Search

The development of sustainable and environmentally friendly industrial processes is becoming very crucial and demanding for the rapid implementation of innovative bio-based technologies. Natural extreme environments harbor the potential for discovering and utilizing highly specific and efficient biocatalysts that are adapted to harsh conditions. This review focuses on extremophilic microorganisms and their enzymes (extremozymes) from various hot springs, shallow marine vents, and other geothermal habitats in Europe and the Caucasus region. These hot environments have been partially investigated and analyzed for microbial diversity and enzymology. Hotspots like Iceland, Italy, and the Azores harbor unique microorganisms, including bacteria and archaea. The latest results demonstrate a great potential for the discovery of new microbial species and unique enzymes that can be explored for the development of Circular Bioeconomy.Different screening approaches have been used to discover enzymes that are active at extremes of temperature (up 120 °C), pH (0.1 to 11), high salt concentration (up to 30%) as well as activity in the presence of solvents (up to 99%). The majority of published enzymes were revealed from bacterial or archaeal isolates by traditional activity-based screening techniques. However, the latest developments in molecular biology, bioinformatics, and genomics have revolutionized life science technologies. Post-genomic era has contributed to the discovery of millions of sequences coding for a huge number of biocatalysts. Both strategies, activity- and sequence-based screening approaches, are complementary and contribute to the discovery of unique enzymes that have not been extensively utilized so far., (© 2023. The Author(s).)

The major global and man-made challenges of our time are the fossil fuel-driven climate change a global plastic pollution and rapidly emerging plant, human and animal infections. To meet the necessary global changes, a dramatic transformation must take place in science and society. This transformation will involve very intense and forward oriented industrial and basic research strongly focusing on (bio)technology and industrial bioprocesses developments towards engineering a zero-carbon sustainable bioeconomy. Within this transition microorganisms-and especially extremophiles-will play a significant and global role as technology drivers. They harbor the keys and blueprints to a sustainable biotechnology in their genomes. Within this article, we outline urgent and important areas of microbial research and technology advancements and that will ultimately make major contributions during the transition from a linear towards a circular bioeconomy., (© 2022. The Author(s).)

Next-generation sequencing and computational biology have facilitated the implementation of new combinatorial screening approaches to discover novel enzymes of biotechnological interest. In this study, we describe the successful establishment of a multi-omic approach for the identification of thermostable hydrolase-encoding genes by determination of gene expression levels. We applied this combinatorial approach using an anaerobic enrichment culture from an Azorean hot spring sample grown on green coffee beans as recalcitrant substrate. An in-depth analysis of the microbial community resulted in microorganisms capable of metabolizing the selected substrate, such as the genera Caloramator, Dictyoglomus and Thermoanaerobacter as active and abundant microorganisms. To discover glycoside hydrolases, 90,342 annotated genes were screened for specific reaction types. A total number of 106 genes encoding cellulases (EC 3.2.1.4), beta-glucosidases (EC 3.2.1.21) and endo-1,4-beta-mannosidases (EC 3.2.1.78) were selected. Mapping of RNA-Seq reads to the related metagenome led to expression levels for each gene. Amongst those, 14 genes, encoding glycoside hydrolases, showed highest expression values, and were used for further cloning. Four proteins were biochemically characterized and were identified as thermoactive glycoside hydrolases with a broad substrate range. This work demonstrated that a combinatory omic approach is a suitable strategy identifying unique thermoactive enzymes from environmental samples.

A metagenomic library from DNA isolated from a biogas plant was constructed and screened for thermoactive endoglucanases to gain insight into the enzymatic diversity involved in plant biomass breakdown at elevated temperatures. Two cellulase-encoding genes were identified and the corresponding proteins showed sequence similarities of 59% for Cel5A to a putative cellulase from Anaerolinea thermolimosa and 99% for Cel5B to a characterized endoglucanase isolated from a biogas plant reactor. The cellulase Cel5A consists of one catalytical domain showing sequence similarities to glycoside hydrolase family 5 and comprises 358 amino acids with a predicted molecular mass of 41.2 kDa. The gene coding for cel5A was successfully cloned and expressed in Escherichia coli C43(DE3). The recombinant protein was purified to homogeneity using affinity chromatography with a specific activity of 182 U/mg, and a yield of 74%. Enzymatic activity was detectable towards cellulose and mannan containing substrates and over a broad temperature range from 40 °C to 70 °C and a pH range from 4.0 to 7.0 with maximal activity at 55 °C and pH 5.0. Cel5A showed high thermostability at 60 °C without loss of activity after 24 h. Due to the enzymatic characteristics, Cel5A is an attractive candidate for the degradation of lignocellulosic material.

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

To obtain new insights into community compositions of hyperthermophilic microorganisms, defined as having optimal growth temperatures of 80 °C and above, sediment and water samples were taken from two shallow marine hydrothermal vents (I and II) with temperatures of 100 °C at Vulcano Island, Italy. A combinatorial approach of denaturant gradient gel electrophoresis (DGGE) and metagenomic sequencing was used for microbial community analyses of the samples. In addition, enrichment cultures, growing anaerobically on selected polysaccharides such as starch and cellulose, were also analyzed by the combinatorial approach. Our results showed a high abundance of hyperthermophilic archaea, especially in sample II, and a comparable diverse archaeal community composition in both samples. In particular, the strains of the hyperthermophilic anaerobic genera Staphylothermus and Thermococcus, and strains of the aerobic hyperthermophilic genus Aeropyrum, were abundant. Regarding the bacterial community, ε-Proteobacteria, especially the genera Sulfurimonas and Sulfurovum, were highly abundant. The microbial diversity of the enrichment cultures changed significantly by showing a high dominance of archaea, particularly the genera Thermococcus and Palaeococcus, depending on the carbon source and the selected temperature.

Two genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87 kDa) and GH36bt enzyme Agal2 (57 kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3 U/mg at 80 °C, pH 6.5, and Agal2 225.4 U/mg at 60-70 °C, pH 6.5. Half-lives of 14 h (Agal1) and 39 h (Agal2) were obtained at 50 °C, and Agal1 showed half-lives of 4 and 2 h at 70 and 80 °C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-D-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides.

Alkaline protease produced by the halotolerant alkaliphilic Bacillus sp. strain NPST-AK15 was purified to homogeneity by the combination of ammonium sulfate precipitation, anion-exchange and gel permeation chromatography. The purified enzyme was a monomeric protein with an estimated molecular weight of 32 kDa. NPST-AK15 protease was highly active and stable over a wide pH range, with a maximal activity at pH 10.5. The enzyme showed optimum activity at 60 °C and was stable at 30-50 °C for at least 1 h. Thermal stability of the purified protease was substantially improved by CaCl2 (1.1- to 6.6-fold). The K m, V max and k cat values for the enzyme were 2.5 mg ml(-1), 42.5 µM min(-1) mg(-1), and 392.46 × 10(3) min(-1), respectively. NPST-AK15 protease activity was strongly inhibited by PMSF, suggesting that the enzyme is a serine protease. The enzyme was highly stable in NaCl up to 20 % (w/v). Moreover, the purified enzyme was stable in several organic solvents such as diethyl ether, benzene, toluene, and chloroform. In addition, it showed high stability and compatibility with a wide range of surfactants and commercial detergents and was slightly activated by hydrogen peroxide. These features of NPST-AK15 protease make this enzyme a promising candidate for application in the laundry and pharmaceutical industries.

There is a considerable potential of cold-active biocatalysts for versatile industrial applications. A psychrophilic bacterial strain, Shewanella arctica 40-3, has been isolated from arctic sea ice and was shown to exhibit pullulan-degrading activity. Purification of a monomeric, 150-kDa pullulanase was achieved using a five-step purification approach. The native enzyme was purified 50.0-fold to a final specific activity of 3.0 U/mg. The enzyme was active at a broad range of temperature (10-50 °C) and pH (5-9). Optimal activity was determined at 45 °C and pH 7. The presence of various metal ions is tolerated by the pullulanase, while detergents resulted in decreased activity. Complete conversion of pullulan to maltotriose as the sole product and N-terminal amino acid sequence indicated that the enzyme is a type-I pullulanase and belongs to rarely characterized pullulan-degrading enzymes from psychrophiles.

Subseafloor sediment samples derived from a sediment core of 60 m length were used to enrich psychrophilic aerobic bacteria on cellulose, xylan, chitin, and starch. A variety of species belonging to Alpha- and Gammaproteobacteria and to Flavobacteria were isolated from sediment depths between 12 and 42 mbsf. Metagenomic DNA purified from the pooled enrichments was sequenced and analyzed for phylogenetic composition and presence of genes encoding carbohydrate-active enzymes. More than 200 open reading frames coding for glycoside hydrolases were identified, and more than 60 of them relevant for enzymatic degradation of lignocellulose. Four genes encoding β-glucosidases with less than 52% identities to characterized enzymes were chosen for recombinant expression in Escherichia coli. In addition one endomannanase, two endoxylanases, and three β-xylosidases were produced recombinantly. All genes could be actively expressed. Functional analysis revealed discrepancies and additional variability for the recombinant enzymes as compared to the sequence-based predictions.

Two hydrothermal springs (AI: 51 °C, pH 3; AIV: 92 °C, pH 8) were analysed to determine prokaryotic community composition. Using pyrosequencing, 93,576 partial 16S rRNA gene sequences amplified with V2/V3-specific primers for Bacteria and Archaea were investigated and compared to 16S rRNA gene sequences from direct metagenome sequencing without prior amplification. The results were evaluated by fluorescence in situ hybridization (FISH). While in site AIV Bacteria and Archaea were detected in similar relative abundances (Bacteria 40 %, Archaea 35 %), the acidic spring AI was dominated by Bacteria (68 %). In spring AIV the combination of 16S rRNA gene sequence analysis and FISH revealed high abundance (>50 %) of heterotrophic bacterial genera like Caldicellulosiruptor, Dictyoglomus, and Fervidobacterium. In addition, chemolithoautotrophic Aquificales were detected in the bacterial community with Sulfurihydrogenibium being the dominant genus. Regarding Archaea, only Crenarchaeota, were detected, dominated by the family Desulfurococcaceae (>50 %). In addition, Thermoproteaceae made up almost 25 %. In the acidic spring (AI) prokaryotic diversity was lower than in the hot, slightly alkaline spring AIV. The bacterial community of site AI was dominated by organisms related to the chemolithoautotrophic genus Acidithiobacillus (43 %), to the heterotrophic Acidicaldus (38 %) and to Anoxybacillus (7.8 %). This study reveals differences in the relative abundance of heterotrophic versus autotrophic microorganisms as compared to other hydrothermal habitats. Furthermore, it shows how different methods to analyse prokaryotic communities in complex ecosystems can complement each other to obtain an in-depth picture of the taxonomic composition and diversity within these hydrothermal springs.

The present study was aimed to localize and characterize hexavalent chromate [Cr(VI)] reductase activity of the extreme alkaliphilic Amphibacillus sp. KSUCr3 (optimal growth pH 10.5). The resting cells were able to reduce about 62 % of the toxic heavy metal Cr(VI) at initial concentration of 200 μM within 30 min. Cell permeabilization resulted in decrease of Cr(VI) reduction in comparison to untreated cells. Enzymatic assays of different sub-cellular fractions of Amphibacillus sp. KSUCr3 demonstrated that the Cr(VI) reductase was mainly associated with the membranous fraction and expressed constitutively. In vitro studies of the crude enzyme indicated that copper ion was essential for Cr(VI) reductase activity. In addition, Ca²⁺ and Mn²⁺ slightly stimulated the chromate reductase activity. Glucose was the best external electron donor, showing enhancement of the enzyme activity by about 3.5-fold. The K (m) and V (max) determined for chromate reductase activity in the membranous fraction were 23.8 μM Cr(VI) and 72 μmol/min/mg of protein, respectively. Cr(VI) reductase activity was maximum at 40 °C and pH 7.0 and it was significantly inhibited in the presence of disulfide reducers (2-mercaptoethanol), ion chelating agent (EDTA), and respiratory inhibitors (CN and Azide). Complete reduction of 100 and 200 μM of Cr(VI) by membrane associated enzyme were observed within 40 and 180 min, respectively. However, it should be noted that biochemical characterization has been done with crude enzyme only, and that final conclusion can only be drawn with the purified enzyme.

A high proportion of microorganisms that colonise cold environments originate from marine sites; hence, they must combine adaptation to low temperature with osmoregulation. However, little or nothing is known about the nature of compatible solutes used by cold-adapted organisms to balance the osmotic pressure of the external medium. We studied the intracellular accumulation of small organic solutes in the Arctic isolate Carnobacterium strain 17-4 as a function of the growth temperature and the NaCl concentration in the medium. Data on 16S rDNA sequence and DNA-DNA hybridisation tests corroborate the assignment of this isolate as a new species of the bacterial genus Carnobacterium. The growth profiles displayed maximal specific growth rate at 30°C in medium without NaCl, and maximal values of final biomass at growth temperatures between 10 and 20°C. Therefore, Carnobacterium strain 17-4 exhibits halotolerant and psychrotolerant behaviours. The solute pool contained glycine-betaine, the main solute used for osmoregulation, and an unknown compound whose structure was identified as α-glucopyranosyl-(1-3)-β-glucopyranosyl-(1-1)-α-glucopyranose (abbreviated as gluconeotrehalose), using nuclear magnetic resonance and mass spectrometry. This unusual solute consistently accumulated to high levels (0.35 ± 0.05 mg/mg cell protein) regardless of the growth temperature or salinity. The efficiency of gluconeotrehalose in the stabilisation of four model enzymes against heat damage was also assessed, and the effects were highly protein dependent. The lack of variation in the gluconeotrehalose content observed under heat stress, osmotic stress, and starvation provides no clue for the physiological role of this rare solute.

The OsmC-region (osmotically induced protein family) of the two-domain esterase EstO from the psychrotolerant bacterium Pseudoalteromonas arctica has been shown to increase thermolability. In an attempt to test if these properties can be conferred to another enzyme, we genetically fused osmC to the 3'-region of the family 8 xylanase encoding gene xyn8 from P. arctica. The chimeric open reading frame xyn8-OsmC was cloned and the chimeric protein was purified after heterologous expression in Escherichia coli. Xyn8 and Xyn8-OsmC showed cold-adapted properties (more than 60% activity at 0°C) using birchwood xylan as the preferred substrate. Maximal catalytic activity is slightly shifted from 15°C (Xyn8) to 20°C for Xyn8-OsmC. Thermostability of Xyn8-OsmC is significantly changed in comparison to wild-type Xyn8. The OsmC-fusion variant showed an apparent decrease in thermostability between 40 and 45°C, while both proteins are highly instable at 50°C.

A gene encoding an esterase (estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus (osmotically induced family of proteins). The highly conserved five-residue motif typical for all alpha/beta hydrolases (G x S x G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser(106), Asp(196), and His(225). Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25 degrees C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40 degrees C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90 degrees C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C(2)-C(8)).

Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni2+ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5-8.5) and temperature range (10-90 degrees C). Highest activity of the esterases was measured at 70 degrees C (EstA) and 55 degrees C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90 degrees C. The determined half-life (t1/2) at 90 degrees C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.

The ability of a psychrotolerant microbial consortium to degrade crude oil at low temperatures was investigated. The enriched arctic microbial community was also tested for its ability to utilize various hydrocarbons, such as long-chain alkanes (n-C24 to n-C34), pristane, (methyl-)naphthalenes, and xylenes, as sole carbon and energy sources. Except for o-xylene and methylnaphthalenes, all tested compounds were metabolized under conditions that are typical for contaminated marine liquid sites, namely at pH 6-9 and at 4-27 degrees C. By applying molecular biological techniques (16S rDNA sequencing, DGGE) nine strains could be identified in the consortium. Five of these strains could be isolated in pure cultures. The involved strains were closely related to the following genera: Pseudoalteromonas (two species), Pseudomonas (two species), Shewanella (two species), Marinobacter (one species), Psychrobacter (one species), and Agreia (one species). Interestingly, the five isolated strains in different combinations were unable to degrade crude oil or its components significantly, indicating the importance of the four unculturable microorganisms in the degradation of single or of complex mixtures of hydrocarbons. The obtained mixed culture showed obvious advantages including stability of the consortium, wide range adaptability for crude oil degradation, and strong degradation ability of crude oil.

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4 degrees C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses alpha and gamma of Proteobacteria, the Bacillus-Clostridium group, the order Actinomycetales, and the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria gamma subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25 degrees C. Only a few strains were psychrophilic, with an optimal growth at 10-15 degrees C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4 degrees C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, alpha-amylase and beta-galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10 degrees C, and almost no production was detected at higher temperatures (20-30 degrees C). Catalytic activity was detected even below the freezing point of water (at -5 degrees C), demonstrating the unique properties of these enzymes.

The fls gene encoding fervidolysin, a keratin-degrading proteolytic enzyme from the thermophilic bacterium Fervidobacterium pennivorans, was isolated using degenerate primers combined with Southern hybridization and inverse polymerase chain reaction. Further sequence characterization demonstrated that the 2.1-kb fls gene encoded a 699-amino-acid preproenzyme showing high homology with the subtilisin family of the serine proteases. It was cloned into a pET9d vector, without its signal sequence, and expressed in Escherichia coli. The heterologously produced fervidolysin was purified by heat incubation followed by ion exchange chromatography and emerged in the soluble fraction as three distinct protein bands, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal-sequence analysis of these bands and their comparison with that determined from biochemically purified keratinase and its predicted protein sequence, identified them as a 73-kDa fervidolysin precursor, a 58-kDa mature fervidolysin, and a 14-kDa fervidolysin propeptide. Using site-directed mutagenesis, the active-site histidine residue at position 79 was replaced by an alanine residue. The resulting fervidolysin showed a single protein band corresponding in size to the 73-kDa fervidolysin precursor, indicating that its proteolytic cleavage resulted from an autoproteolytic process. Knowledge-based modeling experiments showed a distinctive binding region for subtilases, in which binding of the propeptide could take place prior to autoproteolysis. Assays using keratin and other proteinaceous substrates did not display fervidolysin activity, perhaps because of the tight binding of the propeptide in the substrate-binding site, where it could then function as an inhibitor.

The replication of DNA is a fundamental step in the cell cycle, which must be coordinated with cell division to ensure that the daughter cells inherit the same genomic material as the parental cell. The recently published complete genome sequences of some archaeal species together with preliminary biochemical studies suggest that the Archaea quite likely duplicate their chromosome by using replication machinery that seems to be a simplified version of the eukaryotic machinery, although their metabolic facets and their cellular morphology are prokaryotic-like. This review is focused on recent progress on the structural and functional analysis of proteins and enzymes involved in the initiation and elongation steps of DNA replication in Archaea. Differences between the genome replication apparatus of the Euryarchaea and the Crenarchaea (the two main phylogenetic divisions of the Archaea domain) are highlighted.