerjee A, Kaul P, Banerjee UC (2006) Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida. Arch Microbiol 184:407–418
Bengisgarber C, Gutman AL (1989) Selective hydrolysis of dinitriles into cyano-carboxylic acids by Rhodococcus rhodochrous NCIB 11216. Appl Microbiol Biotechnol 32:11–16
Bhalla TC, Miura A, Wakamoto A, Ohba Y, Furuhashi K (1992) Asymmetric hydrolysis of alpha aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34. Appl Microbiol Biotechnol 37:184–190
Brady D, Dube N, Pettersen R (2006) Green chemistry: highly selective biocatalytic hydrolysis of nitrile compounds. S Afr J Sci 102:339–344
Chauhan S, Wu S, Blumerman S, Fallon RD, Gavagan JE, DiCosimo R, Payne MS (2003) Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72 W. Appl Microbiol Biotechnol 61:118–122
DeSantis G, Wong K, Farwell B, Chatman K, Zhu Z, Tomlinson G, Huang H, Tan X, Bibbs L, Chen P, Kretz K, Burk MJ (2003) Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J Am Chem Soc 125:11476–11477
Heinemann U, Engels D, Buerger S, Kiziak C, Mattes R, Stolz A (2003) Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein. Appl Environ Microbiol 69:4359–4366
Kaplan O, Vejvoda V, Plíhal O, Pompach P, Kavan D, Bojarova P, Bezouska K, Mackova M, Cantarella M, Jirku V, Kren V, Martínkova L (2006) Purification and characterization of a nitrilase from Aspergillus niger K10. Appl Microbiol Biotechnol 73:567–575
Kim J, Tiwari MK, Moon H, Jeya M, Ramu T, Oh D, Kim I, Lee J (2009) Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5. Appl Microbiol Biotechnol 83:273–283
Kiziak C, Conradt D, Stolz A, Mattes R, Klein J (2005) Nitrilase from Pseudomonas fluorescens EBC191: cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology 151:3639–3648 (Reading, England)
Kobayashi M, Yanaka N, Nagasawa T, Yamada H (1990) Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles. J Biotechnol 172:4807–4815
Kobayashi M, Komeda H, Yanaka N, Nagasawa T, Yamada H (1992) Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue. J Biol Chem 267:20746–20751
Liu J, Zhang Z, Li A, Pan J, Xu J (2011) Significantly enhanced production of recombinant nitrilase by optimization of culture conditions and glycerol feeding. Appl Microbiol Biotechnol 89:665–672
Mueller P, Egorova K, Vorgias CE, Boutou E, Trauthwein H, Verseck S, Antranikian G (2006) Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi. Protein Expr Purif 47:672–681
Nagasawa T, Mauger J, Yamada H (1990) A novel nitrilase, arylacetonitrilase of Alcaligenes faecalis JM3. Purification and characterization. Eur J Biochem/FEBS 194:765–772
O’Reilly C, Turner PD (2003) The nitrilase family of CN hydrolyzing enzymes-a comparative study. J Appl Microbiol 95:1161–1174
Panova A, Mersinger LJ, Liu Q, Foo T, Roe DC, Spillan WL, Sigmund AE, Ben-Bassat A, Wagner LW, O’Keefe DP, Wu S, Petrillo KL, Payne MS, Breske ST, Gallagher FG, DiCosimo R (2007) Chemoenzymatic synthesis of glycolic acid. Adv Synth Catal 349:1462–1474
Robertson DE, Chaplin JA, DeSantis G, Podar M, Madden M, Chi E, Richardson T, Milan A, Miller M, Weiner DP, Wong K, McQuaid J, Farwell B, Preston LA, Tan X, Snead MA, Keller M, Mathur E, Kretz PL, Burk MJ, Short JM (2004) Exploring nitrilase sequence space for enantioselective catalysis. Appl Environ Microbiol 70:2429–2436
Seffernick JL, Samanta SK, Louie TM, Wackett LP, Subramanian M (2009) Investigative mining of sequence data for novel enzymes: a case study with nitrilases. J Biotechnol 143:17–26
Singh R, Sharma R, Tewari N, Geetanjali Rawat DS (2006) Nitrilase and its application as a ‘green’ catalyst. Chem Biodivers 3:1279–1287
Vejvoda V, Kaplan O, Bezouska K, Pompach P, Sulc M, Cantarella M, Benada O, Uhnakova B, Rinagelova A, Lutz-Wahl S, Fischer L, Kren V, Martinkova L (2008) Purification and characterization of a nitrilase from Fusarium solani O1. J Mol Catal B: Enzymatic 50:99–106
Vorwerk S, Biernacki S, Hillebrand H, Janzik I, Muller A, Weiler EW, Piotrowski M (2001) Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster. Planta 212:508–516
Wang H, Sun H, Wei D (2013) Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction. BMC Biotechnol 13:14
Weatherburn MW (1967) Phenol-hypochlorite reaction for determination of ammonia. Anal Chem 39:971–974
Xie Z, Feng J, Garcia E, Bernett M, Yazbeck D, Tao J (2006) Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid. J Mol Catal B: Enzymatic 41:75–80
Yang CS, Wang XD, Wei DZ (2011) A New nitrilase-producing strain named Rhodobacter sphaeroides LHS-305: biocatalytic characterization and substrate specificity. Appl Biochem Biotechnol 165:1556–1567
Zhang Z, Xu JH, Ouyang L, Liu Y (2011) Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(2)-mandelic acid production. Bioprocess Biosyst Eng 34:315–322
Zhu D, Mukherjee C, Bieh ER, Hua L (2007) Discovery of a mandelonitrile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome mining. J Biotechnol 129:645–650
Zhu D, Mukherjee C, Yang Y, Rios BE, Gallagher DT, Smith NN, Bieh ER, Hua L (2008) A new nitrilase from Bradyrhizobium japonicum USDA 110 Gene cloning, biochemical characterization and substrate specificity. J Biotechnol 133:327–333
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Wang, H., Li, G., Li, M. et al. A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization. World J Microbiol Biotechnol 30, 245–252 (2014). https://doi.org/10.1007/s11274-013-1445-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-013-1445-7