Abstract
An inducible and heterocyclic thermostable nitrilase was obtained from Staphylococcus sp. KP789135.1. The whole cell nitrilase was active at 50 °C temperature and neutral pH with a half-life of 4 h at 50 °C and 2.5 h at 60 °C. Metal ions such as Fe3+, Mg2+, and Ca2+ enhanced the biocatalytic activity while Ag2+, Hg2+, and Cu2+ completely inhibited biocatalysis, which confirmed the presence of thiol group in the catalytic site. Staphylococcus sp. KP789135.1 has broad hydrolytic activity towards heterocyclic nitriles and few amides. It showed high tolerance against 3-cyanopyridine (5 mM - 20 mM). The calculated Km and Vmax were 1.33 mM and 0.98 U, lower km values demonstrated the highest affinity towards 3-cyanopyridine. In silico study revealed 99% similarity with Staphylococcus strain. The 3D structures were deduced from I-TASSER server and validated from PROCHECK, found relatively low % of amino acid residues has phi/psi angles in the disallowed region that suggest the acceptability of Ramachandran plot in the present work. Docking study revealed 3-cyanopyridine has lowest potential energy (potential energy OPLS -7.00745) and thus showed more stable enzyme ligand interaction. This is the first report on biotransformation of 3-cyanopyridine to nicotinic acid using thermostable nitrilase from Staphylococcus sp.
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References
Agarwal A, Nigam VK (2014) Nitrilase mediated conversion of Indole-3-acetonitrile to Indole-3-acetic acid. Biocatal Agric Biotechnol 3(4):351–357. https://doi.org/10.1016/j.bcab.2014.05.005
Agarwal A, Nigam VK, Vidyarthi AS (2012) Nitrilases–an attractive nitrile degrading biocatalyst. Int J Pharm Biol Sci 3:232–246
Almatawah QA, Cowan DA (1999) Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine. Enzym Microbial Technol 25(8-9):718–724. https://doi.org/10.1016/s0141-0229(99)00104-0
Arfi T, Nigam VK (2015) Screening and isolation of different Bacteria for synthesis of nicotinic acid. Res J Pharm Biol Chem Sci 6(2):1750–1758
Arfi A, Nigam VK (2018) Extraction and quantification of nicotinic acid from 3-Cyanopyrdinase catalysed reaction. Res J Chem Environ 22(9):1–6
Badoei-Dalfard A, Karami Z, Ramezani-pour N (2016a) Bench scale production of nicotinic acid using a newly isolated Stenotrophomonas maltophilia AC21 producing highly-inducible and versatile nitrilase. J Mol Catal B Enzym 133:S552–S559. https://doi.org/10.1016/j.molcatb.2016.11.019
Badoei-Dalfard A, Ramezani-Pour N, Karami Z (2016b) Production and characterization of a Nitrilase from Pseudomonas aeruginosa RZ44 and its potential for nitrile biotransformation. Iranian J Biotechnol 14(3):142. https://doi.org/10.15171/ijb.1179
Banerjee A, Kaul P, Banerjee UC (2006) Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis. App Microbiol Biotechnol 72(1):77–87. https://doi.org/10.1007/s00253-005-0255-8
Brandao PF, Bull AT (2003) Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes. Anton Leeuw Int J G 84(2):89
Chen Z, Zhao J, Jiang S et al (2019) Recent research advancements on regioselective nitrilase: fundamental and applicative aspects. Appl Microbiol Biotechnol 103:6393–6405. https://doi.org/10.1007/s00253-019-09915-8
Egelkamp R, Friedrich I, Hertel R, Daniel R (2020) From sequence to function: a new workflow for nitrilase identification. Appl Microbiol Biotechnol 104:4957–4970. https://doi.org/10.1007/s00253-020-10544-9
Fan H, Chen L, Sun H, Wang H, Ren Y, Wei D (2017) A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production. Bioproc Biosyst Eng 40(8):1271–1281. https://doi.org/10.1007/s00449-017-1787-x
Gong JS, Lu ZM, Li H, Zhou ZM, Shi JS, Xu ZH (2013) Metagenomic technology and genome mining: emerging areas for exploring novel nitrilases. Appl Microbiol Biotechnol 97(15):6603–6611. https://doi.org/10.1007/s00253-013-4932-8
Harper DB (1977) Characterization of a nitrilase from Nocardia sp. J Biochem 167(3):685–692
Hartzell GE (2001) Engineering analysis of hazards to life safety in fires: the fire effluent toxicity component. Safe Sci 38(2):147–155. https://doi.org/10.1016/s0925-7535(00)00065-5
Howden AJ, Preston GM (2009) Nitrilase enzymes and their role in plant–microbe interactions. Microb Biotechnol 2(4):441–451. https://doi.org/10.1111/j.1751-7915.2009.00111.x
Jin LQ, Liu ZQ, Xu JM, Zheng YG (2013) Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150. World J Microbiol Biotechnol 29(3):431–440. https://doi.org/10.1007/s11274-012-1195-y
Kumar V, Seth A, Kumari V, Kumar V, Bhalla TC (2015) Purification, characterization and in-silico analysis of nitrilase from Gordonia terrae. Protein Peptide Lett 22(1):52–62. https://doi.org/10.2174/0929866521666140909154537
Li H, Dong W, Zhang Y, Liu K, Zhang W, Zhang M, Ma J, Jiang M (2016) Enhanced catalytic efficiency of nitrilase from Acidovorax facilis 72W and application in bioconversion of 3-cyanopyridine to nicotinic acid. J Mol Catal B Enzym 133:S459–S467. https://doi.org/10.1016/j.molcatb.2017.03.010
Liu G, Kong Y, Fan Y, Geng C, Peng D, Sun M (2017) Whole-genome sequencing of Bacillus velezensis LS69, a strain with a broad inhibitory spectrum against pathogenic bacteria. J Biotechnol 249:20–24. https://doi.org/10.1016/j.jbiotec.2017.03.018
Liu D, Xi L, Han D, Ke D, Su S, Liu J (2019) Cloning, expression, and characterization of a novel nitrilase, PaCNit, from Pannonibacter carbonis Q4.6. Biotechnol Lett 41:583–589. https://doi.org/10.1007/s10529-019-02661-x
Martínková L, Rucká L, Nešvera J, Pátek M (2017) Recent advances and challenges in the heterologous production of microbial nitrilases for biocatalytic applications. World J Microbiol Biotechnol 33(8):1–8. https://doi.org/10.1007/s11274-016-2173-6
Meth-Cohn O, Wang MX (1997) An in-depth study of the biotransformation of nitriles into amides and/ or acids using Rhodococcus rhodochrous AJ270. J Chem Soc 1:1099–1104. https://doi.org/10.1039/a607977f
Nigam VK, Arfi T, Kumar V, Shukla P (2017) Bioengineering of nitrilases towards its use as green catalyst: applications and perspectives. Indian J Microbiol 57(2):131–138. https://doi.org/10.1007/s12088-017-0645-5
Pai O, Banoth L, Ghosh S, Chisti Y, Banerjee UC (2014) Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli. Process Biochem 49(4):655–659. https://doi.org/10.1016/j.procbio.2014.01.023
Pratush A, Seth A, Bhalla TC (2011) Optimization of process parameters for conversion of 3-cyanopyridine to nicotinamide using resting cells of mutant 4D strain of Rhodococcus rhodochrous PA-34. Int J Bioautomation 15:151–158
Pratush A, Seth A, Bhalla TC (2013) Purification and characterization of nitrile ydratase of mutant 4D of Rhodococcus rhodochrous PA-34. 3 Biotech 3(2):165–171. https://doi.org/10.1007/s13205-012-0081-5
Sharma N, Kushwaha R, Sodhi JS, Bhalla TC (2009) In Silico analysis of amino acid sequences in relation to specificity and physiochemical properties of some microbial nitrilases. J Proteomics Bioinform 2:185–192. https://doi.org/10.4172/jpb.1000076
Sharma N, Thakur N, Raj T, Bhalla TC (2017) Mining of microbial genomes for the novel sources of nitrilases. BioMed Res Int 2017:1–14. https://doi.org/10.1155/2017/7039245
Taguchi K, Fukusaki E, Bamba T (2014) Determination of niacin and its metabolites using supercritical fluid chromatography coupled to tandem mass spectrometry. Mass Spectrom 3(1):A0029. https://doi.org/10.5702/massspectrometry.a0029
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. https://doi.org/10.1093/molbev/msr121
Thakur N, Sharma NK, Thakur S, Moinca BTC (2019) Bioprocess Development for the Synthesis of 4-Aminophenylacetic Acid Using Nitrilase Activity of Whole Cells of Alcaligenes faecalis MTCC 12629. Catal Lett 149:2854–2863. https://doi.org/10.1007/s10562-019-02762-2
Thuku RN, Brady D, Benedik MJ, Sewell BT (2009) Microbial nitrilases: versatile, spiral forming, industrial enzymes. J App Microbiol 106(3):703–727. https://doi.org/10.1111/j.1365-2672.2008.03941.x
Wu Y, Gong JS, Lu ZM, Li H, Zhu XY, Li H, Shi JS, Xu ZH (2013) Isolation and characterization of Gibberella intermedia CA3-1, a novel and versatile nitrilase-producing fungus. J Basic Microbiol 53(11):934–941. https://doi.org/10.1002/jobm.201200143
Yang C, Wang X, Wei D (2011) A new nitrilase-producing strain named Rhodobacter sphaeroides LHS-305: biocatalytic characterization and substrate specificity. App Biochem Biotechnol 165(7-8):1556–1567. https://doi.org/10.1007/s12010-011-9375-z
Yusuf F, Rather IA, Jamwal U, Gandhi SG, Chaubey A (2015) Cloning and functional characterization of nitrilase from Fusarium proliferatum AUF-2 for detoxification of nitriles. Functional & Integrative Genomics 15(4):413–424. https://doi.org/10.1007/s10142-014-0430-z
Acknowledgments
The author Tesnim Arfi duly acknowledges Senior Research Fellowship of Moulana Azad National Fellowship, U. G. C, Government of India (F1-17.1/2012-13/MANF-2012-13-MUS-JHA-9035), New Delhi-110002 for pursuing Ph.D. The authors deeply appreciate the central instrumentation facility (CIF) of Birla Institute of Technology, Mesra for providing necessary experimental facilities to carry out this research work.
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Arfi, T., Nigam, V.K. Studies on a thermostable nitrilase from Staphylococcus Sp and its In-silico characterisation. Biologia 75, 2421–2432 (2020). https://doi.org/10.2478/s11756-020-00554-3
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DOI: https://doi.org/10.2478/s11756-020-00554-3