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Thermophilic and Hyperthermophilic Microbes and Enzymes 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 "Molecular Microbiology".

Deadline for manuscript submissions: closed (30 September 2022) | Viewed by 23046

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Special Issue Editors

Prof. Dr. Giuseppe Manco

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Institute of Biochemistry and Cell Biology, National Research Council, Via Pietro Castellino 111, 80131 Naples, Italy
Interests: structure and function of thermostable enzymes; human enzymes and disease; proteomics; bioremediation and detoxification; biosensors; functional materials
Special Issues, Collections and Topics in MDPI journals

Dr. Yannick J. Bomble

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Biosciences Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA
Interests: biomass deconstruction and conversion; enzyme engineering; cell free production of biochemicals
Special Issues, Collections and Topics in MDPI journals

Dr. Elena Porzio

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Institute of Biochemistry and Cell Biology (IBBC), National Research Council (CNR), Via Pietro Castellino 111, 80131 Naples, Italy
Interests: biochemistry & molecular biology; thermostable enzymes; archaeal proteins; bioremediation; protein engineering (in vitro molecular evolution); high throughput screening; antimicrobials

Special Issue Information

Dear Colleagues,

Thermophilic and hyperthermophilic microbes represent a fascinating class of microorganisms, not only because of their resilience to thrive at elevated temperatures and in harsh environments, but also because of the enzymes that they harbor. These microorganisms fall mainly in the bacteria and archaea domains, and exist in many habitats including hot springs, hydrothermal vents, or volcanic ash sediments, among others. These habitats all exhibit thermophilic or hyperthermophilic temperatures, but can also be acidic, alkaline, or contain high levels of salts. In order to thrive in such harsh environments, these microorganisms have evolved robust enzymes that are able to function at peak activity in these harsh conditions. These microorganisms represent a rich source of enzymes with an increased stability, which are purposely modified by protein engineering and can excel in harsh industrial conditions, making them especially appealing for biotechnological applications. Sometimes, they display promiscous activities that represent a peculiar basis for evolution. Bioprospecting for enzymes in these microbes has gained popularity in the last two decades, as they also represent ideal templates and strategies for reengineering essential but less stable enzymes, catalyzing reactions that do not exist in thermophiles.

This Special Issue encourages original research articles, perspectives, and reviews on the topic of thermophilic and hyperthermophilic microbes and enzymes. Topics of interest include, but are not limited to, the following:

Bioprospecting
Enzyme characterization
Microbial phenotypes
Novel metabolic pathways
Novel metabolic enzymes
Bioremediation
Enzyme evolution and engineering
Proteomics approach
Microbial ecology
Microbial consortia

Related Special issue "Thermophilic and Hyperthermophilic Microbes and Enzymes" and previous papers:

Altered Cofactor Preference of Thermostable StDAPDH by a Single Mutation at K159

Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity

Metabolic Adaptation to Sulfur of Hyperthermophilic Palaeococcus pacificus DY20341^T from Deep-Sea Hydrothermal Sediments

Occurrence of Thermophilic Microorganisms in Different Full Scale Biogas Plants

DING Proteins Extend to the Extremophilic World

Effect of Cultivation Parameters on Fermentation and Hydrogen Production in the Phylum Thermotogae

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

Dr. Giuseppe Manco
Dr. Yannick J. Bomble
Dr. Elena Porzio
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. 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.

Related Special Issue

Thermophilic and Hyperthermophilic Microbes and Enzymes 3.0 in International Journal of Molecular Sciences (2 articles)

Published Papers (13 papers)

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Research

Jump to: Review

15 pages, 2908 KiB

Open AccessArticle

Thermostable Lactonases Inhibit Pseudomonas aeruginosa Biofilm: Effect In Vitro and in Drosophila melanogaster Model of Chronic Infection

by Elena Porzio, Davide Andrenacci and Giuseppe Manco

Int. J. Mol. Sci. 2023, 24(23), 17028; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms242317028 - 01 Dec 2023

Viewed by 703

Abstract

Pseudomonas aeruginosa is one of the six antimicrobial-resistant pathogens known as “ESKAPE” that represent a global threat to human health and are considered priority targets for the development of novel antimicrobials and alternative therapeutics. The virulence of P. aeruginosa is regulated by a four-chemicals communication system termed quorum sensing (QS), and one main class of QS signals is termed acylhomoserine lactones (acyl-HSLs), which includes 3-Oxo-dodecanoil homoserine lactone (3-Oxo-C12-HSL), which regulates the expression of genes implicated in virulence and biofilm formation. Lactonases, like Paraoxonase 2 (PON2) from humans and the phosphotriesterase-like lactonases (PLLs) from thermostable microorganisms, are able to hydrolyze acyl-HSLs. In this work, we explored in vitro and in an animal model the effect of some lactonases on the production of Pseudomonas virulence factors. This study presents a model of chronic infection in which bacteria were administered by feeding, and Drosophila adults were treated with enzymes and the antibiotic tobramycin, alone or in combination. In vitro, we observed significant effects of lactonases on biofilm formation as well as effects on bacterial motility and the expression of virulence factors. The treatment in vivo by feeding with the lactonase SacPox allowed us to significantly increase the biocidal effect of tobramycin in chronic infection. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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20 pages, 5677 KiB

Open AccessArticle

Insight into Different Stages of Steroid Degradation in Thermophilic Saccharopolyspora hirsuta VKM Ac-666^T Strain

by Tatyana Lobastova, Victoria Fokina, Irina Pozdnyakova-Filatova, Sergey Tarlachkov, Andrey Shutov and Marina Donova

Int. J. Mol. Sci. 2022, 23(24), 16174; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232416174 - 18 Dec 2022

Cited by 3 | Viewed by 1356

Abstract

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666^T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT–qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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18 pages, 2691 KiB

Open AccessArticle

Structural, Thermodynamic and Enzymatic Characterization of N,N-Diacetylchitobiose Deacetylase from Pyrococcus chitonophagus

by Katarzyna Biniek-Antosiak, Magdalena Bejger, Joanna Śliwiak, Daniel Baranowski, Ahmed S. A. Mohammed, Dmitri I. Svergun and Wojciech Rypniewski

Int. J. Mol. Sci. 2022, 23(24), 15736; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232415736 - 12 Dec 2022

Viewed by 1250

Abstract

Chitin is a major source of energy and macroelements for many organisms. An important step in its degradation is the deacetylation of chitin or its fragments. Deacetylase from the extremophile Pyrococcus chitonophagus has been analyzed by X-ray crystallography, small-angle X-ray scattering, differential scanning calorimetry, isothermal titration calorimetry and NMR to determine its structure, thermodynamics and enzymatic properties. It is a hexameric, zinc-containing metalloenzyme that retains its structural integrity up to temperatures slightly exceeding 100 °C. It removes the acetyl group specifically from the non-reducing end of the sugar substrate. Its main substrate is N,N-diacetylchitobiose but it also active, at a reduced level, toward N-acetyl-d-glucosamine or a trimer of N-acetyl-d-glucosamine units. Crystallographic analysis includes the structure of the enzyme with its main substrate approaching the active site in a monodentate manner, replacing the single water molecule that is bound at the Zn²⁺ cation when the ligand is absent. The Zn²⁺ cation remains tetrahedrally coordinated, with three of its ligands provided by the protein’s conserved His-Asp-His triad. The crystal structures are consistent with the reaction mechanism proceeding via an anhydride intermediate. Hydrolysis as the first step cannot be ruled out in a hydrated environment but no defined ‘hydrolytic water’ site can be identified in the analyzed structures. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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9 pages, 2327 KiB

Open AccessArticle

Understanding Life at High Temperatures: Relationships of Molecular Channels in Enzymes of Methanogenic Archaea and Their Growth Temperatures

by Laura F. Ginsbach and Juan M. Gonzalez

Int. J. Mol. Sci. 2022, 23(23), 15149; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232315149 - 02 Dec 2022

Viewed by 976

Abstract

Analyses of protein structures have shown the existence of molecular channels in enzymes from Prokaryotes. Those molecular channels suggest a critical role of spatial voids in proteins, above all, in those enzymes functioning under high temperature. It is expected that these spaces within the protein structure are required to access the active site and to maximize availability and thermal stability of their substrates and cofactors. Interestingly, numerous substrates and cofactors have been reported to be highly temperature-sensitive biomolecules. Methanogens represent a singular phylogenetic group of Archaea that performs anaerobic respiration producing methane during growth. Methanogens inhabit a variety of environments including the full range of temperatures for the known living forms. Herein, we carry out a dimensional analysis of molecular tunnels in key enzymes of the methanogenic pathway from methanogenic Archaea growing optimally over a broad temperature range. We aim to determine whether the dimensions of the molecular tunnels are critical for those enzymes from thermophiles. Results showed that at increasing growth temperature the dimensions of molecular tunnels in the enzymes methyl-coenzyme M reductase and heterodisulfide reductase become increasingly restrictive and present strict limits at the highest growth temperatures, i.e., for hyperthermophilic methanogens. However, growth at lower temperature allows a wide dimensional range for the molecular spaces in these enzymes. This is in agreement with previous suggestions on a potential major role of molecular tunnels to maintain biomolecule stability and activity of some enzymes in microorganisms growing at high temperatures. These results contribute to better understand archaeal growth at high temperatures. Furthermore, an optimization of the dimensions of molecular tunnels would represent an important adaptation required to maintain the activity of key enzymes of the methanogenic pathway for those methanogens growing optimally at high temperatures. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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15 pages, 3364 KiB

Open AccessArticle

Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides

by Flora Salzano, Martina Aulitto, Gabriella Fiorentino, Emilia Pedone, Patrizia Contursi and Danila Limauro

Int. J. Mol. Sci. 2022, 23(22), 14310; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232214310 - 18 Nov 2022

Cited by 5 | Viewed by 1429

Abstract

A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 °C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 °C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (K_M 0.52 mM, k_cat 1606 s⁻¹, and k_cat/K_M 3088.46 mM⁻¹·s⁻¹) and para-nitrophenyl-α-L-arabinofuranoside (K_M 10.56 mM, k_cat 2395.8 s⁻¹, and k_cat/K_M 226.87 mM⁻¹·s⁻¹). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl is a true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents, or EDTA was observed except for 5 mM Cu²⁺. AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 M by both arabinose and glucose. The high tolerance to organic solvents and monosaccharides together with other characteristics reported above suggests that AmβXyl may have several applications in many industrial fields. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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17 pages, 3614 KiB

Open AccessArticle

Intron-Dependent or Independent Pseudouridylation of Precursor tRNA Containing Atypical Introns in Cyanidioschyzon merolae

by Yasuha Nagato, Chie Tomikawa, Hideyuki Yamaji, Akiko Soma and Kazuyuki Takai

Int. J. Mol. Sci. 2022, 23(20), 12058; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232012058 - 11 Oct 2022

Cited by 3 | Viewed by 1520

Abstract

Eukaryotic precursor tRNAs (pre-tRNAs) often have an intron between positions 37 and 38 of the anticodon loop. However, atypical introns are found in some eukaryotes and archaea. In an early-diverged red alga Cyanidioschyzon merolae, the tRNA^Ile(UAU) gene contains three intron coding regions, located in the D-, anticodon, and T-arms. In this study, we focused on the relationship between the intron removal and formation of pseudouridine (Ψ), one of the most universally modified nucleosides. It had been reported that yeast Pus1 is a multiple-site-specific enzyme that synthesizes Ψ34 and Ψ36 in tRNA^Ile(UAU) in an intron-dependent manner. Unexpectedly, our biochemical experiments showed that the C. merolae ortholog of Pus1 pseudouridylated an intronless tRNA^Ile(UAU) and that the modification position was determined to be 55 which is the target of Pus4 but not Pus1 in yeast. Furthermore, unlike yeast Pus1, cmPus1 mediates Ψ modification at positions 34, 36, and/or 55 only in some specific intron-containing pre-tRNA^Ile(UAU) variants. cmPus4 was confirmed to be a single-site-specific enzyme that only converts U55 to Ψ, in a similar manner to yeast Pus4. cmPus4 did not catalyze the pseudouridine formation in pre-tRNAs containing an intron in the T-arm. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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13 pages, 2039 KiB

Open AccessArticle

Occurrence of Capnophilic Lactic Fermentation in the Hyperthermophilic Anaerobic Bacterium Thermotoga sp. Strain RQ7

by Nunzia Esercizio, Mariamichela Lanzilli, Simone Landi, Lucio Caso, Zhaohui Xu, Genoveffa Nuzzo, Carmela Gallo, Emiliano Manzo, Sergio Esposito, Angelo Fontana and Giuliana d’Ippolito

Int. J. Mol. Sci. 2022, 23(19), 12049; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231912049 - 10 Oct 2022

Cited by 1 | Viewed by 1545

Abstract

Capnophilic lactic fermentation (CLF) is an anaplerotic pathway exclusively identified in the anaerobic hyperthermophilic bacterium Thermotoga neapolitana, a member of the order Thermotogales. The CO₂-activated pathway enables non-competitive synthesis of hydrogen and L-lactic acid at high yields, making it an economically attractive process for bioenergy production. In this work, we discovered and characterized CLF in Thermotoga sp. strain RQ7, a naturally competent strain, opening a new avenue for molecular investigation of the pathway. Evaluation of the fermentation products and expression analyses of key CLF-genes by RT-PCR revealed similar CLF-phenotypes between T. neapolitana and T. sp. strain RQ7, which were absent in the non-CLF-performing strain T. maritima. Key CLF enzymes, such as PFOR, HYD, LDH, RNF, and NFN, are up-regulated in the two CLF strains. Another important finding is the up-regulation of V-ATPase, which couples ATP hydrolysis to proton transport across the membranes, in the two CLF-performing strains. The fact that V-ATPase is absent in T. maritima suggested that this enzyme plays a key role in maintaining the necessary proton gradient to support high demand of reducing equivalents for simultaneous hydrogen and lactic acid synthesis in CLF. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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13 pages, 1856 KiB

Open AccessArticle

Characterization of the Biomass Degrading Enzyme GuxA from Acidothermus cellulolyticus

by Neal N. Hengge, Sam J. B. Mallinson, Patthra Pason, Vladimir V. Lunin, Markus Alahuhta, Daehwan Chung, Michael E. Himmel, Janet Westpheling and Yannick J. Bomble

Int. J. Mol. Sci. 2022, 23(11), 6070; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23116070 - 28 May 2022

Cited by 5 | Viewed by 1756

Abstract

Microbial conversion of biomass relies on a complex combination of enzyme systems promoting synergy to overcome biomass recalcitrance. Some thermophilic bacteria have been shown to exhibit particularly high levels of cellulolytic activity, making them of particular interest for biomass conversion. These bacteria use varying combinations of CAZymes that vary in complexity from a single catalytic domain to large multi-modular and multi-functional architectures to deconstruct biomass. Since the discovery of CelA from Caldicellulosiruptor bescii which was identified as one of the most active cellulase so far identified, the search for efficient multi-modular and multi-functional CAZymes has intensified. One of these candidates, GuxA (previously Acel_0615), was recently shown to exhibit synergy with other CAZymes in C. bescii, leading to a dramatic increase in growth on biomass when expressed in this host. GuxA is a multi-modular and multi-functional enzyme from Acidothermus cellulolyticus whose catalytic domains include a xylanase/endoglucanase GH12 and an exoglucanase GH6, representing a unique combination of these two glycoside hydrolase families in a single CAZyme. These attributes make GuxA of particular interest as a potential candidate for thermophilic industrial enzyme preparations. Here, we present a more complete characterization of GuxA to understand the mechanism of its activity and substrate specificity. In addition, we demonstrate that GuxA exhibits high levels of synergism with E1, a companion endoglucanase from A. cellulolyticus. We also present a crystal structure of one of the GuxA domains and dissect the structural features that might contribute to its thermotolerance. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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15 pages, 3863 KiB

Open AccessArticle

Enhanced Magnetic Hyperthermia of Magnetoferritin through Synthesis at Elevated Temperature

by Jiacheng Yu, Changqian Cao, Fengjiao Fang and Yongxin Pan

Int. J. Mol. Sci. 2022, 23(7), 4012; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23074012 - 04 Apr 2022

Cited by 4 | Viewed by 1925

Abstract

Iron oxide nanoparticles have attracted a great deal of research interest in recent years for magnetic hyperthermia therapy owing to their biocompatibility and superior thermal conversion efficiency. Magnetoferritin is a type of biomimetic superparamagnetic iron oxide nanoparticle in a ferritin cage with good monodispersity, biocompatibility, and natural hydrophilicity. However, the magnetic hyperthermic efficiency of this kind of nanoparticle is limited by the small size of the mineral core as well as its low synthesis temperature. Here, we synthesized a novel magnetoferritin particle by using a recombinant ferritin from the hyperthermophilic archaeon Pyrococcus furiosus as a template with high iron atom loading of 9517 under a designated temperature of 90 °C. Compared with the magnetoferritins synthesized at 45 and 65 °C, the one synthesized at 90 °C displays a larger average magnetite and/or maghemite core size of 10.3 nm. This yields an increased saturation magnetization of up to 49.6 emu g⁻¹ and an enhanced specific absorption rate (SAR) of 805.3 W g⁻¹ in an alternating magnetic field of 485.7 kHz and 49 kA m⁻¹. The maximum intrinsic loss power (ILP) value is 1.36 nHm² kg⁻¹. These results provide new insights into the biomimetic synthesis of magnetoferritins with enhanced hyperthermic efficiency and demonstrate the potential application of magnetoferritin in the magnetic hyperthermia of tumors. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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13 pages, 1585 KiB

Open AccessCommunication

A Homologous Recombination System to Generate Epitope-Tagged Target Genes in Chaetomium thermophilum: A Genetic Approach to Investigate Native Thermostable Proteins

by Nikola Kellner, Sabine Griesel and Ed Hurt

Int. J. Mol. Sci. 2022, 23(6), 3198; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23063198 - 16 Mar 2022

Cited by 2 | Viewed by 1799

Abstract

Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50–52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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Review

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14 pages, 1366 KiB

Open AccessReview

Potential Applications of Thermophilic Bacteriophages in One Health

by Hong Liu, Milad Kheirvari and Ebenezer Tumban

Int. J. Mol. Sci. 2023, 24(9), 8222; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24098222 - 04 May 2023

Cited by 7 | Viewed by 2180

Abstract

Bacteriophages have a wide range of applications such as combating antibiotic resistance, preventing food contamination for food safety, and as biomarkers to indirectly assess the quality of water. Additionally, bacteriophage components (endolysins and coat proteins) have a lot of applications in food processing, vaccine design, and the delivery of cargo to the body. Therefore, bacteriophages/components have a multitude of applications in human, plant/veterinary, and environmental health (One Health). Despite their versatility, bacteriophage/component use is mostly limited to temperatures within 4–40 °C. This limits their applications (e.g., in food processing conditions, pasteurization, and vaccine design). Advances in thermophilic bacteriophage research have uncovered novel thermophilic endolysins (e.g., ΦGVE2 amidase and MMPphg) that can be used in food processing and in veterinary medicine. The endolysins are thermostable at temperatures > 65 °C and have broad antimicrobial activities. In addition to thermophilic endolysins, enzymes (DNA polymerase and ligases) derived from thermophages have different applications in molecular biology/biotechnology: to generate DNA libraries and develop diagnostics for human and animal pathogens. Furthermore, coat proteins from thermophages are being explored to develop virus-like particle platforms with versatile applications in human and animal health. Overall, bacteriophages, especially those that are thermophilic, have a plethora of applications in One Health. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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24 pages, 2732 KiB

Open AccessReview

Thermo-L-Asparaginases: From the Role in the Viability of Thermophiles and Hyperthermophiles at High Temperatures to a Molecular Understanding of Their Thermoactivity and Thermostability

by Maria Dumina and Alexander Zhgun

Int. J. Mol. Sci. 2023, 24(3), 2674; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24032674 - 31 Jan 2023

Cited by 7 | Viewed by 1929

Abstract

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine, food industry, and diagnostics. Among various organisms expressing L-ASNases, thermophiles and hyperthermophiles produce enzymes with superior performances—stable and heat resistant thermo-ASNases. This review is an attempt to take a broader view on the thermo-ASNases. Here we discuss the position of thermo-ASNases in the large family of L-ASNases, their role in the heat-tolerance cellular system of thermophiles and hyperthermophiles, and molecular aspects of their thermoactivity and thermostability. Different types of thermo-ASNases exhibit specific L-asparaginase activity and additional secondary activities. All products of these enzymatic reactions are associated with diverse metabolic pathways and are important for mitigating heat stress. Thermo-ASNases are quite distinct from typical mesophilic L-ASNases based on structural properties, kinetic and activity profiles. Here we attempt to summarize the current understanding of the molecular mechanisms of thermo-ASNases’ thermoactivity and thermostability, from amino acid composition to structural–functional relationships. Research of these enzymes has fundamental and biotechnological significance. Thermo-ASNases and their improved variants, cloned and expressed in mesophilic hosts, can form a large pool of enzymes with valuable characteristics for biotechnological application. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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31 pages, 3643 KiB

Open AccessReview

Thermophilic Nucleic Acid Polymerases and Their Application in Xenobiology

by Guangyuan Wang, Yuhui Du, Xingyun Ma, Fangkai Ye, Yanjia Qin, Yangming Wang, Yuming Xiang, Rui Tao and Tingjian Chen

Int. J. Mol. Sci. 2022, 23(23), 14969; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232314969 - 29 Nov 2022

Cited by 3 | Viewed by 3125

Abstract

Thermophilic nucleic acid polymerases, isolated from organisms that thrive in extremely hot environments, possess great DNA/RNA synthesis activities under high temperatures. These enzymes play indispensable roles in central life activities involved in DNA replication and repair, as well as RNA transcription, and have already been widely used in bioengineering, biotechnology, and biomedicine. Xeno nucleic acids (XNAs), which are analogs of DNA/RNA with unnatural moieties, have been developed as new carriers of genetic information in the past decades, which contributed to the fast development of a field called xenobiology. The broad application of these XNA molecules in the production of novel drugs, materials, and catalysts greatly relies on the capability of enzymatic synthesis, reverse transcription, and amplification of them, which have been partially achieved with natural or artificially tailored thermophilic nucleic acid polymerases. In this review, we first systematically summarize representative thermophilic and hyperthermophilic polymerases that have been extensively studied and utilized, followed by the introduction of methods and approaches in the engineering of these polymerases for the efficient synthesis, reverse transcription, and amplification of XNAs. The application of XNAs facilitated by these polymerases and their mutants is then discussed. In the end, a perspective for the future direction of further development and application of unnatural nucleic acid polymerases is provided. Full article

(This article belongs to the Special Issue Thermophilic and Hyperthermophilic Microbes and Enzymes 2.0)

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