ABSTRACT
Decellularized human pericardium is under study as an allogenic material for cardiovascular applications. The effects of crosslinking on the mechanical properties of decellularized pericardium were determined with a uniaxial tensile test, and the effects of crosslinking on the collagen structure of decellularized pericardium were determined by multiphoton microscopy. The viability of human umbilical vein endothelial cells seeded on decellularized human pericardium and on pericardium strongly and weakly crosslinked with glutaraldehyde and with genipin was evaluated by means of an MTS assay. The viability of the cells, measured by their metabolic activity, decreased considerably when the pericardium was crosslinked with glutaraldehyde. Conversely, the cell viability increased when the pericardium was crosslinked with genipin. Coating both non-modified pericardium and crosslinked pericardium with a fibrin mesh or with a mesh containing attached heparin and/or fibronectin led to a significant increase in cell viability. The highest degree of viability was attained for samples that were weakly crosslinked with genipin and modified by means of a fibrin and fibronectin coating. The results indicate a method by which in vivo endothelialization of human cardiac allografts or xenografts could potentially be encouraged.
Subject(s)
Biocompatible Materials , Pericardium/transplantation , Allografts , Animals , Biocompatible Materials/chemistry , Biomechanical Phenomena , Cell Survival , Collagen/chemistry , Collagen/ultrastructure , Cross-Linking Reagents , Fibrin , Fibronectins , Glutaral , Heterografts , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Iridoids , Materials Testing , Microscopy, Fluorescence, Multiphoton , Pericardium/chemistry , Pericardium/ultrastructure , Surface Plasmon Resonance , Tensile StrengthABSTRACT
Long-term performance of implanted cardiovascular grafts can be ensured if living endothelium overgrows their surface. Surface modifications to implants are therefore being sought that can encourage endothelialization while preventing thrombus formation until the natural endothelium is formed. In the present study, heparin was covalently attached to a fibrin mesh grown from a polyvinyl chloride (PVC) substrate surface by the catalytic action of surface immobilized thrombin on a fibrinogen solution. The coating prevented platelet activation, thrombin generation and clot formation, and reduced inflammatory reactions when exposed to fresh human whole blood circulating in a Chandler loop model. In addition, in vitro seeded human umbilical vein and human saphenous vein endothelial cells showed considerably enhanced attachment and proliferation on the coating. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2995-3005, 2017.
Subject(s)
Anticoagulants/chemistry , Anticoagulants/pharmacology , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacology , Heparin/chemistry , Heparin/pharmacology , Thrombosis/prevention & control , Blood Coagulation/drug effects , Blood Vessel Prosthesis/adverse effects , Cell Adhesion/drug effects , Cells, Cultured , Endothelial Cells/cytology , Endothelial Cells/drug effects , Fibrin/chemistry , Hematocrit , Human Umbilical Vein Endothelial Cells , Humans , Platelet Activation/drug effects , Saphenous Vein/cytology , Thrombosis/blood , Thrombosis/etiologyABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0163697.].
ABSTRACT
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.
Subject(s)
Escherichia coli/metabolism , Molecular Chaperones/metabolism , Recombinant Fusion Proteins/metabolism , Vascular Endothelial Growth Factor A/metabolism , Amino Acid Sequence , Cells, Cultured , Chromatography, Affinity/methods , Cloning, Molecular , Fibrin/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Plasmids/metabolism , Protein Folding , Solubility , Thioredoxins/metabolismABSTRACT
The aim of this study was to discover new nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic nitrilase, arylacetonitrilase, aliphatic nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetonitrilases and an aromatic nitrilase, respectively.
Subject(s)
Aminohydrolases/metabolism , Nitriles/metabolism , Aminohydrolases/genetics , Cloning, Molecular , Computational Biology , Data Mining , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.
Subject(s)
Amidohydrolases/metabolism , Gene Expression Regulation, Bacterial , Hydro-Lyases/metabolism , Hydroxylamines/metabolism , Nitriles/metabolism , Rhodococcus/enzymology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Gene Expression Profiling , Multigene Family , Rhodococcus/genetics , Sequence Analysis, DNA , Substrate Specificity , Transcription, GeneticABSTRACT
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.
Subject(s)
Aminohydrolases/chemistry , Genome, Fungal , Hydro-Lyases/chemistry , Aminohydrolases/genetics , Aminohydrolases/metabolism , Escherichia coli/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungi/enzymology , Fungi/genetics , Genomics , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
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.
Subject(s)
Aminohydrolases/isolation & purification , Aminohydrolases/metabolism , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Fungi/enzymology , Aminohydrolases/chemistry , Aminohydrolases/genetics , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungi/genetics , Gene Expression , Hydrogen-Ion Concentration , Kinetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , TemperatureABSTRACT
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.
Subject(s)
Aminohydrolases/metabolism , Aspergillus niger/enzymology , Bacterial Proteins/metabolism , Cloning, Molecular/methods , Recombinant Proteins/metabolism , Amino Acid Sequence , Aminohydrolases/biosynthesis , Aminohydrolases/genetics , Aminohydrolases/isolation & purification , Aspergillus niger/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , DNA, Complementary , Enzyme Stability , Light , Molecular Dynamics Simulation , Molecular Sequence Data , Polymerase Chain Reaction , Protein Folding , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Scattering, Radiation , Sequence Alignment , Sequence Analysis, DNAABSTRACT
PURPOSE OF WORK: our aim is to describe new fungal nitrilases whose sequences were published but whose catalytic properties were unknown. We adapted for expression in E. coli three of the genes and confirmed that the enzymes acted on organic nitriles. The genome mining approach was used to search for nitrilases in filamentous fungi. Synthetic genes encoding nitrilases in Aspergillus niger, Gibberella moniliformis and Neurospora crassa were expressed in Escherichia coli. This is the first heterologous expression of fungal enzymes of this type. The recombinant enzyme derived from G. moniliformis was an aromatic nitrilase with an activity of 390 U l(-1) culture with benzonitrile as substrate. This was much less than the activities of the recombinant enzymes derived from A. niger and N. crassa that had activities of 2500 and 2700 U l(-1) culture, respectively, with phenylacetonitrile as substrate.
Subject(s)
Aminohydrolases/metabolism , Aspergillus niger/enzymology , Fungal Proteins/metabolism , Genome, Fungal , Gibberella/enzymology , Neurospora crassa/enzymology , Aminohydrolases/genetics , Aspergillus niger/genetics , Cloning, Molecular , Computational Biology/methods , Escherichia coli/genetics , Fungal Proteins/genetics , Gene Expression , Gibberella/genetics , Neurospora crassa/genetics , Nitriles/metabolism , Organic Chemicals/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 degrees C with a deactivation constant (k (d)) and enzyme half-life of 0.014 h(-1) and 50 h, respectively, but the latter exhibited an even higher stability characterized by k (d) = 0.008 h(-1) and half-life of 87 h at 40 degrees C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.
Subject(s)
Aminohydrolases/metabolism , Aspergillus niger/enzymology , Fusarium/enzymology , Nitriles/metabolism , Pyridines/metabolism , Aminohydrolases/chemistry , Aminohydrolases/isolation & purification , Bioreactors , Biotransformation , Enzyme Stability , Isonicotinic Acids/metabolism , Kinetics , Substrate SpecificityABSTRACT
Of the numerous putative fungal nitrilases available from protein databases only a few enzymes were purified and characterized. The purified nitrilases from Fusarium solani, Fusarium oxysporum f. sp. melonis and Aspergillus niger share a preference for (hetero)aromatic nitriles, temperature optima between 40 and 50 degrees C and pH optima in the slightly alkaline region. On the other hand, they differ in their chemoselectivity, i.e. their tendency to produce amides as by-products. The production of fungal nitrilases is increased by up to three orders of magnitude on the addition of 2-cyanopyridine to the culture media. The whole-cell and subcellular biocatalysts were immobilized by various methods (LentiKats(R); adsorption on hydrophobic or ion exchange resins; cross-linked enzyme aggregates). Operational stability was examined using continuous stirred membrane bioreactors. Fungal nitrilases appear promising for biocatalytic applications and biodegradation of nitrile environmental contaminants.
Subject(s)
Aminohydrolases/metabolism , Fungi/enzymology , Biocatalysis , Enzyme Stability , Fungi/classification , Species Specificity , Substrate SpecificityABSTRACT
2,6-Pyridinedicarbonitrile (1a) and 2,4-pyridinedicarbonitrile (2a) were hydrated by Rhodococcus erythropolis A4 to 6-cyanopyridine-2-carboxamide (1b; 83% yield) and 2-cyanopyridine-4-carboxamide (2b; 97% yield), respectively, after 10 min. After 118 h, the intermediates 1b or 2b were transformed into 2,6-pyridinedicarboxamide (1c; 35% yield) and 2,6-pyridinedicarboxylic acid (1d; 60% yield) or 2-cyanopyridine-4-carboxylic acid (2c; 64% yield), respectively. The nitrilase from Fusarium solani afforded cyanocarboxylic acids 1e and 2c after 118 h (yields 95 and 62%, respectively). 3,4-Pyridinedicarbonitrile (3a) and 2,3-pyrazinedicarbonitrile (4a) were inferior substrates of nitrile hydratase and nitrilase.
Subject(s)
Aminohydrolases/metabolism , Fungi/enzymology , Heterocyclic Compounds/metabolism , Nitriles/metabolism , Rhodococcus/enzymology , Amides/metabolism , Biotransformation , Chromatography, High Pressure Liquid , Heterocyclic Compounds/chemistry , Hydrolysis , Nitriles/chemistry , Substrate SpecificityABSTRACT
Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg(-1)) at 45 degrees C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of D: -sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.
Subject(s)
Aminohydrolases/chemistry , Aminohydrolases/isolation & purification , Aspergillus niger/enzymology , Amino Acid Sequence , Aspergillus fumigatus/enzymology , Enzyme Stability , Hydrogen-Ion Concentration , Molecular Sequence Data , Sequence Homology, Amino Acid , Substrate Specificity , TemperatureABSTRACT
2-Cyanopyridine proved to act as a powerful nitrilase inducer in Aspergillus niger K10, Fusarium solani O1, Fusarium oxysporum CCF 1414, Fusarium oxysporum CCF 483 and Penicillium multicolor CCF 2244. Valeronitrile also enhanced the nitrilase activity in most of the strains. The highest nitrilase activities were produced by fungi cultivated in a Czapek-Dox medium with both 2-cyanopyridine and valeronitrile. The specific nitrilase activities of these cultures were two to three orders of magnitude higher than those of cultures grown on other nitriles such as 3-cyanopyridine or 4-cyanopyridine.