Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from Trametes versicolor and NitAb from Agaricus bisporus were purified and characterized as the representatives of this type of fungal NLase. Both enzymes were slightly more similar to NIT4 type than to NIT1/NIT2/NIT3 type of plant NLases in terms of their amino acid sequences. Expression of the synthetic genes in Escherichia coli Origami B (DE3) was induced with 0.02 mM isopropyl β-D-1-thiogalactopyranoside at 20 °C. Purification of NitTv1 and NitAb by cobalt affinity chromatography gave ca. 6.6 mg and 9.6 mg of protein per 100 mL of culture medium, respectively. Their activities were determined with 25 mM of nitriles in 50 mM Tris/HCl buffer, pH 8.0, at 30 °C. NitTv1 and NitAb transformed β-cyano-L-alanine (β-CA) with the highest specific activities (ca. 132 and 40 U mg-1, respectively) similar to plant NLase NIT4. β-CA was transformed into Asn and Asp as in NIT4 but at lower Asn:Asp ratios. The fungal NLases also exhibited significant activities for (aryl)aliphatic nitriles such as 3-phenylpropionitrile, cinnamonitrile and fumaronitrile (substrates of NLase NIT1). NitTv1 was more stable than NitAb (at pH 5-9 vs. pH 5-7). These NLases may participate in plant-fungus interactions by detoxifying plant nitriles and/or producing plant hormones. Their homology models elucidated the molecular interactions with various nitriles in their active sites.

• Klíčová slova
• Agaricus bisporus, Trametes versicolor, arylaliphatic nitriles, fumaronitrile, fungi, homology modeling, plant nitrilase homologues, plant-fungus interactions, substrate specificity, β-cyano-L-alanine,
• MeSH
• Agaricus * enzymologie   genetika   MeSH
• aminohydrolasy * genetika   metabolismus   MeSH
• asparagin genetika   metabolismus   MeSH
• fungální proteiny * genetika   metabolismus   MeSH
• fylogeneze * MeSH
• kyselina asparagová genetika   metabolismus   MeSH
• Polyporaceae enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminohydrolasy * MeSH
• asparagin MeSH
• fungální proteiny * MeSH
• kyselina asparagová MeSH
• nitrilase MeSH Prohlížeč

The aim of this study is to review the current state of and highlight the challenges in the production of microbial nitrilases as catalysts for the mild hydrolysis of industrially important nitriles. Together with aldoxime dehydratase, the nitrile-hydrolyzing enzymes (nitrilase, nitrile hydratase) are key enzymes in the aldoxime-nitrile pathway which is widely distributed in bacteria and fungi. The availability of nitrilases has grown significantly over the past decade due to the use of metagenomic and database-mining approaches. Databases contain plenty of putative enzymes of this type, whose overproduction may improve the spectrum and the industrial utility of nitrilases. By exploiting this resource, the number of experimentally verified nitrilases has recently increased to several hundred. We especially focus on the efficient heterologous expression systems that are applicable for the overproduction of wild-type nitrilases and their artificial variants. Biocatalyst forms with industrial potential are also highlighted. The potential industrial applications of nitrilases are classified according to their target products (α-hydroxy acids, α- and β-amino acids, cyano acids, amides). The emerging uses of nitrilases and their subtypes (cyanide hydratases, cyanide dihydratases) in bioremediation is also summarized. The integration of nitrilases with other enzymes into artificial multienzymatic and chemoenzymatic pathways is considered a promising strategy for future applications.

• Klíčová slova
• Aldoxime–nitrile pathway, Biocatalytic applications, Database mining, Heterologous production, Metagenome mining, Nitrilase,
• MeSH
• aminohydrolasy genetika   metabolismus   MeSH
• Bacteria enzymologie   genetika   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• biodegradace MeSH
• biokatalýza MeSH
• databáze proteinů MeSH
• fungální proteiny genetika   metabolismus   MeSH
• houby enzymologie   genetika   MeSH
• metagenomika MeSH
• nitrily metabolismus   MeSH
• proteinové inženýrství metody   MeSH
• rekombinantní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• aminohydrolasy MeSH
• bakteriální proteiny MeSH
• fungální proteiny MeSH
• nitrilase MeSH Prohlížeč
• nitrily MeSH
• rekombinantní proteiny MeSH

We describe the production of a highly-active mutant VEGF variant, α2-PI1-8-VEGF121, which contains a substrate sequence for factor XIIIa at the aminoterminus designed for incorporation into a fibrin gel. The α2-PI1-8-VEGF121 gene was synthesized, cloned into a pET-32a(+) vector and expressed in Escherichia coli Origami B (DE3) host cells. To increase the protein folding and the solubility, the resulting thioredoxin-α2-PI1-8-VEGF121 fusion protein was co-expressed with recombinant molecular chaperones GroES/EL encoded by independent plasmid pGro7. The fusion protein was purified from the soluble fraction of cytoplasmic proteins using affinity chromatography. After cleavage of the thioredoxin fusion part with thrombin, the target protein was purified by a second round of affinity chromatography. The yield of purified α2-PI1-8-VEGF121 was 1.4 mg per liter of the cell culture. The α2-PI1-8-VEGF121 expressed in this work increased the proliferation of endothelial cells 3.9-8.7 times in comparison with commercially-available recombinant VEGF121. This very high mitogenic activity may be caused by co-expression of the growth factor with molecular chaperones not previously used in VEGF production. At the same time, α2-PI1-8-VEGF121 did not elicit considerable inflammatory activation of human endothelial HUVEC cells and human monocyte-like THP-1 cells.

• MeSH
• chromatografie afinitní metody   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• Escherichia coli metabolismus   MeSH
• fibrin metabolismus   MeSH
• klonování DNA MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární chaperony metabolismus   MeSH
• plazmidy metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• rozpustnost MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• thioredoxiny metabolismus   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fibrin MeSH
• molekulární chaperony MeSH
• rekombinantní fúzní proteiny MeSH
• thioredoxiny MeSH
• vaskulární endoteliální růstový faktor A MeSH
• VEGFA protein, human MeSH Prohlížeč

The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.

• MeSH
• aminohydrolasy chemie   genetika   metabolismus   MeSH
• Arthrodermataceae enzymologie   MeSH
• Aspergillus niger enzymologie   MeSH
• druhová specificita MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• fylogeneze MeSH
• koncentrace vodíkových iontů MeSH
• kyseliny mandlové metabolismus   MeSH
• Nectria enzymologie   MeSH
• Neurospora crassa enzymologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminohydrolasy MeSH
• fungální proteiny MeSH
• kyseliny mandlové MeSH
• mandelic acid MeSH Prohlížeč

Escherichia coli strains expressing different nitrilases transformed nitriles or KCN. Six nitrilases (from Aspergillus niger (2), A. oryzae, Neurospora crassa, Arthroderma benhamiae, and Nectria haematococca) were arylacetonitrilases, two enzymes (from A. niger and Penicillium chrysogenum) were cyanide hydratases and the others (from P. chrysogenum, P. marneffei, Gibberella moniliformis, Meyerozyma guilliermondi, Rhodococcus rhodochrous, and R. ruber) preferred (hetero)aromatic nitriles as substrates. Promising nitrilases for the transformation of industrially important substrates were found: the nitrilase from R. ruber for 3-cyanopyridine, 4-cyanopyridine and bromoxynil, the nitrilases from N. crassa and A. niger for (R,S)-mandelonitrile, and the cyanide hydratase from A. niger for KCN and 2-cyanopyridine.

• MeSH
• aminohydrolasy chemie   genetika   metabolismus   MeSH
• dehydratasy chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• fungální proteiny chemie   genetika   metabolismus   MeSH
• genom fungální * MeSH
• genomika MeSH
• houby enzymologie   genetika   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aminohydrolasy MeSH
• cyanide hydratase MeSH Prohlížeč
• dehydratasy MeSH
• fungální proteiny MeSH
• nitrilase MeSH Prohlížeč
• rekombinantní proteiny MeSH