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Purification and characterization of heterologously expressed nitrilases from filamentous fungi

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An Erratum to this article was published on 17 September 2013

Abstract

Nitrilases from Aspergillus niger CBS 513.88, A. niger K10, Gibberella moniliformis, Neurospora crassa OR74A, and Penicillium marneffei ATCC 18224 were expressed in Escherichia coli BL21-Gold (DE3) after IPTG induction. N. crassa nitrilase exhibited the highest yield of 69,000 U L−1 culture. Co-expression of chaperones (GroEL/ES in G. moniliformis and P. marneffei; GroEL/ES and trigger factor in N. crassa and A. niger CBS 513.88) enhanced the enzyme solubility. Specific activities of strains expressing the former two enzymes increased approximately fourfold upon co-expression of GroEL/ES. The enzyme from G. moniliformis (co-purified with GroEL) preferred benzonitrile as substrate (K m of 0.41 mM, V max of 9.7 μmol min−1 mg−1 protein). The P. marneffei enzyme (unstable in its purified state) exhibited the highest V max of 7.3 μmol min−1 mg−1 protein in cell-free extract, but also a high K m of 15.4 mM, for 4-cyanopyridine. The purified nitrilases from A. niger CBS 513.88 and N. crassa acted preferentially on phenylacetonitrile (K m of 3.4 and 2.0 mM, respectively; V max of 10.6 and 17.5 μmol min−1 mg−1 protein, respectively), and hydrolyzed also (R,S)-mandelonitrile with higher K m values. Significant amounts of amides were only formed by the G. moniliformis nitrilase from phenylacetonitrile and 4-cyanopyridine.

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Acknowledgments

The authors wish to thank Hynek Mrazek MSc. for his technical help with nitrilase purification. Financial support via projects P504/11/0394, 305/09/H008 (Czech Science Foundation), IAA500200708 (Grant Agency of the Academy of Sciences of the Czech Republic), LC06010, OC09046 (Ministry of Education of the Czech Republic), COST/ESF CM0701 (STSM fellowships COST-STSM-CM0701-4765 and −4766 to A. Malandra) and Institutional Research Concept AV0Z50200510 (Institute of Microbiology) is gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Vídeňská 1083, CZ-142 20, Prague, Czech Republic

    Alena Petříčková, Alicja Barbara Veselá, Ondřej Kaplan, David Kubáč, Bronislava Uhnáková, Anna Malandra, Anna Rinágelová, Philip Weyrauch, Vladimír Křen, Karel Bezouška & Ludmila Martínková

  2. Institute of Microbiology, Centre for DNA Sequencing, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20, Prague, Czech Republic

    Jürgen Felsberg

  3. Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-128 40, Prague, Czech Republic

    Alena Petříčková, Alicja Barbara Veselá & Karel Bezouška

  4. Department of Chemistry, Chemical Engineering and Materials, University of L’Aquila, Via Campo di Pile - Zona industriale di Pile, 67100, L’Aquila, Italy

    Anna Malandra

  5. Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms-University Münster, Correnstrasse 3, 48149, Münster, Germany

    Philip Weyrauch

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  1. Alena Petříčková
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Correspondence to Ludmila Martínková.

Additional information

Alena Petříčková and Alicja B. Veselá contributed equally to this work.

An erratum to this article is available at http://dx.doi.org/10.1007/s00253-013-5204-3.

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Fig. S1

Nucleotide sequences of the nitrilase genes from P. marneffei ATCC18224 (GenBank JN012233) and A. niger K10 (GenBank JN243351) adapted for expression in E. coli using GeneArt (Regensburg, Germany) software. (DOC 29 kb)

Fig. S2

Phylogenetic tree of fungal nitrilase/cyanide hydratase enzymes. The search was performed with the sequence of nitrilase from A. niger K10 (Kaplan et al. 2011a; marked with asterisks) as the query using BLASTP. Nitrilases expressed and characterized by us are highlighted (DOC 693 kb)

Fig. S3

Multiple sequence alignment using ClustalW software (Thompson et al. 1994) of nitrilases from Gibberella moniliformis (GiMon Nit; GenBank ABF83489), Neurospora crassa OR74A (NeCra Nit; GenBank CAD70472), Aspergillus niger K10 (AsNig Nit1; GenBank ABX75546; Kaplan et al. 2011a), Penicillium marneffei ATCC18224 (PeMar Nit; GenBank XP_002144951), and Aspergillus niger CBS 513.88 (AsNig Nit2; GenBank XP_001397369). Clustal co clustal consensus, asterisk identical aa residues, colon similar aa residues, dot less similar aa residues (DOC 36 kb)

Table S4

Screening of chaperone effects on nitrilase activity in E. coli BL21-Gold(DE3) strains (DOC 68.5 kb)

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Petříčková, A., Veselá, A.B., Kaplan, O. et al. Purification and characterization of heterologously expressed nitrilases from filamentous fungi. Appl Microbiol Biotechnol 93, 1553–1561 (2012). https://doi.org/10.1007/s00253-011-3525-7

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