Mining thermophiles for biotechnologically relevant enzymes: evaluating the potential of European and Caucasian hot springs.

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.

Characterization of a thermoactive endoglucanase isolated from a biogas plant metagenome.

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.

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates.

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.

Extremely thermoactive archaeal endoglucanase from a shallow marine hydrothermal vent from Vulcano Island.

Already-characterized microbial cellulases have proven to be highly useful for industrial processes, since they can withstand harsh industrial conditions with characteristics such as high thermo- and acid stability. These properties provide promising features for the process of plant biomass degradation and biofuel generation. Nevertheless, the number of known extremely thermoactive archaeal cellulases is low. Hence, the discovery of archaeal cellulases with different characteristics is crucial for the development of efficient and sustainable biorefinery. In this work, the metagenome of a high-temperature enrichment culture from marine environment of Vulcano Island was screened for the presence of novel endoglucanase-encoding genes of archaeal origin. The ORF vul_cel5A was detected, and the deduced protein was characterized as the most thermoactive endoglucanase described to date. Vul_Cel5A was identified as a thermoactive glycoside hydrolase family 5 endoglucanase, with the highest sequence identity (72-75%) to putative endoglucanases from archaeal genera. Vul_Cel5A showed the highest activity at notable 115 °C towards barley ß-glucan (210.7 U/mg), and lichenan (209.9 U/mg), and further towards carboxymethyl cellulose (38.6 U/mg) and locust bean gum (83.0 U/mg). The endoglucanase exhibited a half-life time of 46 min at 100 °C and did not show any loss of activity after incubation for 48 h at 75 °C. Furthermore, Vul_Cel5A showed high affinity to barley ß-glucan with a Km of 0.52 mg/mL and showed tolerance against various chemical reagents. Due to the outstanding high thermoactivity and thermostability and tolerance to acidic conditions, Vul_Cel5A represents a promising novel archaeal endo-ß-glucanase for application in biorefineries for an efficient biomass pre-treatment.

Carbohydrate-active enzymes identified by metagenomic analysis of deep-sea sediment bacteria.

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 ß-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 ß-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.

Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region.

NAD(P)(+)-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co(2+). Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.

Structural and biochemical characterisation of a NAD⁺-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications.

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni(2+)-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni(2+), activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD(+)/NADH, whereas no activity has been observed with NADP(+)/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn(2+). The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni(2+)-dependent activity towards 1,3-propanediol.

Comparative analysis of two members of the metal ion-containing group III-alcohol dehydrogenases from Dickeya zeae.

PURPOSE OF WORK: A pair of NAD(+)- and NADP(+)-dependent group III-alcohol dehydrogenases was characterized from the enterobacterium, Dickeya zeae, to expand our understanding of the distribution and biochemical properties of this interesting group of enzymes. Two putative group III-alcohol dehydrogenases (ADHs) were identified in the genome of Dickeya zeae. Amino acid alignments and phylogenetic analysis revealed that Adh3.1 and Adh3.2 are only distantly related (~25 % identity at the protein level). Both proteins were purified to homogeneity after heterologous expression in E. coli. A specific activity of 1.8 U/mg was measured for the NAD(+)-dependent enzyme Adh3.1 with ethanol used as substrate, while NADPH-dependent Adh3.2 preferred butanal (29.1 U/mg) as substrate. Maximum activity for Adh3.1 was at 50 °C and pH 10 and for Adh3.2 at 70 °C and pH 6. Cell viability assays were used to confirm activity towards butanal and glyoxals. Biochemical characterization and phylogenetic analyses led to the hypothesis that Adh3.1 and Adh3.2 are probably the result of an ancient gene duplication event followed by functional diversification.

A novel thermostable and glucose-tolerant ß-glucosidase from Fervidobacterium islandicum.

An open reading frame (ORF) encoding the enzyme ß-glucosidase from the extremely thermophilic bacterium Fervidobacterium islandicum has been identified, cloned and sequenced. The bgl1A gene was cloned in a pET-Blue1 vector and transformed in Escherichia coli, resulting in high-level expression of ß-glucosidase FiBgl1A that was purified to homogeneity in a two-step purification. FiBgl1A is composed of 459 amino acid residues and showed high homology to glycoside hydrolase family 1 proteins. It exhibited highest activity towards p-nitrophenyl-ß-D: -glucopyranoside with an optimum activity at pH 6.0 and 7.0 and at 90 °C. The enzyme is resistant to glucose inhibition. Furthermore, it did not require divalent cations for activity, nor was it affected by the addition of p-chloromercuribenzoate (10 mM), EDTA (10 mM), urea (10 mM) or dithiothreitol (10 mM). Addition of surfactants (with the exception of SDS) and a number of solvents enhanced the activity of FiBgl1A. It also displayed remarkable activity across a broad temperature range (80-100 °C). The thermoactivity and thermostability of FiBgl1A and its resistance to denaturing and reducing agents make this enzyme a potential candidate for industrial applications.

Complete genome sequences of Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4, polysaccharide-degrading members of the family Flavobacteriaceae.

Two members of the family Flavobacteriaceae were isolated from subseafloor sediments using artificial seawater with cellulose, xylan, and chitin as the sole carbon and energy sources. Here, we present the complete genome sequences of Krokinobacter sp. strain 4H-3-7-5 and Lacinutrix sp. strain 5H-3-7-4, which both encode putatively novel enzymes involved in cellulose, hemicellulose, and chitin metabolism.

Complete genome sequence of the marine cellulose- and xylan-degrading bacterium Glaciecola sp. strain 4H-3-7+YE-5.

Glaciecola sp. strain 4H-3-7+YE-5 was isolated from subseafloor sediments at Suruga Bay in Japan and is capable of efficiently hydrolyzing cellulose and xylan. The complete genome sequence of Glaciecola sp. 4H-3-7+YE-5 revealed several genes encoding putatively novel glycoside hydrolases, offering a high potential for plant biomass degradation.

Complete genome sequence of Carnobacterium sp. 17-4.

Members of the carnobacteria have been extensively studied as probiotic cultures in aquacultures and protective cultures in seafood, diary, and meat. We report on the finished genome sequence of Carnobacterium sp. 17-4, which has been isolated from permanently cold seawater. The genetic information reveals a new circular bacteriocin biosynthesis cluster.

Gluconeotrehalose is the principal organic solute in the psychrotolerant bacterium Carnobacterium strain 17-4.

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)-ß-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.