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Special Issue "Nitrilases and Nitrile Hydratases"

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

Deadline for manuscript submissions: 30 June 2020.

Special Issue Editors

Prof. Dr. Ludmila Martínková
Website
Guest Editor
Institute of Microbiology of the Czech Academy of Sciences, Czech
Interests: biotransformation, biocatalysis; gene expression; protein purification; immobilization; nitrilases; tyrosinases; laccases
Dr. Margit Winkler
Website
Co-Guest Editor
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

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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 2000 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 (3 papers)

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Research

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Open AccessArticle
Novel Chaperones RrGroEL and RrGroES for Activity and Stability Enhancement of Nitrilase in Escherichia coli and Rhodococcus ruber
Molecules 2020, 25(4), 1002; https://doi.org/10.3390/molecules25041002 - 24 Feb 2020
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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Open AccessArticle
Substrate Profiling of the Cobalt Nitrile Hydratase from Rhodococcus rhodochrous ATCC BAA 870
Molecules 2020, 25(1), 238; https://doi.org/10.3390/molecules25010238 - 06 Jan 2020
Cited by 1
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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Open AccessReview
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
Molecules 2019, 24(23), 4232; https://doi.org/10.3390/molecules24234232 - 21 Nov 2019
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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