Skip to main content
SpringerLink
Log in

Continuous hydrolysis of 4-cyanopyridine by nitrilases from Fusarium solani O1 and Aspergillus niger K10

  • Biotechnological Products and Process Engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 °C with a deactivation constant (k d) and enzyme half-life of 0.014 h−1 and 50 h, respectively, but the latter exhibited an even higher stability characterized by k d = 0.008 h−1 and half-life of 87 h at 40 °C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Alfani F, Cantarella M, Spera A, Viparelli P (2001) Operational stability of Brevibacterium imperialis CBS 489–74 nitrile hydratase. J Mol Catal B-Enzym 11:687–697

    Article  CAS  Google Scholar 

  • Almatawah QA, Cowan DA (1999) Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine. Enzyme Microb Technol 25:718–724

    Article  CAS  Google Scholar 

  • Almatawah OA, Cramp R, Cowan DA (1999) Characterization of an inducible nitrilase from a thermophilic bacillus. Extremophiles 3:283–291

    Article  CAS  PubMed  Google Scholar 

  • Banerjee A, Sharma R, Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brady D, Beeton A, Zeevaart J, Kgaje C, van Rantwijk F, Sheldon RA (2004) Characterization of nitrilase and nitrile hydratase biocatalytic systems. Appl Microbiol Biotechnol 64:76–85

    CAS  PubMed  Google Scholar 

  • Cantarella M, Cantarella L, Gallifuoco A, Frezzini R, Spera A, Alfani F (2004) A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498–74 resting cells for propionamide production. J Mol Catal B-Enzym 29:105–113

    Article  CAS  Google Scholar 

  • Cantarella M, Cantarella L, Gallifuoco A, Spera A (2006) Use of a UF-membrane reactor for controlling selectively the nitrile hydratase-amidase system in Microbacterium imperiale CBS 498–74 resting cells—case study: benzonitrile conversion. Enzyme Microb Technol 38:126–134

    Article  CAS  Google Scholar 

  • Cantarella M, Cantarella L, Gallifuoco A, Intellini R, Kaplan O, Spera A, Martínková L (2008) Amidase-catalyzed production of nicotinic acid in batch and continuous stirred membrane reactors. Enzyme Microb Technol 42:222–229

    Article  CAS  Google Scholar 

  • Chaplin JA, Levin MD, Morgan B, Farid N, Li J, Zhu Z, McQuaid J, Nicholson LW, Rand CA, Burk MJ (2004) Chemoenzymatic approaches to the dynamic kinetic asymmetric synthesis of aromatic amino acids. Tetrahedron: Asymmetry 15:2793–2796

    Article  CAS  Google Scholar 

  • Fernandes BCM, Mateo C, Kiziak C, Chmura A, Wacker J, van Rantwijk F, Stolz A, Sheldon RA (2006) Nitrile hydratase activity of a recombinant nitrilase. Adv Synth Catal 348:2597–2603

    Article  CAS  Google Scholar 

  • Harper DB (1977) Fungal degradation of aromatic nitriles. Enzymology of C–N cleavage by Fusarium solani. Biochem J 167:685–692

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kaplan O, Nikolaou K, Pišvejcová A, Martínková L (2006a) Hydrolysis of nitriles and amides by filamentous fungi. Enzyme Microb Technol 38:260–264

    Article  CAS  Google Scholar 

  • Kaplan O, Vejvoda V, Charvátová-Pišvejcová A, Martínková L (2006b) Hyperinduction of nitrilases in filamentous fungi. J Ind Microbiol Biotechnol 33:891–896

    Article  CAS  PubMed  Google Scholar 

  • Kaplan O, Vejvoda V, Plíhal O, Pompach P, Kavan D, Bojarová P, Bezouška K, Macková M, Cantarella M, Jirků V, Křen V, Martínková L (2006c) Purification and characterization of a nitrilase from Aspergillus niger K10. Appl Microbiol Biotechnol 73:567–575

    Article  CAS  PubMed  Google Scholar 

  • Kaul P, Stolz A, Banerjee UC (2007) Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis. Adv Synth Catal 349:2167–2176

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi M, Nagasawa T, Yamada H (1989) Nitrilase of Rhodococcus rhodochrous J1. Purification and characterization. Eur J Biochem 182:349–356

    Article  CAS  PubMed  Google Scholar 

  • Kubáč D, Kaplan O, Elišáková V, Pátek M, Vejvoda V, Slámová K, Tóthová A, Lemaire M, Gallienne E, Lutz-Wahl S, Fischer L, Kuzma M, Pelantová H, van Pelt S, Bolte J, Křen V, Martínková L (2008) Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4. J Mol Catal B-Enzym 50:107–113

    Article  Google Scholar 

  • Liese A, Seelbach K, Wandrey C (2000) Industrial biotransformations. Weinheim, WILEY-VCH pp 317–321

    Book  Google Scholar 

  • Mathew CD, Nagasawa T, Kobayashi M, Yamada H (1988) Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl Environ Microbiol 54:1030–1032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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. Prot Express Purif 47:672–681

    Article  CAS  Google Scholar 

  • Oßwald S, Wajant H, Effenberger F (2002) Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana. Eur J Biochem 269:680–687

    Article  Google Scholar 

  • Prasad S, Misra A, Jangir VP, Awasthi A, Raj J, Bhalla TC (2007) A propionitrile-induced nitrilase of Rhodococcus sp. NDB 1165 and its application in nicotinic acid synthesis. World J Microbiol Biotechnol 23:345–353

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom APG, van Rantwijk F, van der Wielen LAM, Sheldon RA (2004) Preparation, optimization, and structures, of cross-linked enzyme aggregates (CLEAs). Biotechnol Bioeng 87:754–762

    Article  CAS  PubMed  Google Scholar 

  • Sharma NN, Sharma M, Kumar H, Bhalla TC (2006) Nocardia globerula NHB-2: bench scale production of nicotinic acid. Proc Biochem 41:2078–2081

    Article  CAS  Google Scholar 

  • Šnajdrová R, Kristová-Mylerová V, Crestia D, Nikolaou K, Kuzma M, Lemaire M, Gallienne E, Bolte J, Bezouška K, Křen V, Martínková L (2004) Nitrile biotransformation by Aspergillus niger. J Mol Catal B-Enzym 29:227–232

    Article  Google Scholar 

  • Stevenson DE, Feng R, Dumas F, Groleau D, Mihoc A, Storer AC (1992) Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnol Appl Biochem 15:283–302

    CAS  PubMed  Google Scholar 

  • Vaughan PA, Knowles CJ, Cheetham PSJ (1989) Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodochrous LL100–21. Enzyme Microb Technol 11:815–823

    Article  CAS  Google Scholar 

  • Vejvoda V, Kaplan O, Kubáč D, Křen V, Martínková L (2006) Immobilization of fungal nitrilase and bacterial amidase—two enzymes working in accord. Biocatal Biotransform 24:414–418

    Article  CAS  Google Scholar 

  • Vejvoda V, Kaplan O, Bezouška K, Pompach P, Šulc M, Cantarella M, Benada O, Uhnáková B, Rinágelová A, Lutz-Wahl S, Fischer L, Křen V, Martínková L (2008) Purification and characterization of a nitrilase from Fusarium solani O1. J Mol Catal B-Enzym 50:99–106

    Article  CAS  Google Scholar 

  • Winkler M, Kaplan O, Vejvoda V, Klempier N, Martínková L (2009) Biocatalytic application of nitrilases from Fusarium solani O1 and Aspergillus niger K10. J Mol Catal B-Enzym 59:243–247

    Article  CAS  Google Scholar 

  • Yamamoto K, Komatsu K (1991) Purification and characterization of nitrilase responsible for the enantioselective hydrolysis from Acinetobacter sp. AK 226. Agric Biol Chem 55:1459–1466

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Financial support via projects IAA500200708 (Grant Agency of the Academy of Sciences of the Czech Republic), LC06010 (Ministry of Education of the Czech Republic), COST/ESF CM0701, OC09046, FT-TA5/043 (Ministry of Education and Trade of the Czech Republic), 305/09/H008 (Czech Science Foundation), the institutional research concept AV0Z50200510 (Institute of Microbiology) and the LLP-Erasmus Fellowship to A. Malandra are gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of Chemistry, Chemical Engineering and Materials, University of L’Aquila, Monteluco di Roio, 67040, L’Aquila, Italy

    Anna Malandra & Maria Cantarella

  2. Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20, Prague, Czech Republic

    Anna Malandra, Ondřej Kaplan, Vojtěch Vejvoda, Bronislava Uhnáková, Barbora Štěpánková, David Kubáč & Ludmila Martínková

Authors
  1. Anna Malandra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Cantarella
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ondřej Kaplan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vojtěch Vejvoda
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bronislava Uhnáková
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Barbora Štěpánková
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. David Kubáč
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ludmila Martínková
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ludmila Martínková.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Malandra, A., Cantarella, M., Kaplan, O. et al. Continuous hydrolysis of 4-cyanopyridine by nitrilases from Fusarium solani O1 and Aspergillus niger K10. Appl Microbiol Biotechnol 85, 277–284 (2009). https://doi.org/10.1007/s00253-009-2073-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-009-2073-x

Keywords

Search

Navigation