Lignins are the most abundant biopolymers that consist of aromatic units. Lignins are obtained by fractionation of lignocellulose in the form of "technical lignins". The depolymerization (conversion) of lignin and the treatment of depolymerized lignin are challenging processes due to the complexity and resistance of lignins. Progress toward mild work-up of lignins has been discussed in numerous reviews. The next step in the valorization of lignin is the conversion of lignin-based monomers, which are limited in number, into a wider range of bulk and fine chemicals. These reactions may need chemicals, catalysts, solvents, or energy from fossil resources. This is counterintuitive to green, sustainable chemistry. Therefore, in this review, we focus on biocatalyzed reactions of lignin monomers, e.g., vanillin, vanillic acid, syringaldehyde, guaiacols, (iso)eugenol, ferulic acid, p-coumaric acid, and alkylphenols. For each monomer, its production from lignin or lignocellulose is summarized, and, mainly, its biotransformations that provide useful chemicals are discussed. The technological maturity of these processes is characterized based on, e.g., scale, volumetric productivities, or isolated yields. The biocatalyzed reactions are compared with their chemically catalyzed counterparts if the latter are available.
Nitrilases participate in the nitrile metabolism in microbes and plants. They are widely used to produce carboxylic acids from nitriles. Nitrilases were described in bacteria, Ascomycota and plants. However, they remain unexplored in Basidiomycota. Yet more than 200 putative nitrilases are found in this division via GenBank. The majority of them occur in the subdivision Agaricomycotina. In this work, we analyzed their sequences and classified them into phylogenetic clades. Members of clade 1 (61 proteins) and 2 (25 proteins) are similar to plant nitrilases and nitrilases from Ascomycota, respectively, with sequence identities of around 50%. The searches also identified five putative cyanide hydratases (CynHs). Representatives of clade 1 and 2 (NitTv1 from Trametes versicolor and NitAg from Armillaria gallica, respectively) and a putative CynH (NitSh from Stereum hirsutum) were overproduced in Escherichia coli. The substrates of NitTv1 were fumaronitrile, 3-phenylpropionitrile, β-cyano-l-alanine and 4-cyanopyridine, and those of NitSh were hydrogen cyanide (HCN), 2-cyanopyridine, fumaronitrile and benzonitrile. NitAg only exhibited activities for HCN and fumaronitrile. The substrate specificities of these nitrilases were largely in accordance with substrate docking in their homology models. The phylogenetic distribution of each type of nitrilase was determined for the first time.
- MeSH
- aminohydrolasy chemie genetika metabolismus MeSH
- Basidiomycota klasifikace enzymologie genetika MeSH
- fumaráty metabolismus MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- fylogeneze MeSH
- kyanovodík metabolismus MeSH
- pyridiny metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Almost 100 genes within the genus Bradyrhizobium are known to potentially encode aldoxime dehydratases (Oxds), but none of the corresponding proteins have been characterized yet. Aldoximes are natural substances involved in plant defense and auxin synthesis, and Oxds are components of enzymatic cascades enabling bacteria to transform, utilize and detoxify them. The aim of this work was to characterize a representative of the highly conserved Oxds in Bradyrhizobium spp. which include both plant symbionts and members of the soil communities. The selected oxd gene from Bradyrhizobium sp. LTSPM299 was expressed in Escherichia coli, and the corresponding gene product (OxdBr1; GenBank: WP_044589203) was obtained as an N-His6-tagged protein (monomer, 40.7 kDa) with 30-47% identity to Oxds characterized previously. OxdBr1 was most stable at pH ca. 7.0-8.0 and at up to 30 °C. As substrates, the enzyme acted on (aryl)aliphatic aldoximes such as E/Z-phenylacetaldoxime, E/Z-2-phenylpropionaldoxime, E/Z-3-phenylpropionaldoxime, E/Z-indole-3-acetaldoxime, E/Z-propionaldoxime, E/Z-butyraldoxime, E/Z-valeraldoxime and E/Z-isovaleraldoxime. Some of the reaction products of OxdBr1 are substrates of nitrilases occurring in the same genus. Regions upstream of the oxd gene contained genes encoding a putative aliphatic nitrilase and its transcriptional activator, indicating the participation of OxdBr1 in the metabolic route from aldoximes to carboxylic acids.
The aim of this study is to summarize the current progress in the design of biocatalytic processes applicable for the production of optically pure mandelic acids and their analogues. These compounds are used as building blocks for pharmaceutical chemistry and as chiral resolving agents. Their enzymatic syntheses mainly employed nitrile hydrolysis with nitrilases, ester hydrolysis, ammonolysis or esterification with lipases or esterases, and ketone reduction or alcohol oxidation with dehydrogenases. Each of these methods will be characterized in terms of its product concentrations, enantioselectivities, and the types of catalysts used. This review will focus on the dynamic kinetic resolution of mandelonitrile and analogues by nitrilases resulting in the production of high concentrations of (R)-mandelic acid or (R)-2-chloromandelic acid with excellent e.e. Currently, there is no comparable process for (S)-mandelic acids. However, the coupling of the S-selective cyanation of benzaldehyde with the enantioretentive hydrolysis of (S)-mandelonitrile thus obtained is a promising strategy. The major product can be changed from (S)-acid to (S)-amide using nitrilase mutants. The competitiveness of the biocatalytic and chemical processes will be assessed. This review covers the literature published within 2003-2017.
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.
- 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
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.
- MeSH
- aminohydrolasy genetika metabolismus MeSH
- data mining MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- klonování DNA MeSH
- nitrily metabolismus MeSH
- rekombinantní proteiny izolace a purifikace metabolismus MeSH
- substrátová specifita MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this study was to design an effective method for the bioremediation of coking wastewaters, specifically for the concurrent elimination of their highly toxic components - cyanide and phenols. Almost full degradation of free cyanide (0.32-20 mM; 8.3-520 mg L(-1)) in the model and the real coking wastewaters was achieved by using a recombinant cyanide hydratase in the first step. The removal of cyanide, a strong inhibitor of tyrosinase, enabled an effective degradation of phenols by this enzyme in the second step. Phenol (16.5 mM, 1,552 mg L(-1)) was completely removed from a real coking wastewater within 20 h and cresols (5.0 mM, 540 mg L(-1)) were removed by 66% under the same conditions. The integration of cyanide hydratase and tyrosinase open up new possibilities for the bioremediation of wastewaters with complex pollution.
- MeSH
- fenol metabolismus MeSH
- fenoly metabolismus MeSH
- koks MeSH
- kyanidy metabolismus MeSH
- odpadní voda * MeSH
- tyrosinasa MeSH
- Publikační typ
- časopisecké články MeSH
Tyrosinases act in the development of organoleptic properties of tea, raisins, etc., but also cause unwanted browning of fruits, vegetables, and mushrooms. The tyrosinase from Agaricus bisporus has been used as a model to study tyrosinase inhibitors, which are also indispensable in the treatment of skin pigmentation disorders. However, this model has disadvantages such as side enzyme activities and the presence of multiple isoenzymes. Therefore, we aimed to introduce a new tyrosinase model. The pro-tyrosinase from Polyporus arcularius was overproduced in Escherichia coli. Trypsin digestion led to a cleavage after R388 and hence enzyme activation. The tyrosinase was a homodimer and transformed L-DOPA and tert-butylcatechol preferentially. Various aurons were examined as effectors of this enzyme. 2'- and 3'-hydroxyaurones acted as its activators and 2',4'-dihydroxyaurone as an inhibitor, whereas 4'-hydroxyaurones were its substrates. The enzyme is a promising model for tyrosinase effector studies, being a single isoenzyme and void of side enzyme activities.
- MeSH
- benzofurany metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- kinetika MeSH
- Polyporus enzymologie MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- tyrosinasa chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The purpose of this study is to summarize the current knowledge of the enzymes which are involved in the hydrolysis of cyanide, i.e., cyanide hydratases (CHTs; EC 4.2.1.66) and cyanide dihydratases (CynD; EC 3.5.5.1). CHTs are probably exclusively produced by filamentous fungi and widely occur in these organisms; in contrast, CynDs were only found in a few bacterial genera. CHTs differ from CynDs in their reaction products (formamide vs. formic acid and ammonia, respectively). Several CHTs were also found to transform nitriles but with lower relative activities compared to HCN. Mutants of CynDs and CHTs were constructed to study the structure-activity relationships in these enzymes or to improve their catalytic properties. The effect of the C-terminal part of the protein on the enzyme activity was determined by constructing the corresponding deletion mutants. CynDs are less active at alkaline pH than CHTs. To improve its bioremediation potential, CynD from Bacillus pumilus was engineered by directed evolution combined with site-directed mutagenesis, and its operation at pH 10 was thus enabled. Some of the enzymes have been tested for their potential to eliminate cyanide from cyanide-containing wastewaters. CynDs were also used to construct cyanide biosensors.
- MeSH
- Bacteria enzymologie MeSH
- biosenzitivní techniky * MeSH
- biotransformace MeSH
- dehydratasy chemie genetika metabolismus MeSH
- houby enzymologie MeSH
- hydrolasy chemie genetika metabolismus MeSH
- hydrolýza MeSH
- koncentrace vodíkových iontů MeSH
- kyanidy analýza metabolismus MeSH
- látky znečišťující životní prostředí analýza metabolismus MeSH
- mutační analýza DNA MeSH
- mutantní proteiny genetika metabolismus MeSH
- proteinové inženýrství MeSH
- stabilita enzymů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
