Editorial

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2 pages, 182 KiB

Open AccessEditorial

Editorial for the Special Issue: Thermophiles and Thermozymes

by

María-Isabel González-Siso

Microorganisms 2019, 7(3), 62; https://doi.org/10.3390/microorganisms7030062 - 27 Feb 2019

Cited by 5 | Viewed by 2288

Abstract

Heat-loving microorganisms or thermophiles arouse noticeable scientific interest nowadays, not only with the aim to elucidate the mystery of life at high temperatures, but also due to the huge field of biotechnological applications of the enzymes they produce or thermozymes, able to function [...] Read more.

Heat-loving microorganisms or thermophiles arouse noticeable scientific interest nowadays, not only with the aim to elucidate the mystery of life at high temperatures, but also due to the huge field of biotechnological applications of the enzymes they produce or thermozymes, able to function under industrial harsh conditions [...] Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

Research

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15 pages, 2275 KiB

Open AccessArticle

Into the Thermus Mobilome: Presence, Diversity and Recent Activities of Insertion Sequences Across Thermus spp.

by

Alba Blesa

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Mercedes Sánchez

,

Eva Sacristán-Horcajada

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Sandra González-de la Fuente

,

Ramón Peiró

and

José Berenguer

Microorganisms 2019, 7(1), 25; https://doi.org/10.3390/microorganisms7010025 - 21 Jan 2019

Cited by 15 | Viewed by 3899

Abstract

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning [...] Read more.

A high level of transposon-mediated genome rearrangement is a common trait among microorganisms isolated from thermal environments, probably contributing to the extraordinary genomic plasticity and horizontal gene transfer (HGT) observed in these habitats. In this work, active and inactive insertion sequences (ISs) spanning the sequenced members of the genus Thermus were characterized, with special emphasis on three T. thermophilus strains: HB27, HB8, and NAR1. A large number of full ISs and fragments derived from different IS families were found, concentrating within megaplasmids present in most isolates. Potentially active ISs were identified through analysis of transposase integrity, and domestication-related transposition events of ISTth7 were identified in laboratory-adapted HB27 derivatives. Many partial copies of ISs appeared throughout the genome, which may serve as specific targets for homologous recombination contributing to genome rearrangement. Moreover, recruitment of IS1000 32 bp segments as spacers for CRISPR sequence was identified, pointing to the adaptability of these elements in the biology of these thermophiles. Further knowledge about the activity and functional diversity of ISs in this genus may contribute to the generation of engineered transposons as new genetic tools, and enrich our understanding of the outstanding plasticity shown by these thermophiles. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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10 pages, 889 KiB

Open AccessArticle

Molecular Tunnels in Enzymes and Thermophily: A Case Study on the Relationship to Growth Temperature

by

Juan Miguel Gonzalez

Microorganisms 2018, 6(4), 109; https://doi.org/10.3390/microorganisms6040109 - 20 Oct 2018

Cited by 3 | Viewed by 3063

Abstract

Developments in protein expression, analysis and computational capabilities are decisively contributing to a better understanding of the structure of proteins and their relationship to function. Proteins are known to be adapted to the growth rate of microorganisms and some microorganisms (named (hyper)thermophiles) thrive [...] Read more.

Developments in protein expression, analysis and computational capabilities are decisively contributing to a better understanding of the structure of proteins and their relationship to function. Proteins are known to be adapted to the growth rate of microorganisms and some microorganisms (named (hyper)thermophiles) thrive optimally at high temperatures, even above 100 °C. Nevertheless, some biomolecules show great instability at high temperatures and some of them are universal and required substrates and cofactors in multiple enzymatic reactions for all (both mesophiles and thermophiles) living cells. Only a few possibilities have been pointed out to explain the mechanisms that thermophiles use to successfully thrive under high temperatures. As one of these alternatives, the role of molecular tunnels or channels in enzymes has been suggested but remains to be elucidated. This study presents an analysis of channels in proteins (i.e., substrate tunnels), comparing two different protein types, glutamate dehydrogenase and glutamine phosphoribosylpyrophosphate amidotransferase, which are supposed to present a different strategy on the requirement for substrate tunnels with low and high needs for tunneling, respectively. The search and comparison of molecular tunnels in these proteins from microorganisms thriving optimally from 15 °C to 100 °C suggested that those tunnels in (hyper)thermophiles are required and optimized to specific dimensions at high temperatures for the enzyme glutamine phosphoribosylpyrophosphate amidotransferase. For the enzyme glutamate dehydrogenase, a reduction of empty spaces within the protein could explain the optimization at increasing temperatures. This analysis provides further evidence on molecular channeling as a feasible mechanism in hyperthermophiles with multiple relevant consequences contributing to better understand how they live under those extreme conditions. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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13 pages, 1356 KiB

Open AccessArticle

Contribution of the Oligomeric State to the Thermostability of Isoenzyme 3 from Candida rugosa

by

María-Efigenia Álvarez-Cao

,

Roberto González

,

María A. Pernas

and

María Luisa Rúa

Microorganisms 2018, 6(4), 108; https://doi.org/10.3390/microorganisms6040108 - 19 Oct 2018

Cited by 11 | Viewed by 2608

Abstract

Thermophilic proteins have evolved different strategies to maintain structure and function at high temperatures; they have large, hydrophobic cores, and feature increased electrostatic interactions, with disulfide bonds, salt-bridging, and surface charges. Oligomerization is also recognized as a mechanism for protein stabilization to confer [...] Read more.

Thermophilic proteins have evolved different strategies to maintain structure and function at high temperatures; they have large, hydrophobic cores, and feature increased electrostatic interactions, with disulfide bonds, salt-bridging, and surface charges. Oligomerization is also recognized as a mechanism for protein stabilization to confer a thermophilic adaptation. Mesophilic proteins are less thermostable than their thermophilic homologs, but oligomerization plays an important role in biological processes on a wide variety of mesophilic enzymes, including thermostabilization. The mesophilic yeast Candida rugosa contains a complex family of highly related lipase isoenzymes. Lip3 has been purified and characterized in two oligomeric states, monomer (mLip3) and dimer (dLip3), and crystallized in a dimeric conformation, providing a perfect model for studying the effects of homodimerization on mesophilic enzymes. We studied kinetics and stability at different pHs and temperatures, using the response surface methodology to compare both forms. At the kinetic level, homodimerization expanded Lip3 specificity (serving as a better catalyst on soluble substrates). Indeed, dimerization increased its thermostability by more than 15 °C (maximum temperature for dLip3 was out of the experimental range; >50 °C), and increased the pH stability by nearly one pH unit, demonstrating that oligomerization is a viable strategy for the stabilization of mesophilic enzymes. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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25 pages, 4777 KiB

Open AccessArticle

Thermostable Xylanase Production by Geobacillus sp. Strain DUSELR13, and Its Application in Ethanol Production with Lignocellulosic Biomass

by

Mohit Bibra

,

Venkat Reddy Kunreddy

and

Rajesh K. Sani

Microorganisms 2018, 6(3), 93; https://doi.org/10.3390/microorganisms6030093 - 05 Sep 2018

Cited by 47 | Viewed by 5560

Abstract

The aim of the current study was to optimize the production of xylanase, and its application for ethanol production using the lignocellulosic biomass. A highly thermostable crude xylanase was obtained from the Geobacillus sp. strain DUSELR13 isolated from the deep biosphere of Homestake [...] Read more.

The aim of the current study was to optimize the production of xylanase, and its application for ethanol production using the lignocellulosic biomass. A highly thermostable crude xylanase was obtained from the Geobacillus sp. strain DUSELR13 isolated from the deep biosphere of Homestake gold mine, Lead, SD. Geobacillus sp. strain DUSELR13 produced 6 U/mL of the xylanase with the beechwood xylan. The xylanase production was improved following the optimization studies, with one factor at a time approach, from 6 U/mL to 19.8 U/mL with xylan. The statistical optimization with response surface methodology further increased the production to 31 U/mL. The characterization studies revealed that the crude xylanase complex had an optimum pH of 7.0, with a broad pH range of 5.0–9.0, and an optimum temperature of 75 °C. The ~45 kDa xylanase protein was highly thermostable with t_1/2 of 48, 38, and 13 days at 50, 60, and 70 °C, respectively. The xylanase activity increased with the addition of Cu^+2, Zn⁺², K^+, and Fe⁺² at 1 mM concentration, and Ca⁺², Zn⁺², Mg⁺², and Na⁺ at 10 mM concentration. The comparative analysis of the crude xylanase against its commercial counterpart Novozymes Cellic HTec and Dupont, Accellerase XY, showed that it performed better at higher temperature, hydrolyzing 65.4% of the beechwood at 75 °C. The DUSEL R13 showed the mettle to hydrolyze, and utilize the pretreated, and untreated lignocellulosic biomass: prairie cord grass (PCG), and corn stover (CS) as the substrate, and gave a maximum yield of 20.5 U/mL with the untreated PCG. When grown in co-culture with Geobacillus thermoglucosidasius, it produced 3.53 and 3.72 g/L ethanol, respectively with PCG, and CS. With these characteristics the xylanase under study could be an industrial success for the high temperature bioprocesses. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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21 pages, 4946 KiB

Open AccessArticle

Genome Analysis of Vallitalea guaymasensis Strain L81 Isolated from a Deep-Sea Hydrothermal Vent System

by

Anders Schouw

,

Francesca Vulcano

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Irene Roalkvam

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William Peter Hocking

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,

and

Microorganisms 2018, 6(3), 63; https://doi.org/10.3390/microorganisms6030063 - 04 Jul 2018

Cited by 9 | Viewed by 4506

Abstract

Abyssivirga alkaniphila strain L81^T, recently isolated from a black smoker biofilm at the Loki’s Castle hydrothermal vent field, was previously described as a mesophilic, obligately anaerobic heterotroph able to ferment carbohydrates, peptides, and aliphatic hydrocarbons. The strain was classified as a [...] Read more.

Abyssivirga alkaniphila strain L81^T, recently isolated from a black smoker biofilm at the Loki’s Castle hydrothermal vent field, was previously described as a mesophilic, obligately anaerobic heterotroph able to ferment carbohydrates, peptides, and aliphatic hydrocarbons. The strain was classified as a new genus within the family Lachnospiraceae. Herein, its genome is analyzed and A. alkaniphila is reassigned to the genus Vallitalea as a new strain of V. guaymasensis, designated V. guaymasensis strain L81. The 6.4 Mbp genome contained 5651 protein encoding genes, whereof 4043 were given a functional prediction. Pathways for fermentation of mono-saccharides, di-saccharides, peptides, and amino acids were identified whereas a complete pathway for the fermentation of n-alkanes was not found. Growth on carbohydrates and proteinous compounds supported methane production in co-cultures with Methanoplanus limicola. Multiple confurcating hydrogen-producing hydrogenases, a putative bifurcating electron-transferring flavoprotein—butyryl-CoA dehydrogenase complex, and a Rnf-complex form a basis for the observed hydrogen-production and a putative reverse electron-transport in V. guaymasensis strain L81. Combined with the observation that n-alkanes did not support growth in co-cultures with M. limicola, it seemed more plausible that the previously observed degradation patterns of crude-oil in strain L81 are explained by unspecific activation and may represent a detoxification mechanism, representing an interesting ecological function. Genes encoding a capacity for polyketide synthesis, prophages, and resistance to antibiotics shows interactions with the co-occurring microorganisms. This study enlightens the function of the fermentative microorganisms from hydrothermal vents systems and adds valuable information on the bioprospecting potential emerging in deep-sea hydrothermal systems. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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16 pages, 11233 KiB

Open AccessArticle

Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring

by

Mohamed Amine Gomri

,

Agustín Rico-Díaz

,

Juan-José Escuder-Rodríguez

,

Tedj El Moulouk Khaldi

,

María-Isabel González-Siso

and

Karima Kharroub

Microorganisms 2018, 6(2), 31; https://doi.org/10.3390/microorganisms6020031 - 12 Apr 2018

Cited by 9 | Viewed by 5692

Abstract

Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production [...] Read more.

Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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Review

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46 pages, 1965 KiB

Open AccessReview

Transfer RNA Modification Enzymes from Thermophiles and Their Modified Nucleosides in tRNA

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and

Microorganisms 2018, 6(4), 110; https://doi.org/10.3390/microorganisms6040110 - 20 Oct 2018

Cited by 27 | Viewed by 6589

Abstract

To date, numerous modified nucleosides in tRNA as well as tRNA modification enzymes have been identified not only in thermophiles but also in mesophiles. Because most modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles, they are considered [...] Read more.

To date, numerous modified nucleosides in tRNA as well as tRNA modification enzymes have been identified not only in thermophiles but also in mesophiles. Because most modified nucleosides in tRNA from thermophiles are common to those in tRNA from mesophiles, they are considered to work essentially in steps of protein synthesis at high temperatures. At high temperatures, the structure of unmodified tRNA will be disrupted. Therefore, thermophiles must possess strategies to stabilize tRNA structures. To this end, several thermophile-specific modified nucleosides in tRNA have been identified. Other factors such as RNA-binding proteins and polyamines contribute to the stability of tRNA at high temperatures. Thermus thermophilus, which is an extreme-thermophilic eubacterium, can adapt its protein synthesis system in response to temperature changes via the network of modified nucleosides in tRNA and tRNA modification enzymes. Notably, tRNA modification enzymes from thermophiles are very stable. Therefore, they have been utilized for biochemical and structural studies. In the future, thermostable tRNA modification enzymes may be useful as biotechnology tools and may be utilized for medical science. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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13 pages, 869 KiB

Open AccessReview

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering

by

Anthony J. Finch

and

Jin Ryoun Kim

Microorganisms 2018, 6(4), 97; https://doi.org/10.3390/microorganisms6040097 - 25 Sep 2018

Cited by 28 | Viewed by 7166

Abstract

Literature from the past two decades has outlined the existence of a trade-off between protein stability and function. This trade-off creates a unique challenge for protein engineers who seek to introduce new functionality to proteins. These engineers must carefully balance the mutation-mediated creation [...] Read more.

Literature from the past two decades has outlined the existence of a trade-off between protein stability and function. This trade-off creates a unique challenge for protein engineers who seek to introduce new functionality to proteins. These engineers must carefully balance the mutation-mediated creation and/or optimization of function with the destabilizing effect of those mutations. Subsequent research has shown that protein stability is positively correlated with “evolvability” or the ability to support mutations which bestow new functionality on the protein. Since the ultimate goal of protein engineering is to create and/or optimize a protein’s function, highly stable proteins are preferred as potential scaffolds for protein engineering. This review focuses on the application potential for thermophilic proteins as scaffolds for protein engineering. The relatively high inherent thermostability of these proteins grants them a great deal of mutational robustness, making them promising scaffolds for various protein engineering applications. Comparative studies on the evolvability of thermophilic and mesophilic proteins have strongly supported the argument that thermophilic proteins are more evolvable than mesophilic proteins. These findings indicate that thermophilic proteins may represent the scaffold of choice for protein engineering in the future. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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26 pages, 925 KiB

Open AccessReview

Cellulases from Thermophiles Found by Metagenomics

by

Juan-José Escuder-Rodríguez

,

María-Eugenia DeCastro

,

María-Esperanza Cerdán

,

Esther Rodríguez-Belmonte

,

Manuel Becerra

and

María-Isabel González-Siso

Microorganisms 2018, 6(3), 66; https://doi.org/10.3390/microorganisms6030066 - 10 Jul 2018

Cited by 44 | Viewed by 6805

Abstract

Cellulases are a heterogeneous group of enzymes that synergistically catalyze the hydrolysis of cellulose, the major component of plant biomass. Such reaction has biotechnological applications in a broad spectrum of industries, where they can provide a more sustainable model of production. As a [...] Read more.

Cellulases are a heterogeneous group of enzymes that synergistically catalyze the hydrolysis of cellulose, the major component of plant biomass. Such reaction has biotechnological applications in a broad spectrum of industries, where they can provide a more sustainable model of production. As a prerequisite for their implementation, these enzymes need to be able to operate in the conditions the industrial process requires. Thus, cellulases retrieved from extremophiles, and more specifically those of thermophiles, are likely to be more appropriate for industrial needs in which high temperatures are involved. Metagenomics, the study of genes and gene products from the whole community genomic DNA present in an environmental sample, is a powerful tool for bioprospecting in search of novel enzymes. In this review, we describe the cellulolytic systems, we summarize their biotechnological applications, and we discuss the strategies adopted in the field of metagenomics for the discovery of new cellulases, focusing on those of thermophilic microorganisms. Full article

(This article belongs to the Special Issue Thermophiles and Thermozymes)

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