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Special Issue "Molecular Biocatalysis 2.0"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: 7 March 2020.

Special Issue Editors

Prof. Dr. Vladimír Křen
E-Mail Website
Guest Editor
Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Biotransformation, National Centre of Biocatalysis and Biotransformation, Videnska 1083, CZ 142 20 Praha 4, Czech Republic
Interests: Biocatalysis and biotransformation; immobilized microbial cells, their use in production and biotransformation of natural products; biotransformation of natural products by enzymes and microorganisms; preparation of glycosidases of microbial origin and their use for glycosylation of natural compounds: glycoconjugates, multivalent compounds, ergot alkaloids, flavonoids, antioxidants and chemoprotectants
Special Issues and Collections in MDPI journals
Dr. Pavla Bojarová
E-Mail Website
Guest Editor
Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Biotransformation, National Centre of Biocatalysis and Biotransformation, Videnska 1083, CZ 142 20 Praha 4, Czech Republic
Interests: biocatalysis and biotransformation by enzymes and whole cells; glycosidases; glycosyltransferases; sulfatases and sulfotransferases; nitrilases; site-directed mutagenesis and directed evolution of enzymes; engineering of enzymatic reactions; enzymatic synthesis of oligosaccharides; glycoconjugates; glycobiomaterials; glycopolymers; enzyme inhibitors and time-dependent inactivators; analysis of enzyme active site

Special Issue Information

Dear colleagues,

For over a century, biocatalysis has been a prominent area of biotechnology. This year, its utmost importance has been highlighted by the award of the Nobel Prize in Chemistry to Frances H. Arnold for the directed evolution of enzymes. Advanced enzymatic synthesis performed by cascades of tailored catalysts or by programmed whole-cell factories can yield specific products with astonishing bioactivities. The strategy for designing these new platforms involves the analysis of enzymatic systems in vivo, the development and discovery of new biocatalysts to fill the enzyme toolbox for chemistry, and their in vitro assembly into novel synthetic reaction pathways. These tasks are highly multidisciplinary, covering microbiology, protein chemistry, molecular biology, bioinformatics, and data mining, as well as chemistry of acquired products. Topics for this Special Issue include the discovery of new biocatalysts, the biocatalyst engineering for alteration and optimization of their function, use of site-directed mutagenesis and directed evolution for the construction of novel catalytic tools, biocatalytic preparation of new bioactive compounds, cascade, one-pot, and telescoping reactions, biotechnologically attractive enzyme reactions.

Major requirements for papers submitted to this Special Issue are (i) clear novelty; (ii) reproducibility; (iii) molecular bases of reactions and processes; and (iv) defined chemical reactions and structures. Publication of primary sequences of respective proteins used is strongly encouraged. Papers dealing with minor optimizations of known procedures, those using poorly defined organisms or organisms unavailable to other researchers or containing poorly defined substrates and products will be returned without further review.

Prof. Dr. Vladimír Křen
Dr. Pavla Bojarová
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. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. 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

  • Biocatalyst discovery
  • Biocatalyst development, design and engineering
  • Cofactor regeneration and multienzyme processes
  • Fundamentals of biocatalysis
  • New solvents and special processes for biocatalysis
  • Integration of biocatalysis into chemical processes

Published Papers (7 papers)

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Research

Open AccessArticle
Engineered Glucose Oxidase Capable of Quasi-Direct Electron Transfer after a Quick-and-Easy Modification with a Mediator
Int. J. Mol. Sci. 2020, 21(3), 1137; https://doi.org/10.3390/ijms21031137 - 08 Feb 2020
Abstract
Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the [...] Read more.
Glucose oxidase (GOx) has been widely utilized for monitoring glycemic levels due to its availability, high activity, and specificity toward glucose. Among the three generations of electrochemical glucose sensor principles, direct electron transfer (DET)-based third-generation sensors are considered the ideal principle since the measurements can be carried out in the absence of a free redox mediator in the solution without the impact of oxygen and at a low enough potential for amperometric measurement to avoid the effect of electrochemically active interferences. However, natural GOx is not capable of DET. Therefore, a simple and rapid strategy to create DET-capable GOx is desired. In this study, we designed engineered GOx, which was made readily available for single-step modification with a redox mediator (phenazine ethosulfate, PES) on its surface via a lysine residue rationally introduced into the enzyme. Thus, PES-modified engineered GOx showed a quasi-DET response upon the addition of glucose. This strategy and the obtained results will contribute to the further development of quasi-DET GOx-based glucose monitoring dedicated to precise and accurate glycemic control for diabetic patient care. Full article
(This article belongs to the Special Issue Molecular Biocatalysis 2.0)
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Open AccessArticle
Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation
Int. J. Mol. Sci. 2019, 20(24), 6181; https://doi.org/10.3390/ijms20246181 - 07 Dec 2019
Abstract
Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. [...] Read more.
Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure–activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data. Full article
(This article belongs to the Special Issue Molecular Biocatalysis 2.0)
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Open AccessArticle
Structure-Based Redesign of a Self-Sufficient Flavin-Containing Monooxygenase towards Indigo Production
Int. J. Mol. Sci. 2019, 20(24), 6148; https://doi.org/10.3390/ijms20246148 - 05 Dec 2019
Abstract
Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this [...] Read more.
Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this paper we show that the bacterial mFMO can be adapted to improve its ability to convert indole into indigo. The improvement was achieved by a combination of computational and structure-inspired enzyme redesign. We showed that the thermostability and the kcat for indole could be improved 1.5-fold by screening a relatively small number of enzyme mutants. This project not only resulted in an improved biocatalyst but also provided an improved understanding of the structural elements that determine the activity of mFMO and provides hints for further improvement of the monooxygenase as biocatalyst. Full article
(This article belongs to the Special Issue Molecular Biocatalysis 2.0)
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Open AccessArticle
Genetic and Functional Diversity of Nitrilases in Agaricomycotina
Int. J. Mol. Sci. 2019, 20(23), 5990; https://doi.org/10.3390/ijms20235990 - 28 Nov 2019
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Molecular Biocatalysis 2.0)
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Open AccessArticle
Key Factors for a One-Pot Enzyme Cascade Synthesis of High Molecular Weight Hyaluronic Acid
Int. J. Mol. Sci. 2019, 20(22), 5664; https://doi.org/10.3390/ijms20225664 - 12 Nov 2019
Abstract
In the last decades, interest in medical or cosmetic applications of hyaluronic acid (HA) has increased. Size and dispersity are key characteristics of biological function. In contrast to extraction from animal tissue or bacterial fermentation, enzymatic in vitro synthesis is the choice to [...] Read more.
In the last decades, interest in medical or cosmetic applications of hyaluronic acid (HA) has increased. Size and dispersity are key characteristics of biological function. In contrast to extraction from animal tissue or bacterial fermentation, enzymatic in vitro synthesis is the choice to produce defined HA. Here we present a one-pot enzyme cascade with six enzymes for the synthesis of HA from the cheap monosaccharides glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). The combination of two enzyme modules, providing the precursors UDP–GlcA and UDP–GlcNAc, respectively, with hyaluronan synthase from Pasteurella multocida (PmHAS), was optimized to meet the kinetic requirements of PmHAS for high HA productivity and molecular weight. The Mg2+ concentration and the pH value were found as key factors. The HA product can be tailored by different conditions: 25 mM Mg2+ and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-NaOH pH 8 result into an HA product with high Mw HA (1.55 MDa) and low dispersity (1.05). Whereas with 15 mM Mg2+ and HEPES–NaOH pH 8.5, we reached the highest HA concentration (2.7 g/L) with a yield of 86.3%. Our comprehensive data set lays the basis for larger scale enzymatic HA synthesis. Full article
(This article belongs to the Special Issue Molecular Biocatalysis 2.0)
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Open AccessArticle
Bioproduction of Quercetin and Rutinose Catalyzed by Rutinosidase: Novel Concept of “Solid State Biocatalysis”
Int. J. Mol. Sci. 2019, 20(5), 1112; https://doi.org/10.3390/ijms20051112 - 05 Mar 2019
Cited by 3
Abstract
Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during [...] Read more.
Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This “solid-state” enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of “bio-quality.” Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the “Solid-State-Catalysis” concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility. Full article
(This article belongs to the Special Issue Molecular Biocatalysis 2.0)
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Open AccessArticle
Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis
Int. J. Mol. Sci. 2019, 20(4), 916; https://doi.org/10.3390/ijms20040916 - 20 Feb 2019
Cited by 3
Abstract
Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein [...] Read more.
Horseradish peroxidase (HRP) is an intensely studied enzyme with a wide range of commercial applications. Traditionally, HRP is extracted from plant; however, recombinant HRP (rHRP) production is a promising alternative. Here, non-glycosylated rHRP was produced in Escherichia coli as a DsbA fusion protein including a Dsb signal sequence for translocation to the periplasm and a His tag for purification. The missing N-glycosylation results in reduced catalytic activity and thermal stability, therefore enzyme engineering was used to improve these characteristics. The amino acids at four N-glycosylation sites, namely N13, N57, N255 and N268, were mutated by site-directed mutagenesis and combined to double, triple and quadruple enzyme variants. Subsequently, the rHRP fusion proteins were purified by immobilized metal affinity chromatography (IMAC) and biochemically characterized. We found that the quadruple mutant rHRP N13D/N57S/N255D/N268D showed 2-fold higher thermostability and 8-fold increased catalytic activity with 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as reducing substrate when compared to the non-mutated rHRP benchmark enzyme. Full article
(This article belongs to the Special Issue Molecular Biocatalysis 2.0)
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