title: Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10.
abstract: BACKGROUND
  Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult.
  
  
  RESULTS
  A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn₃₅₆) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp₂₉₈-Val₃₁₃ peptide was shortened to Asp₂₉₈-Arg₃₁₀ in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution.
  
  
  CONCLUSIONS
  The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Kaplan
authors.forename: Karel
authors.initials: K
authors.lastname: Bezouška
authors.forename: Ondřej
authors.initials: O
authors.lastname: Plíhal
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Ettrich
authors.forename: Natallia
authors.initials: N
authors.lastname: Kulik
authors.forename: Ondřej
authors.initials: O
authors.lastname: Vaněk
authors.forename: Daniel
authors.initials: D
authors.lastname: Kavan
authors.forename: Oldřich
authors.initials: O
authors.lastname: Benada
authors.forename: Anna
authors.initials: A
authors.lastname: Malandra
authors.forename: Ondřej
authors.initials: O
authors.lastname: Sveda
authors.forename: Alicja B
authors.initials: AB
authors.lastname: Veselá
authors.forename: Anna
authors.initials: A
authors.lastname: Rinágelová
authors.forename: Kristýna
authors.initials: K
authors.lastname: Slámová
authors.forename: Maria
authors.initials: M
authors.lastname: Cantarella
authors.forename: Jürgen
authors.initials: J
authors.lastname: Felsberg
authors.forename: Jarmila
authors.initials: J
authors.lastname: Dušková
authors.forename: Jan
authors.initials: J
authors.lastname: Dohnálek
authors.forename: Michael
authors.initials: M
authors.lastname: Kotik
authors.forename: Vladimír
authors.initials: V
authors.lastname: Křen
authors.forename: Ludmila
authors.initials: L
authors.lastname: Martínková
mesh_terms: D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004795:Enzyme Stability; D008027:Light; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012542:Scattering, Radiation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase