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Nitrilases and Nitrile Hydratases

A special issue of Molecules (ISSN 1420-3049).

Deadline for manuscript submissions: closed (30 June 2020) | Viewed by 21062

Special Issue Editors

Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic
Interests: biotransformation; biocatalysis; gene expression; protein purification; immobilization; nitrilases; tyrosinases; laccases
Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria
Interests: biocatalysis; organic chemistry; protein overproduction; enzymology; nitrilases; nitrile hydratases; carboxylate reductases; hydroxynitrile lyases; CYP and non-CYP drug metabolizing enzymes; formation and biotransformation of nitriles; multistep reactions

Special Issue Information

Dear Colleagues,

The biotechnological impact of nitrilases and nitrile hydratases has become widely acknowledged since their discovery a few decades ago. The past 10 years or so have witnessed a tremendous increase in the number of the biochemically characterized enzymes of these types, but also in the number of sequences coding for their putative homologs. The long-lasting trend in the investigation of these enzymes is their improvement towards higher activities, selectivities, and stabilities, as well as exploring new resources of enzymes. Additionally, new biocatalytic uses are being constantly identified. All these approaches are necessary for a more intensive exploitation of the enzymes in the production of fine chemicals for the chemical, pharmaceutical, and food industries. This Special Issue will collect contributions on enhancing the biocatalytic potential of nitrilases and nitrile hydratases through, e.g., protein engineering, genome mining, metagenomic libraries screening, and new substrate and/or product identification.

We welcome submissions meeting the following criteria: (i) clear novelty; (ii) reproducibility; (iii) use of defined organisms and enzymes; (iv) focus on molecular aspects of the reactions. Papers which merely optimize known procedures or fail to clearly define the enzymes, substrates, and products will not be considered for publication. Publishing primary sequences of new enzymes is strongly encouraged.

Prof. Dr. Ludmila Martínková
Dr. Margit Winkler
Guest Editors

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Molecules is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Nitrilase
  • Nitrile hydratase
  • Genome mining
  • Metagenomic libraries
  • Protein engineering
  • Biocatalysis

Published Papers (6 papers)

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Research

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18 pages, 6644 KiB  
Article
Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability
by Zhongyi Cheng, Yao Lan, Junling Guo, Dong Ma, Shijin Jiang, Qianpeng Lai, Zhemin Zhou and Lukasz Peplowski
Molecules 2020, 25(20), 4806; https://doi.org/10.3390/molecules25204806 - 19 Oct 2020
Cited by 21 | Viewed by 2884
Abstract
High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction [...] Read more.
High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant. Full article
(This article belongs to the Special Issue Nitrilases and Nitrile Hydratases)
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20 pages, 3819 KiB  
Article
Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus
by Lenka Rucká, Natalia Kulik, Petr Novotný, Anastasia Sedova, Lucie Petrásková, Romana Příhodová, Barbora Křístková, Petr Halada, Miroslav Pátek and Ludmila Martínková
Molecules 2020, 25(17), 3861; https://doi.org/10.3390/molecules25173861 - 25 Aug 2020
Cited by 5 | Viewed by 2124
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Nitrilases and Nitrile Hydratases)
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11 pages, 1592 KiB  
Article
Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases
by Birgit Grill, Maximilian Glänzer, Helmut Schwab, Kerstin Steiner, Daniel Pienaar, Dean Brady, Kai Donsbach and Margit Winkler
Molecules 2020, 25(11), 2521; https://doi.org/10.3390/molecules25112521 - 28 May 2020
Cited by 3 | Viewed by 2330
Abstract
Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 [...] Read more.
Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were—in addition—able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts. Full article
(This article belongs to the Special Issue Nitrilases and Nitrile Hydratases)
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14 pages, 2476 KiB  
Article
Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber
by Chunmeng Xu, Lingjun Tang, Youxiang Liang, Song Jiao, Huimin Yu and Hui Luo
Molecules 2020, 25(4), 1002; https://doi.org/10.3390/molecules25041002 - 24 Feb 2020
Cited by 11 | Viewed by 3471
Abstract
For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus [...] Read more.
For large-scale bioproduction, thermal stability is a crucial property for most industrial enzymes. A new method to improve both the thermal stability and activity of enzymes is of great significance. In this work, the novel chaperones RrGroEL and RrGroES from Rhodococcus ruber, a nontypical actinomycete with high organic solvent tolerance, were evaluated and applied for thermal stability and activity enhancement of a model enzyme, nitrilase. Two expression strategies, namely, fusion expression and co-expression, were compared in two different hosts, E. coli and R. ruber. In the E. coli host, fusion expression of nitrilase with either RrGroES or RrGroEL significantly enhanced nitrilase thermal stability (4.8-fold and 10.6-fold, respectively) but at the expense of enzyme activity (32–47% reduction). The co-expression strategy was applied in R. ruber via either a plasmid-only or genome-plus-plasmid method. Through integration of the nitrilase gene into the R. ruber genome at the site of nitrile hydratase (NHase) gene via CRISPR/Cas9 technology and overexpression of RrGroES or RrGroEL with a plasmid, the engineered strains R. ruber TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroES) and TH3 dNHase::RrNit (pNV18.1-Pami-RrNit-Pami-RrGroEL) were constructed and showed remarkably enhanced nitrilase activity and thermal stability. In particular, the RrGroEL and nitrilase co-expressing mutant showed the best performance, with nitrilase activity and thermal stability 1.3- and 8.4-fold greater than that of the control TH3 (pNV18.1-Pami-RrNit), respectively. These findings are of great value for production of diverse chemicals using free bacterial cells as biocatalysts. Full article
(This article belongs to the Special Issue Nitrilases and Nitrile Hydratases)
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15 pages, 4164 KiB  
Article
Substrate Profiling of the Cobalt Nitrile Hydratase from Rhodococcus rhodochrous ATCC BAA 870
by Adelaide R. Mashweu, Varsha P. Chhiba-Govindjee, Moira L. Bode and Dean Brady
Molecules 2020, 25(1), 238; https://doi.org/10.3390/molecules25010238 - 06 Jan 2020
Cited by 16 | Viewed by 6659
Abstract
The aromatic substrate profile of the cobalt nitrile hydratase from Rhodococcus rhodochrous ATCC BAA 870 was evaluated against a wide range of nitrile containing compounds (>60). To determine the substrate limits of this enzyme, compounds ranging in size from small (90 Da) to [...] Read more.
The aromatic substrate profile of the cobalt nitrile hydratase from Rhodococcus rhodochrous ATCC BAA 870 was evaluated against a wide range of nitrile containing compounds (>60). To determine the substrate limits of this enzyme, compounds ranging in size from small (90 Da) to large (325 Da) were evaluated. Larger compounds included those with a bi-aryl axis, prepared by the Suzuki coupling reaction, Morita–Baylis–Hillman adducts, heteroatom-linked diarylpyridines prepared by Buchwald–Hartwig cross-coupling reactions and imidazo[1,2-a]pyridines prepared by the Groebke–Blackburn–Bienaymé multicomponent reaction. The enzyme active site was moderately accommodating, accepting almost all of the small aromatic nitriles, the diarylpyridines and most of the bi-aryl compounds and Morita–Baylis–Hillman products but not the Groebke–Blackburn–Bienaymé products. Nitrile conversion was influenced by steric hindrance around the cyano group, the presence of electron donating groups (e.g., methoxy) on the aromatic ring, and the overall size of the compound. Full article
(This article belongs to the Special Issue Nitrilases and Nitrile Hydratases)
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Review

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21 pages, 5815 KiB  
Review
Comparative Analysis of the Conversion of Mandelonitrile and 2-Phenylpropionitrile by a Large Set of Variants Generated from a Nitrilase Originating from Pseudomonas fluorescens EBC191
by Andreas Stolz, Erik Eppinger, Olga Sosedov and Christoph Kiziak
Molecules 2019, 24(23), 4232; https://doi.org/10.3390/molecules24234232 - 21 Nov 2019
Cited by 12 | Viewed by 2669
Abstract
The arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts [...] Read more.
The arylacetonitrilase from the bacterium Pseudomonas fluorescens EBC191 has been intensively studied as a model to understand the molecular basis for the substrate-, reaction-, and enantioselectivity of nitrilases. The nitrilase converts various aromatic and aliphatic nitriles to the corresponding acids and varying amounts of the corresponding amides. The enzyme has been analysed by site-specific mutagenesis and more than 50 different variants have been generated and analysed for the conversion of (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile. These comparative analyses demonstrated that single point mutations are sufficient to generate enzyme variants which hydrolyse (R,S)-mandelonitrile to (R)-mandelic acid with an enantiomeric excess (ee) of 91% or to (S)-mandelic acid with an ee-value of 47%. The conversion of (R,S)-2-phenylpropionitrile by different nitrilase variants resulted in the formation of either (S)- or (R)-2-phenylpropionic acid with ee-values up to about 80%. Furthermore, the amounts of amides that are produced from (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile could be changed by single point mutations between 2%–94% and <0.2%–73%, respectively. The present study attempted to collect and compare the results obtained during our previous work, and to obtain additional general information about the relationship of the amide forming capacity of nitrilases and the enantiomeric composition of the products. Full article
(This article belongs to the Special Issue Nitrilases and Nitrile Hydratases)
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