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[RETRACTED] Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10
O. Kaplan, K. Bezouška, O. Plíhal, R. Ettrich, N. Kulik, O. Vaněk, D. Kavan, O. Benada, A. Malandra, O. Sveda, AB. Veselá, A. Rinágelová, K. Slámová, M. Cantarella, J. Felsberg, J. Dušková, J. Dohnálek, M. Kotik, V. Křen, L. Martínková
Status odvoláno Jazyk angličtina Země Anglie, Velká Británie
Typ dokumentu časopisecké články, práce podpořená grantem, publikace stažené z tisku
NLK
BioMedCentral
od 2001-12-01
BioMedCentral Open Access
od 2001
Directory of Open Access Journals
od 2000
Free Medical Journals
od 2001
PubMed Central
od 2001
Europe PubMed Central
od 2001
ProQuest Central
od 2009-01-01
Open Access Digital Library
od 2000-01-01
Open Access Digital Library
od 2001-01-01
Open Access Digital Library
od 2001-04-01
Medline Complete (EBSCOhost)
od 2001-01-01
Health & Medicine (ProQuest)
od 2009-01-01
ROAD: Directory of Open Access Scholarly Resources
od 2001
Springer Nature OA/Free Journals
od 2001-12-01
PubMed
21210990
DOI
10.1186/1472-6750-11-2
Knihovny.cz E-zdroje
- MeSH
- aminohydrolasy biosyntéza genetika izolace a purifikace metabolismus MeSH
- Aspergillus niger enzymologie genetika MeSH
- bakteriální proteiny genetika izolace a purifikace metabolismus MeSH
- klonování DNA metody MeSH
- komplementární DNA MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce MeSH
- radiační rozptyl MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- sbalování proteinů MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza DNA MeSH
- sekvenční seřazení MeSH
- simulace molekulární dynamiky MeSH
- stabilita enzymů MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- publikace stažené z tisku MeSH
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.
Department of Biochemistry Faculty of Science Charles University Prague
Department of Chemistry Chemical Engineering and Materials University of L'Aquila
Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic
Institute of Microbiology Academy of Sciences of the Czech Republic
Institute of Microbiology Academy of Sciences of the Czech Republic Prague Czech Republic
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