Editorial

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Open AccessEditorial

Special Issue on “Enzymes as Biocatalysts: Current Research Trends and Applications”

by Sérgio F. Sousa

Int. J. Mol. Sci. 2022, 23(24), 16209; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232416209 - 19 Dec 2022

Cited by 1 | Viewed by 1168

Enzymes are able to catalyze a wide diversity of chemical reactions in nature, and they do it at an amazing level [...] Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

Research

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14 pages, 7439 KiB

Open AccessArticle

Insights into Mechanisms of Damage Recognition and Catalysis by APE1-like Enzymes

by Anatoly A. Bulygin, Olga S. Fedorova and Nikita A. Kuznetsov

Int. J. Mol. Sci. 2022, 23(8), 4361; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23084361 - 14 Apr 2022

Cited by 5 | Viewed by 1314

Abstract

Apurinic/apyrimidinic (AP) endonucleases are the key DNA repair enzymes in the base excision repair (BER) pathway, and are responsible for hydrolyzing phosphodiester bonds on the 5′ side of an AP site. The enzymes can recognize not only AP sites but also some types [...] Read more.

Apurinic/apyrimidinic (AP) endonucleases are the key DNA repair enzymes in the base excision repair (BER) pathway, and are responsible for hydrolyzing phosphodiester bonds on the 5′ side of an AP site. The enzymes can recognize not only AP sites but also some types of damaged bases, such as 1,N⁶-ethenoadenosine, α-adenosine, and 5,6-dihydrouridine. Here, to elucidate the mechanism underlying such a broad substrate specificity as that of AP endonucleases, we performed a computational study of four homologous APE1-like endonucleases: insect (Drosophila melanogaster) Rrp1, amphibian (Xenopus laevis) APE1 (xAPE1), fish (Danio rerio) APE1 (zAPE1), and human APE1 (hAPE1). The contact between the amino acid residues of the active site of each homologous APE1-like enzyme and the set of damaged DNA substrates was analyzed. A comparison of molecular dynamic simulation data with the known catalytic efficiency of these enzymes allowed us to gain a deep insight into the differences in the efficiency of the cleavage of various damaged nucleotides. The obtained data support that the amino acid residues within the “damage recognition” loop containing residues Asn222–Ala230 significantly affect the catalytic-complex formation. Moreover, every damaged nucleotide has its unique position and a specific set of interactions with the amino acid residues of the active site. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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12 pages, 2492 KiB

Open AccessArticle

Screening Collagenase Activity in Bacterial Lysate for Directed Enzyme Applications

by Ran Tohar, Tamar Ansbacher, Inbal Sher, Livnat Afriat-Jurnou, Evgeny Weinberg and Maayan Gal

Int. J. Mol. Sci. 2021, 22(16), 8552; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168552 - 09 Aug 2021

Cited by 4 | Viewed by 3018

Abstract

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants [...] Read more.

Collagenases are essential enzymes capable of digesting triple-helical collagen under physiological conditions. These enzymes play a key role in diverse physiological and pathophysiological processes. Collagenases are used for diverse biotechnological applications, and it is thus of major interest to identify new enzyme variants with improved characteristics such as expression yield, stability, or activity. The engineering of new enzyme variants often relies on either rational protein design or directed enzyme evolution. The latter includes screening of a large randomized or semirational genetic library, both of which require an assay that enables the identification of improved variants. Moreover, the assay should be tailored for microplates to allow the screening of hundreds or thousands of clones. Herein, we repurposed the previously reported fluorogenic assay using 3,4-dihydroxyphenylacetic acid for the quantitation of collagen, and applied it in the detection of bacterial collagenase activity in bacterial lysates. This enabled the screening of hundreds of E. coli colonies expressing an error-prone library of collagenase G from C. histolyticum, in 96-well deep-well plates, by measuring activity directly in lysates with collagen. As a proof-of-concept, a single variant exhibiting higher activity than the starting-point enzyme was expressed, purified, and characterized biochemically and computationally. This showed the feasibility of this method to support medium-high throughput screening based on direct evaluation of collagenase activity. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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17 pages, 2589 KiB

Open AccessArticle

Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

by Daniela P. Herrera, Andrea M. Chánique, Ascensión Martínez-Márquez, Roque Bru-Martínez, Robert Kourist, Loreto P. Parra and Andreas Schüller

Int. J. Mol. Sci. 2021, 22(9), 4345; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22094345 - 21 Apr 2021

Cited by 9 | Viewed by 3353

Abstract

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). [...] Read more.

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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14 pages, 2464 KiB

Open AccessArticle

Optimisation of Recombinant Myrosinase Production in Pichia pastoris

by Zuzana Rosenbergová, Kristína Kántorová, Martin Šimkovič, Albert Breier and Martin Rebroš

Int. J. Mol. Sci. 2021, 22(7), 3677; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22073677 - 01 Apr 2021

Cited by 11 | Viewed by 3408

Abstract

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this [...] Read more.

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (Mut^S) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/L_medium/h was achieved, which was 102.5-fold higher compared to flask cultivations. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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16 pages, 4271 KiB

Open AccessArticle

Rational Design of Adenylate Kinase Thermostability through Coevolution and Sequence Divergence Analysis

by Jian Chang, Chengxin Zhang, Huaqiang Cheng and Yan-Wen Tan

Int. J. Mol. Sci. 2021, 22(5), 2768; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22052768 - 09 Mar 2021

Cited by 6 | Viewed by 2442

Abstract

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or [...] Read more.

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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Review

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16 pages, 3741 KiB

Open AccessReview

Stereoselective Promiscuous Reactions Catalyzed by Lipases

by Angela Patti and Claudia Sanfilippo

Int. J. Mol. Sci. 2022, 23(5), 2675; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052675 - 28 Feb 2022

Cited by 22 | Viewed by 2327

Abstract

The ability of lipases to display activity beyond their physiological reactions, so-called “catalytic promiscuity”, has gained increasing interest in the last two decades as an important tool for expanding the application of these enzymes in organic synthesis. Some lipases have been shown to [...] Read more.

The ability of lipases to display activity beyond their physiological reactions, so-called “catalytic promiscuity”, has gained increasing interest in the last two decades as an important tool for expanding the application of these enzymes in organic synthesis. Some lipases have been shown to be effective in catalyzing a variety of C-C bond formation reactions and most of the investigations have been directed to the optimization of the products yield through a careful tuning of the experimental parameters. Despite the fact that new stereogenic carbons are formed in many of the tested reactions, the target products have been often obtained in racemic form and examples of an efficient asymmetric induction by the used lipases are quite limited. The aim of this review, mainly focused on those lipase-catalyzed promiscuous reactions in which optically active products have been obtained, is to offer a current state of art together with a perspective in this field of asymmetric synthesis. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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18 pages, 2829 KiB

Open AccessReview

A Review of Advanced Molecular Engineering Approaches to Enhance the Thermostability of Enzyme Breakers: From Prospective of Upstream Oil and Gas Industry

by Muhammad Naeem, Amjad Bajes Khalil, Zeeshan Tariq and Mohamed Mahmoud

Int. J. Mol. Sci. 2022, 23(3), 1597; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23031597 - 30 Jan 2022

Cited by 4 | Viewed by 2684

Abstract

During the fracture stimulation of oil and gas wells, fracturing fluids are used to create fractures and transport the proppant into the fractured reservoirs. The fracturing fluid viscosity is responsible for proppant suspension, the viscosity can be increased through the incorporation of guar [...] Read more.

During the fracture stimulation of oil and gas wells, fracturing fluids are used to create fractures and transport the proppant into the fractured reservoirs. The fracturing fluid viscosity is responsible for proppant suspension, the viscosity can be increased through the incorporation of guar polymer and cross-linkers. After the fracturing operation, the fluid viscosity is decreased by breakers for efficient oil and gas recovery. Different types of enzyme breakers have been engineered and employed to reduce the fracturing fluid′s viscosity, but thermal stability remains the major constraint for the use of enzymes. The latest enzyme engineering approaches such as direct evolution and rational design, have great potential to increase the enzyme breakers’ thermostability against high temperatures of reservoirs. In this review article, we have reviewed recently advanced enzyme molecular engineering technologies and how these strategies could be used to enhance the thermostability of enzyme breakers in the upstream oil and gas industry. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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63 pages, 8538 KiB

Open AccessReview

Perspectives on the Role of Enzymatic Biocatalysis for the Degradation of Plastic PET

by Rita P. Magalhães, Jorge M. Cunha and Sérgio F. Sousa

Int. J. Mol. Sci. 2021, 22(20), 11257; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222011257 - 19 Oct 2021

Cited by 35 | Viewed by 8242

Abstract

Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering [...] Read more.

Plastics are highly durable and widely used materials. Current methodologies of plastic degradation, elimination, and recycling are flawed. In recent years, biodegradation (the usage of microorganisms for material recycling) has grown as a valid alternative to previously used methods. The evolution of bioengineering techniques and the discovery of novel microorganisms and enzymes with degradation ability have been key. One of the most produced plastics is PET, a long chain polymer of terephthalic acid (TPA) and ethylene glycol (EG) repeating monomers. Many enzymes with PET degradation activity have been discovered, characterized, and engineered in the last few years. However, classification and integrated knowledge of these enzymes are not trivial. Therefore, in this work we present a summary of currently known PET degrading enzymes, focusing on their structural and activity characteristics, and summarizing engineering efforts to improve activity. Although several high potential enzymes have been discovered, further efforts to improve activity and thermal stability are necessary. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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46 pages, 41500 KiB

Open AccessReview

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions

by Ilya Lyagin and Elena Efremenko

Int. J. Mol. Sci. 2021, 22(4), 1761; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22041761 - 10 Feb 2021

Cited by 21 | Viewed by 2639

Abstract

Organophosphorus compounds (OPCs) are able to interact with various biological targets in living organisms, including enzymes. The binding of OPCs to enzymes does not always lead to negative consequences for the body itself, since there are a lot of natural biocatalysts that can [...] Read more.

Organophosphorus compounds (OPCs) are able to interact with various biological targets in living organisms, including enzymes. The binding of OPCs to enzymes does not always lead to negative consequences for the body itself, since there are a lot of natural biocatalysts that can catalyze the chemical transformations of the OPCs via hydrolysis or oxidation/reduction and thereby provide their detoxification. Some of these enzymes, their structural differences and identity, mechanisms, and specificity of catalytic action are discussed in this work, including results of computational modeling. Phylogenetic analysis of these diverse enzymes was specially realized for this review to emphasize a great area for future development(s) and applications. Full article

(This article belongs to the Special Issue Enzymes as Biocatalysts: Current Research Trends and Applications)

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