Heterologous expression, purification and characterization of nitrilase from Aspergillus nigerK10

BMC Biotechnology

Research Article

Natallia Kulik¹  Rüdiger Ettrich¹  Maria Cantarella²  Jan Dohnálek³  Jarmila Dušková³  Ondřej Kaplan⁴  Ondřej Šveda⁴  Michael Kotik⁴  Oldřich Benada⁴  Vladimír Křen⁴  Kristýna Slámová⁴  Jürgen Felsberg⁴  Ludmila Martínková⁴  Anna Rinágelová⁴  Alicja B Veselá⁴  Ondřej Plíhal⁴  Karel Bezouška⁵  Daniel Kavan⁵  Ondřej Vaněk⁵  Anna Malandra⁶ 

[1] Centre of Biocatalysis and Biotransformation, Institute of Systems Biology and Ecology, Academy of Sciences of the Czech Republic, Zámek 136, 373 33, Nové Hrady, Czech Republic;Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, Via Campo di Pile - Zona industriale di Pile, I-67100, L'Aquila, Italy;Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského náměstí 2, CZ-162 06, Prague, Czech Republic;Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20, Prague, Czech Republic;Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20, Prague, Czech Republic;Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-128 40, Prague, Czech Republic;Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20, Prague, Czech Republic;Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, Via Campo di Pile - Zona industriale di Pile, I-67100, L'Aquila, Italy;

关键词: Benzonitrile;    Cyanide Hydratases;    Nitrilase Activity;    Nitrilase Gene;    Picolinamide;   

DOI  :  10.1186/1472-6750-11-2

 received in 2010-02-23, accepted in 2011-01-06,  发布年份 2011

来源: Springer

【 摘 要 】

BackgroundNitrilases 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.ResultsA 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.ConclusionsThe 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.

【 授权许可】

Unknown   
© Kaplan et al; licensee BioMed Central Ltd. 2011. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.