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Molecular Biocatalysis 2.0

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

Deadline for manuscript submissions: closed (7 March 2020) | Viewed by 48613

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Prof. Dr. Vladimír Křen

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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
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Dr. Pavla Bojarová

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Guest Editor

1. 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
2. Faculty of Biomedical Engineering, Czech Technical University in Prague, Kladno, 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 Issues, Collections and Topics in MDPI journals

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

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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

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Published Papers (11 papers)

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Editorial

Jump to: Research

3 pages, 207 KiB

Open AccessEditorial

Biocatalysis: “A Jack of all Trades...”

by Vladimír Křen and Pavla Bojarová

Int. J. Mol. Sci. 2020, 21(14), 5115; https://doi.org/10.3390/ijms21145115 - 20 Jul 2020

Cited by 1 | Viewed by 2008

Abstract

Biotransformation has accompanied mankind since the Neolithic community, when people settled down and began to engage in agriculture [...] Full article

(This article belongs to the Special Issue Molecular Biocatalysis 2.0)

Research

Jump to: Editorial

13 pages, 1986 KiB

Open AccessArticle

2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis

by Xiao-Ran Jing, Huan Liu, Yao Nie and Yan Xu

Int. J. Mol. Sci. 2020, 21(15), 5347; https://doi.org/10.3390/ijms21155347 - 28 Jul 2020

Cited by 5 | Viewed by 2732

Abstract

Fe(II)/2-ketoglutarate-dependent dioxygenase (Fe(II)/2-KG DO)-mediated hydroxylation is a critical type of C–H bond functionalization for synthesizing hydroxy amino acids used as pharmaceutical raw materials and precursors. However, DO activity requires 2-ketoglutarate (2-KG), lack of which reduces the efficiency of Fe(II)/2-KG DO-mediated hydroxylation. Here, we conducted multi-enzymatic syntheses of hydroxy amino acids. Using (2s,3r,4s)-4-hydroxyisoleucine (4-HIL) as a model product, we coupled regio- and stereo-selective hydroxylation of l-Ile by the dioxygenase IDO with 2-KG generation from readily available l-Glu by l-glutamate oxidase (LGOX) and catalase (CAT). In the one-pot system, H₂O₂ significantly inhibited IDO activity and elevated Fe²⁺ concentrations of severely repressed LGOX. A sequential cascade reaction was preferable to a single-step process as CAT in the former system hydrolyzed H₂O₂. We obtained 465 mM 4-HIL at 93% yield in the two-step system. Moreover, this process facilitated C–H hydroxylation of several hydrophobic aliphatic amino acids to produce hydroxy amino acids, and C–H sulfoxidation of sulfur-containing l-amino acids to yield l-amino acid sulfoxides. Thus, we constructed an efficient cascade reaction to produce 4-HIL by providing prerequisite 2-KG from cheap and plentiful l-Glu and developed a strategy for creating enzymatic systems catalyzing 2-KG-dependent reactions in sustainable bioprocesses that synthesize other functional compounds. Full article

(This article belongs to the Special Issue Molecular Biocatalysis 2.0)

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17 pages, 2661 KiB

Open AccessArticle

Cell Wall Anchoring of a Bacterial Chitosanase in Lactobacillus plantarum Using a Food-Grade Expression System and Two Versions of an LP × TG Anchor

by Mai-Lan Pham, Anh-Minh Tran, Geir Mathiesen, Hoang-Minh Nguyen and Thu-Ha Nguyen

Int. J. Mol. Sci. 2020, 21(11), 3773; https://doi.org/10.3390/ijms21113773 - 27 May 2020

Cited by 7 | Viewed by 3128

Abstract

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products. Full article

(This article belongs to the Special Issue Molecular Biocatalysis 2.0)

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12 pages, 1804 KiB

Open AccessArticle

Manganese(II) Oxidizing Bacteria as Whole-Cell Catalyst for β-Keto Ester Oxidation

by Juan Guo, Huan Guo, Jin Liu, Fangrui Zhong and Yuzhou Wu

Int. J. Mol. Sci. 2020, 21(5), 1709; https://doi.org/10.3390/ijms21051709 - 2 Mar 2020

Cited by 6 | Viewed by 4655

Abstract

Manganese oxidizing bacteria can produce biogenic manganese oxides (BMO) on their cell surface and have been applied in the fields of agriculture, bioremediation, and drinking water treatment to remove toxic contaminants based on their remarkable chemical reactivity. Herein, we report for the first time the synthetic application of the manganese oxidizing bacteria, Pseudomonas putida MnB1 as a whole-cell biocatalyst for the effective oxidation of β-keto ester with excellent yield. Differing from known chemical protocols toward this transformation that generally necessitate the use of organic solvents, stoichiometric oxygenating agents and complex chemical catalysts, our strategy can accomplish it simply under aqueous and mild conditions with higher efficiency than that provided by chemical manganese oxides. Moreover, the live MnB1 bacteria are capable of continuous catalysis for this C-O bond forming reaction for several cycles and remain proliferating, highlighting the favorable merits of this novel protocol for sustainable chemistry and green synthesis. Full article

(This article belongs to the Special Issue Molecular Biocatalysis 2.0)

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10 pages, 1950 KiB

Open AccessArticle

Engineered Glucose Oxidase Capable of Quasi-Direct Electron Transfer after a Quick-and-Easy Modification with a Mediator

by Nanami Suzuki, Jinhee Lee, Noya Loew, Yuka Takahashi-Inose, Junko Okuda-Shimazaki, Katsuhiro Kojima, Kazushige Mori, Wakako Tsugawa and Koji Sode

Int. J. Mol. Sci. 2020, 21(3), 1137; https://doi.org/10.3390/ijms21031137 - 8 Feb 2020

Cited by 50 | Viewed by 6509

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 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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17 pages, 2590 KiB

Open AccessArticle

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

by Cecilia Garcia-Oliva, Pilar Hoyos, Lucie Petrásková, Natalia Kulik, Helena Pelantová, Alfredo H. Cabanillas, Ángel Rumbero, Vladimír Křen, María J. Hernáiz and Pavla Bojarová

Int. J. Mol. Sci. 2019, 20(24), 6181; https://doi.org/10.3390/ijms20246181 - 7 Dec 2019

Cited by 13 | Viewed by 2825

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. 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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9 pages, 5110 KiB

Open AccessArticle

Structure-Based Redesign of a Self-Sufficient Flavin-Containing Monooxygenase towards Indigo Production

by Nikola Lončar, Hugo L. van Beek and Marco W. Fraaije

Int. J. Mol. Sci. 2019, 20(24), 6148; https://doi.org/10.3390/ijms20246148 - 5 Dec 2019

Cited by 17 | Viewed by 4892

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 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 k_cat 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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19 pages, 6203 KiB

Open AccessArticle

Genetic and Functional Diversity of Nitrilases in Agaricomycotina

by Lenka Rucká, Martin Chmátal, Natalia Kulik, Lucie Petrásková, Helena Pelantová, Petr Novotný, Romana Příhodová, Miroslav Pátek and Ludmila Martínková

Int. J. Mol. Sci. 2019, 20(23), 5990; https://doi.org/10.3390/ijms20235990 - 28 Nov 2019

Cited by 13 | Viewed by 3715

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 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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19 pages, 2648 KiB

Open AccessArticle

Key Factors for a One-Pot Enzyme Cascade Synthesis of High Molecular Weight Hyaluronic Acid

by Johannes Gottschalk, Henning Zaun, Anna Eisele, Jürgen Kuballa and Lothar Elling

Int. J. Mol. Sci. 2019, 20(22), 5664; https://doi.org/10.3390/ijms20225664 - 12 Nov 2019

Cited by 19 | Viewed by 5502

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 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 M_w 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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15 pages, 2679 KiB

Open AccessArticle

Bioproduction of Quercetin and Rutinose Catalyzed by Rutinosidase: Novel Concept of “Solid State Biocatalysis”

by Jana Kapešová, Lucie Petrásková, Kristína Markošová, Martin Rebroš, Michael Kotik, Pavla Bojarová and Vladimír Křen

Int. J. Mol. Sci. 2019, 20(5), 1112; https://doi.org/10.3390/ijms20051112 - 5 Mar 2019

Cited by 35 | Viewed by 5003

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 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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14 pages, 564 KiB

Open AccessArticle

Improving the Performance of Horseradish Peroxidase by Site-Directed Mutagenesis

by Diana Humer and Oliver Spadiut

Int. J. Mol. Sci. 2019, 20(4), 916; https://doi.org/10.3390/ijms20040916 - 20 Feb 2019

Cited by 37 | Viewed by 6751

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 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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