This document describes the characterization of enzymes from a metagenomic library generated from environmental DNA collected from hydrothermal vent sediments in Vulcano Island, Italy. Over 9,600 fosmid clones were screened for various enzyme activities. Four enzymes were selected for further characterization: three carboxylesterases and one α-arabinopyranosidase. The carboxylesterases showed distinct substrate specificities and were thermophilic and tolerant of organic solvents and metal ions. One carboxylesterase was active even after incubation at 90°C. The α-arabinopyranosidase selectively hydrolyzed p-nitrophenol-α-L-arabinopyranose. Analysis of the fosmid
1) Abscisic acid (ABA) induces stomatal closure in pea plants by raising both the cytosolic pH and nitric oxide (NO) levels in guard cells.
2) The rise in cytosolic pH occurs earlier than the increase in NO, suggesting that pH increases are upstream of NO production during ABA-induced stomatal closure.
3) Modulators that raise cytosolic pH like methylamine enhance stomatal closure and NO production by ABA, while agents that lower pH like butyrate prevent the effects of ABA.
Rolfe, RICE et al 2012 Lag Phase of Salmonella Typhimurium - J. Bacteriol.Chris Rice
This study analyzed the lag phase growth period of Salmonella enterica serovar Typhimurium using functional genomics and physiological techniques. The results showed:
1) Adaptation began within 4 minutes as genes for phosphate uptake were expressed.
2) The main lag phase transcriptional program started at 20 minutes, upregulating 945 genes for processes like transcription, translation and iron-sulfur protein assembly.
3) RNA polymerase was not poised upstream of bacterial genes rapidly induced at the start of lag phase, suggesting de novo partitioning of RNA polymerase to transcribe 522 genes within 4 minutes of leaving stationary phase.
4) Inductively coupled plasma mass spectrometry revealed accumulation of iron, calcium and manganese
Response of aquatic fern(Azolla), to watercontaminationKavitha Cingam
The document summarizes a study on the response of the aquatic fern Azolla to contamination by the antibiotic ciprofloxacin (Cipro). The study investigated Azolla's ability to uptake Cipro and the effects on its nitrogen-fixing process. The results showed that Azolla is able to uptake concentrations of Cipro above levels that classify plants as hyperaccumulators. Exposure to Cipro negatively impacted the heterocyst/vegetative cell ratio, nitrogenase activity, total nitrogen, and disrupted photosynthesis. This suggests that Cipro is toxic not only to the nitrogen-fixing cyanobacteria in Azolla but also to Azolla itself, demonstrating its potential use in phytoremediation of Cipro contamination
Grimmett et al., growth rate hypothesisIvan Grimmett
The study tested whether the growth rate hypothesis applies to five species of aquatic hyphomycetes grown in broth cultures. Samples were taken from the cultures over 56 days and analyzed for biomass accumulation, ergosterol concentration (indicator of fungal biomass), and concentrations of carbon, nitrogen, phosphorus, RNA, and DNA. Growth curves followed a rectangular hyperbola pattern. There were no consistent trends in carbon, nitrogen, phosphorus, or ergosterol concentrations related to culture age or growth rate. RNA and DNA concentrations and their ratio decreased with culture age. Only RNA concentrations were positively correlated with growth rate, supporting the growth rate hypothesis for aquatic hyphomycetes.
2009,planta, nitric oxide ros chitosan, srivastava et alNupur Srivastava
1) The study found that nitric oxide (NO) production occurs downstream of reactive oxygen species (ROS) in guard cells during chitosan-induced stomatal closure in pea plants.
2) Using fluorescent probes, the study showed that chitosan exposure led to increased ROS levels within 5 minutes, while NO levels rose after 10 minutes. Inhibiting ROS prevented the rise in both ROS and NO, but inhibiting NO only prevented the rise in NO.
3) The results suggest that chitosan signaling leads to ROS production, which then triggers NO production, and both ROS and NO are required for chitosan-induced stomatal closure.
This document discusses directed evolution of enzymes. It describes how Frances Arnold developed directed evolution techniques including error-prone PCR, DNA shuffling, and StEP to improve enzyme functionality for desired traits like activity, stability, selectivity. Her techniques combine random mutagenesis and recombination with selection to evolve enzymes, allowing optimization and redesigning of enzyme function. Directed evolution has led to enzymes catalyzing new reactions and expanded our understanding of what reactions enzymes can catalyze.
This document summarizes laboratory experiments on Pseudo-nitzschia species isolated from Monterey Bay, California. The experiments tested the effects of nutrient stress and culturing methods on toxin production and photosynthetic performance. Results showed substantial variability between clones in growth rates and toxicity levels under identical conditions. Toxin levels increased under silicon limitation and decreased with higher growth rates in chemostat experiments. Variable fluorescence measurements indicated nutrient stress impaired photosystem II and negatively correlated with toxin accumulation.
Van Houten, 2009, Desulfovibrio Paquesii Sp. Nov., A Hydrogenotrophic Sulfate...roelmeulepas
This document describes the isolation and characterization of a novel sulfate-reducing bacterium, strain SB1T, isolated from a synthesis-gas-fed bioreactor treating zinc- and sulfate-rich wastewater. 16S rRNA gene sequencing showed that strain SB1T was most closely related to Desulfovibrio gigas, with 97.5% sequence similarity. However, DNA-DNA hybridization showed only 56% relatedness between the two strains, below the threshold for species designation. Phenotypic characterization also differentiated strain SB1T from D. gigas. Therefore, a new species, Desulfovibrio paquesii sp. nov., is proposed to accommodate strain SB1T.
1) Abscisic acid (ABA) induces stomatal closure in pea plants by raising both the cytosolic pH and nitric oxide (NO) levels in guard cells.
2) The rise in cytosolic pH occurs earlier than the increase in NO, suggesting that pH increases are upstream of NO production during ABA-induced stomatal closure.
3) Modulators that raise cytosolic pH like methylamine enhance stomatal closure and NO production by ABA, while agents that lower pH like butyrate prevent the effects of ABA.
Rolfe, RICE et al 2012 Lag Phase of Salmonella Typhimurium - J. Bacteriol.Chris Rice
This study analyzed the lag phase growth period of Salmonella enterica serovar Typhimurium using functional genomics and physiological techniques. The results showed:
1) Adaptation began within 4 minutes as genes for phosphate uptake were expressed.
2) The main lag phase transcriptional program started at 20 minutes, upregulating 945 genes for processes like transcription, translation and iron-sulfur protein assembly.
3) RNA polymerase was not poised upstream of bacterial genes rapidly induced at the start of lag phase, suggesting de novo partitioning of RNA polymerase to transcribe 522 genes within 4 minutes of leaving stationary phase.
4) Inductively coupled plasma mass spectrometry revealed accumulation of iron, calcium and manganese
Response of aquatic fern(Azolla), to watercontaminationKavitha Cingam
The document summarizes a study on the response of the aquatic fern Azolla to contamination by the antibiotic ciprofloxacin (Cipro). The study investigated Azolla's ability to uptake Cipro and the effects on its nitrogen-fixing process. The results showed that Azolla is able to uptake concentrations of Cipro above levels that classify plants as hyperaccumulators. Exposure to Cipro negatively impacted the heterocyst/vegetative cell ratio, nitrogenase activity, total nitrogen, and disrupted photosynthesis. This suggests that Cipro is toxic not only to the nitrogen-fixing cyanobacteria in Azolla but also to Azolla itself, demonstrating its potential use in phytoremediation of Cipro contamination
Grimmett et al., growth rate hypothesisIvan Grimmett
The study tested whether the growth rate hypothesis applies to five species of aquatic hyphomycetes grown in broth cultures. Samples were taken from the cultures over 56 days and analyzed for biomass accumulation, ergosterol concentration (indicator of fungal biomass), and concentrations of carbon, nitrogen, phosphorus, RNA, and DNA. Growth curves followed a rectangular hyperbola pattern. There were no consistent trends in carbon, nitrogen, phosphorus, or ergosterol concentrations related to culture age or growth rate. RNA and DNA concentrations and their ratio decreased with culture age. Only RNA concentrations were positively correlated with growth rate, supporting the growth rate hypothesis for aquatic hyphomycetes.
2009,planta, nitric oxide ros chitosan, srivastava et alNupur Srivastava
1) The study found that nitric oxide (NO) production occurs downstream of reactive oxygen species (ROS) in guard cells during chitosan-induced stomatal closure in pea plants.
2) Using fluorescent probes, the study showed that chitosan exposure led to increased ROS levels within 5 minutes, while NO levels rose after 10 minutes. Inhibiting ROS prevented the rise in both ROS and NO, but inhibiting NO only prevented the rise in NO.
3) The results suggest that chitosan signaling leads to ROS production, which then triggers NO production, and both ROS and NO are required for chitosan-induced stomatal closure.
This document discusses directed evolution of enzymes. It describes how Frances Arnold developed directed evolution techniques including error-prone PCR, DNA shuffling, and StEP to improve enzyme functionality for desired traits like activity, stability, selectivity. Her techniques combine random mutagenesis and recombination with selection to evolve enzymes, allowing optimization and redesigning of enzyme function. Directed evolution has led to enzymes catalyzing new reactions and expanded our understanding of what reactions enzymes can catalyze.
This document summarizes laboratory experiments on Pseudo-nitzschia species isolated from Monterey Bay, California. The experiments tested the effects of nutrient stress and culturing methods on toxin production and photosynthetic performance. Results showed substantial variability between clones in growth rates and toxicity levels under identical conditions. Toxin levels increased under silicon limitation and decreased with higher growth rates in chemostat experiments. Variable fluorescence measurements indicated nutrient stress impaired photosystem II and negatively correlated with toxin accumulation.
Van Houten, 2009, Desulfovibrio Paquesii Sp. Nov., A Hydrogenotrophic Sulfate...roelmeulepas
This document describes the isolation and characterization of a novel sulfate-reducing bacterium, strain SB1T, isolated from a synthesis-gas-fed bioreactor treating zinc- and sulfate-rich wastewater. 16S rRNA gene sequencing showed that strain SB1T was most closely related to Desulfovibrio gigas, with 97.5% sequence similarity. However, DNA-DNA hybridization showed only 56% relatedness between the two strains, below the threshold for species designation. Phenotypic characterization also differentiated strain SB1T from D. gigas. Therefore, a new species, Desulfovibrio paquesii sp. nov., is proposed to accommodate strain SB1T.
2008 - Molecular microbial and chemical investigation of the bioremediation o...WALEBUBLÉ
The document describes a study that used laboratory-scale bioreactors to investigate the biodegradation of two-phase olive mill waste (TPOMW) by its indigenous microbiota under different conditions. The effects of nutrient addition and aeration on bioremediation and microbial community changes were evaluated. Analysis found that nutrient addition and aeration led to greater decreases in polyphenolic content and increases in the fungal to bacterial ratio. Molecular identification of bacteria and fungi in the bioreactors identified several genera present, with fungi like Penicillium and Candida dominant.
Meulepas, 2009, Enrichment Of Anaerobic Methanotrophs In Sulfate Reducing Mem...roelmeulepas
This document summarizes a study that used membrane bioreactors to enrich anaerobic methanotrophs (methane-oxidizing archaea) by coupling their metabolism to sulfate reduction. The researchers inoculated reactors with sediment from the Baltic Sea and operated them at 15°C or 30°C, with one 30°C reactor also containing granular sludge. They monitored volumetric methane oxidation and sulfate reduction rates over time. After 884 days, a 15°C reactor achieved rates of 1.0 mmol/g VSS/day, indicating enrichment. 16S rRNA gene analysis showed the archaea ANME-2a became dominant. The membrane bioreactor system effectively enriched the slow-growing methanotroph
This document describes the development of a genetically encoded fluorescent biosensor for 2-oxoglutarate (2OG) based on fluorescence resonance energy transfer (FRET). The researchers constructed the biosensor by fusing yellow fluorescent protein (YFP), the 2OG-binding GAF domain of the NifA protein, and cyan fluorescent protein (CFP) between restriction sites in a plasmid vector. They tested the biosensor in vitro and found it responded to 2OG concentrations within the physiological range observed in E. coli cells. They also optimized the peptide linker between domains. In vivo, the biosensor detected changes in 2OG levels in E. coli cells in response to different carbon sources added. The biosensor could image
- Experiments tested the effect of extracellular self-DNA (exDNA) and heterologous DNA on the growth of 6 species from different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects.
- Treatments with conspecific exDNA produced a concentration-dependent growth inhibition in all species, whereas heterologous DNA did not cause inhibition except in one bacterial species.
- The results suggest exDNA may have a general inhibitory effect on biological systems, providing a potential mechanism for self-inhibition and negative feedback observed in different organisms. Further investigation is needed to understand the molecular mechanisms of this effect.
This document describes research identifying a new laccase gene (lcc1A) in the fungus Trametes sp. I-62 using PCR-RFLP analysis. It confirms the intron/exon structures of three previously identified laccase genes (lcc1, lcc2, lcc3) by sequencing their cDNAs. Lcc1A showed 99.6% identity to lcc1 at the protein level, suggesting they are allelic variants. The identification of this laccase gene family contributes to understanding the biochemical diversity of laccases in fungi.
ABSTRACT- An experimental study was performed with viviparous animal Heterometrous fulvipes to access the cumulative effect of chronic heavy metals exposure on the activity levels of the enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Chronic heavy metal exposure resulted in variation in the enzymes levels with increase in AST and decreases in ALT, contributed to the stress induced by the heavy metals. These changes in enzymatic activity of the maternal and embryonic tissue of H. fulvipes under the influence of heavy metal, mercury and lead is suggestive of the specific impact of mercury and lead on the enzymatic pathway, prompting a further study to consolidate the finding in human study. It is pertinent that the heavy metal toxicity be well documented and appropriate precaution taken in mother and fetus to decrease its detrimental effects. Key-words- Heavy Metals, Animal models, Hepatic Enzymes, Viviparous
This document summarizes research on natural clays that exhibit antibacterial properties. Key findings include:
- Certain clays from hydrothermally altered volcanic environments can eliminate a wide range of bacteria, including antibiotic-resistant strains.
- The most effective antibacterial clay studied comes from an open pit mine in the Cascade Mountains and contains nanoscale illite-smectite and reduced iron phases.
- When hydrated, this clay releases soluble constituents like iron that are toxic to bacteria but below published minimum inhibitory concentrations. The clay appears to buffer pH and oxidation states in a way that promotes soluble iron release.
2017 - Comparison of nitrifying microbial communities of two full-scale membr...WALEBUBLÉ
Barbarroja, P., Moreno-Mesonero, L., Zornoza, A., Fernández-Navarro, J., Alonso, J.L., Muñagorri, F., García, C., Álvarez, C. (2017) Comparison of nitrifying microbial communities of two full-scale membrane bioreactors treating wastewaters from municipal solid wastes using 16S rDNA gene amplicon sequencing. 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
1) Mice were orally administered gold-core/silver-shell nanoparticles (Au/AgNPs) or a control over 7 days to examine genotoxicity.
2) Peripheral blood was collected at 7 days, 14 days, and 21 days and analyzed using biomarkers for DNA damage (γ-H2AX foci and 8-oxoG) and chromosomal damage (micronuclei).
3) Results showed an increase in γ-H2AX foci, a marker for double-strand DNA breaks, in treated mice at 14 days, but no differences in the other biomarkers between treated and control mice.
When yeast cells are exposed to anoxia (no oxygen) on a non-fermentable carbon source, they enter a state of suspended animation where all observable life processes reversibly halt until oxygen is restored. Transcriptional profiling revealed differences in gene expression between yeast exposed to carbon monoxide (CO) gas versus nitrogen (N2) gas. CO can mimic oxygen binding and led to derepression of aerobic metabolism genes compared to N2 exposure. Mutants lacking components of the mitochondrial retrograde signaling pathway recovered normally from CO but not N2 exposure, indicating its importance in the cellular response to different anoxic conditions. The study establishes yeast as a model for investigating suspended animation and oxygen-regulated gene expression.
This document summarizes a presentation on using bioassays to evaluate the genotoxicity of hospital wastewater in Jaipur, India at different concentrations. It describes how hospital wastewater contains various hazardous materials like drugs, chemicals, and pathogens. The Allium cepa (onion) test was used to analyze wastewater samples at concentrations of 100% and 75%. Various parameters like pH, acidity, and alkalinity were measured. Onion root tips exposed to wastewater samples showed increased mitotic abnormalities and chromosomal aberrations compared to controls. The study concludes that direct disposal of untreated hospital wastewater poses genotoxic and carcinogenic risks, so proper treatment is needed before disposal to protect
This document summarizes a study that analyzed changes in bacterial and archaeal communities during thermophilic composting of cattle manure using PhyloChip microarray technology. Samples were collected from different areas of a compost pile based on age and temperature. Total DNA was extracted from the samples and the 16S rRNA gene was amplified and hybridized to a PhyloChip array. Statistical analysis showed differences in microbial populations between raw manure samples and compost samples. During composting, archaeal phyla Thaumarchaeota and Thermoprotei and bacterial phyla Actinobacteria and Firmicutes increased in diversity. Ammonia-oxidizing and methanogenic microbes increased under thermophilic conditions. The
1) The olsA gene mediates the synthesis of ornithine lipids in P. aeruginosa under phosphate-limiting conditions. Ornithine lipids are phosphate-free and their production allows the bacteria to reduce phosphate utilization in its membranes.
2) Mass spectrometry analysis confirmed the production and structure of ornithine lipids in P. aeruginosa, which contain mainly C16:0 and C18:1 fatty acid chains.
3) While resistance to antimicrobial peptides increases under phosphate limitation, ornithine lipid production was found to not be required for this increased resistance or for virulence in a C. elegans infection model.
This document summarizes a study that identified a novel reactive chlorine species (RCS) defense mechanism in the bacterium Azospira suillum. The study found:
1) A sigma factor (SigF) and its anti-sigma factor (NrsF) regulate expression of genes involved in RCS defense, including a methionine-rich periplasmic protein (MrpX) and a methionine sulfoxide reductase (YedY1).
2) MrpX is strongly induced by RCS like hypochlorite and acts to scavenge it through methionine oxidation, while YedY1 regenerates reduced MrpX.
Microbial fuel cells could be used to the study growth rates of aerobic microbial species on the basis of voltage produced by them
in the microbial fuel cell assembly. A fresh culture of Rhizobium leguminosarum was added in the anode chamber of a microbial
fuel cell assembly and subsequent voltage produced by it was recorded after every fifteen minutes. The 24 ml/hr of air was
pumped in the anode chamber to maintain the dissolved oxygen level and resistance of 12 ohm was applied across the electrodes.
This process was studied in triplicates and voltage data was recorded. The graph plotted of voltage against time suggested the
growth curve of the species in the microbial fuel cell system. It was found that voltage gradually increased with time ranging from
50 mV to 190 mV with a supply of oxygen in the anode, but it declines gradually to zero in absence of aeration with time and
depletion of nutrients.
2019 - Profiling of filamentous bacteria in activated sludge by 16s RNA ampli...WALEBUBLÉ
Abstract: In this study, filamentous bacteria in the activated sludge of a WWTP were investigated throughout a one-year period using high-throughput short-read (Illumina) and full-length (PacBio) 16S rRNA gene amplicon sequencing. The results showed that a total of 28 filamentous bacteria genera
were identified using Illumina sequencing. Also, we found 25 species using PacBio sequencing, belonging to Curvibacter, Mycobacterium, Haliscomenobacter, Defluvicoccus, Sphaerotilus, Thiothrix, Leptothrix, Gordonia and Tetrasphaera genera. Active Volatile Suspended Solids (AVSS) were
calculated from ATP data contained in living microorganisms, this parameter represents the living biomass concentration, and the food/microorganisms ratio (F/M ratio) was calculated using AVSS instead of MLVSS. To assess the contribution of the F/M ratio to the variability observed in the filamentous bacteria structure we carried out distance-based linear models (DISTLM) and distancebased redundancy analysis (dbRDA).
The document contains slides from a course on microbial phylogenomics taught by Jonathan Eisen at UC Davis in winter 2016. The slides discuss various topics relating to metagenomics including the environmental genome shotgun sequencing of the Sargasso Sea, methods for binning sequences from metagenomic data like aligning to reference genomes or assembly, and examples of projects that used shotgun sequencing like the Wolbachia and glassy-winged sharpshooter projects. It also discusses challenges with assembly for metagenomic data due to variations in coverage and the DeLong lab's early work characterizing uncultured marine microbes.
Wurzbacher, Grimmett, Bärlocher, 2016. Metabarcoding fungal diversity Ivan Grimmett
This document summarizes a study that characterized fungal communities on coarse particulate organic matter (CPOM) like leaves and fine particulate organic matter (FPOM) in a stream in Nova Scotia, Canada using metabarcoding. Maple leaves were exposed in the stream for 4 weeks and 4 FPOM size fractions were collected over the same period by filtration. Pyrosequencing of the samples identified a total of 821 fungal operational taxonomic units, with 726 exclusive to particle samples and 47 to leaf samples. The study aimed to shed light on fungal processing of organic matter in streams and broaden understanding of freshwater fungal diversity.
1) The document describes the green synthesis and optimization of polyhedral gold nanoparticles using Acinetobacter sp. SW30 isolated from activated sewage sludge.
2) Various physiological and physicochemical parameters that influence the synthesis of gold nanoparticles were optimized, including culture age, cell density, gold salt concentration, temperature, and pH.
3) Polyhedral gold nanoparticles averaging 20 ± 10 nm were synthesized using a 24 hour old culture with a cell density of 2.4 × 109 cfu/ml, at 50°C and pH 9 in 0.5 mM HAuCl4 solution.
This study examines two species of photosynthetic sea slugs, Plakobranchus ocellatus and Elysia timida, that are able to retain functional chloroplasts from consumed algae for extended periods. The researchers sequenced expressed mRNA from actively photosynthesizing individuals of both species and found no evidence that nuclear genes specific to photosynthesis had been transferred from the algal food source to the slugs, despite the long-term maintenance of plastid function. This suggests the molecular basis for plastid longevity in these species does not involve lateral transfer of algal nuclear genes.
New microsoft office power point presentationKashyap Kumar
This document discusses Ectocarpus, a filamentous brown algae, as a model marine organism. Ectocarpus is found in temperate coastal regions worldwide and is used to study brown algal biology, including life cycle regulation, sex determination, morphogenesis, ecology, and stress response. Its genome was sequenced, revealing over 16,000 genes and repeated sequences that may play a role in silencing transposable elements. The sequencing also found an integrated DNA virus sequence that is silenced in some Ectocarpus strains. Ectocarpus has adapted well to survive in the intertidal zone through complex photosynthesis genes and enzymes that help with stress responses.
2008 - Molecular microbial and chemical investigation of the bioremediation o...WALEBUBLÉ
The document describes a study that used laboratory-scale bioreactors to investigate the biodegradation of two-phase olive mill waste (TPOMW) by its indigenous microbiota under different conditions. The effects of nutrient addition and aeration on bioremediation and microbial community changes were evaluated. Analysis found that nutrient addition and aeration led to greater decreases in polyphenolic content and increases in the fungal to bacterial ratio. Molecular identification of bacteria and fungi in the bioreactors identified several genera present, with fungi like Penicillium and Candida dominant.
Meulepas, 2009, Enrichment Of Anaerobic Methanotrophs In Sulfate Reducing Mem...roelmeulepas
This document summarizes a study that used membrane bioreactors to enrich anaerobic methanotrophs (methane-oxidizing archaea) by coupling their metabolism to sulfate reduction. The researchers inoculated reactors with sediment from the Baltic Sea and operated them at 15°C or 30°C, with one 30°C reactor also containing granular sludge. They monitored volumetric methane oxidation and sulfate reduction rates over time. After 884 days, a 15°C reactor achieved rates of 1.0 mmol/g VSS/day, indicating enrichment. 16S rRNA gene analysis showed the archaea ANME-2a became dominant. The membrane bioreactor system effectively enriched the slow-growing methanotroph
This document describes the development of a genetically encoded fluorescent biosensor for 2-oxoglutarate (2OG) based on fluorescence resonance energy transfer (FRET). The researchers constructed the biosensor by fusing yellow fluorescent protein (YFP), the 2OG-binding GAF domain of the NifA protein, and cyan fluorescent protein (CFP) between restriction sites in a plasmid vector. They tested the biosensor in vitro and found it responded to 2OG concentrations within the physiological range observed in E. coli cells. They also optimized the peptide linker between domains. In vivo, the biosensor detected changes in 2OG levels in E. coli cells in response to different carbon sources added. The biosensor could image
- Experiments tested the effect of extracellular self-DNA (exDNA) and heterologous DNA on the growth of 6 species from different taxonomic groups, including bacteria, fungi, algae, plants, protozoa and insects.
- Treatments with conspecific exDNA produced a concentration-dependent growth inhibition in all species, whereas heterologous DNA did not cause inhibition except in one bacterial species.
- The results suggest exDNA may have a general inhibitory effect on biological systems, providing a potential mechanism for self-inhibition and negative feedback observed in different organisms. Further investigation is needed to understand the molecular mechanisms of this effect.
This document describes research identifying a new laccase gene (lcc1A) in the fungus Trametes sp. I-62 using PCR-RFLP analysis. It confirms the intron/exon structures of three previously identified laccase genes (lcc1, lcc2, lcc3) by sequencing their cDNAs. Lcc1A showed 99.6% identity to lcc1 at the protein level, suggesting they are allelic variants. The identification of this laccase gene family contributes to understanding the biochemical diversity of laccases in fungi.
ABSTRACT- An experimental study was performed with viviparous animal Heterometrous fulvipes to access the cumulative effect of chronic heavy metals exposure on the activity levels of the enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Chronic heavy metal exposure resulted in variation in the enzymes levels with increase in AST and decreases in ALT, contributed to the stress induced by the heavy metals. These changes in enzymatic activity of the maternal and embryonic tissue of H. fulvipes under the influence of heavy metal, mercury and lead is suggestive of the specific impact of mercury and lead on the enzymatic pathway, prompting a further study to consolidate the finding in human study. It is pertinent that the heavy metal toxicity be well documented and appropriate precaution taken in mother and fetus to decrease its detrimental effects. Key-words- Heavy Metals, Animal models, Hepatic Enzymes, Viviparous
This document summarizes research on natural clays that exhibit antibacterial properties. Key findings include:
- Certain clays from hydrothermally altered volcanic environments can eliminate a wide range of bacteria, including antibiotic-resistant strains.
- The most effective antibacterial clay studied comes from an open pit mine in the Cascade Mountains and contains nanoscale illite-smectite and reduced iron phases.
- When hydrated, this clay releases soluble constituents like iron that are toxic to bacteria but below published minimum inhibitory concentrations. The clay appears to buffer pH and oxidation states in a way that promotes soluble iron release.
2017 - Comparison of nitrifying microbial communities of two full-scale membr...WALEBUBLÉ
Barbarroja, P., Moreno-Mesonero, L., Zornoza, A., Fernández-Navarro, J., Alonso, J.L., Muñagorri, F., García, C., Álvarez, C. (2017) Comparison of nitrifying microbial communities of two full-scale membrane bioreactors treating wastewaters from municipal solid wastes using 16S rDNA gene amplicon sequencing. 7th congress of European microbiologists FEMS 2017, Valencia, Spain, 9-13 July 2017.
1) Mice were orally administered gold-core/silver-shell nanoparticles (Au/AgNPs) or a control over 7 days to examine genotoxicity.
2) Peripheral blood was collected at 7 days, 14 days, and 21 days and analyzed using biomarkers for DNA damage (γ-H2AX foci and 8-oxoG) and chromosomal damage (micronuclei).
3) Results showed an increase in γ-H2AX foci, a marker for double-strand DNA breaks, in treated mice at 14 days, but no differences in the other biomarkers between treated and control mice.
When yeast cells are exposed to anoxia (no oxygen) on a non-fermentable carbon source, they enter a state of suspended animation where all observable life processes reversibly halt until oxygen is restored. Transcriptional profiling revealed differences in gene expression between yeast exposed to carbon monoxide (CO) gas versus nitrogen (N2) gas. CO can mimic oxygen binding and led to derepression of aerobic metabolism genes compared to N2 exposure. Mutants lacking components of the mitochondrial retrograde signaling pathway recovered normally from CO but not N2 exposure, indicating its importance in the cellular response to different anoxic conditions. The study establishes yeast as a model for investigating suspended animation and oxygen-regulated gene expression.
This document summarizes a presentation on using bioassays to evaluate the genotoxicity of hospital wastewater in Jaipur, India at different concentrations. It describes how hospital wastewater contains various hazardous materials like drugs, chemicals, and pathogens. The Allium cepa (onion) test was used to analyze wastewater samples at concentrations of 100% and 75%. Various parameters like pH, acidity, and alkalinity were measured. Onion root tips exposed to wastewater samples showed increased mitotic abnormalities and chromosomal aberrations compared to controls. The study concludes that direct disposal of untreated hospital wastewater poses genotoxic and carcinogenic risks, so proper treatment is needed before disposal to protect
This document summarizes a study that analyzed changes in bacterial and archaeal communities during thermophilic composting of cattle manure using PhyloChip microarray technology. Samples were collected from different areas of a compost pile based on age and temperature. Total DNA was extracted from the samples and the 16S rRNA gene was amplified and hybridized to a PhyloChip array. Statistical analysis showed differences in microbial populations between raw manure samples and compost samples. During composting, archaeal phyla Thaumarchaeota and Thermoprotei and bacterial phyla Actinobacteria and Firmicutes increased in diversity. Ammonia-oxidizing and methanogenic microbes increased under thermophilic conditions. The
1) The olsA gene mediates the synthesis of ornithine lipids in P. aeruginosa under phosphate-limiting conditions. Ornithine lipids are phosphate-free and their production allows the bacteria to reduce phosphate utilization in its membranes.
2) Mass spectrometry analysis confirmed the production and structure of ornithine lipids in P. aeruginosa, which contain mainly C16:0 and C18:1 fatty acid chains.
3) While resistance to antimicrobial peptides increases under phosphate limitation, ornithine lipid production was found to not be required for this increased resistance or for virulence in a C. elegans infection model.
This document summarizes a study that identified a novel reactive chlorine species (RCS) defense mechanism in the bacterium Azospira suillum. The study found:
1) A sigma factor (SigF) and its anti-sigma factor (NrsF) regulate expression of genes involved in RCS defense, including a methionine-rich periplasmic protein (MrpX) and a methionine sulfoxide reductase (YedY1).
2) MrpX is strongly induced by RCS like hypochlorite and acts to scavenge it through methionine oxidation, while YedY1 regenerates reduced MrpX.
Microbial fuel cells could be used to the study growth rates of aerobic microbial species on the basis of voltage produced by them
in the microbial fuel cell assembly. A fresh culture of Rhizobium leguminosarum was added in the anode chamber of a microbial
fuel cell assembly and subsequent voltage produced by it was recorded after every fifteen minutes. The 24 ml/hr of air was
pumped in the anode chamber to maintain the dissolved oxygen level and resistance of 12 ohm was applied across the electrodes.
This process was studied in triplicates and voltage data was recorded. The graph plotted of voltage against time suggested the
growth curve of the species in the microbial fuel cell system. It was found that voltage gradually increased with time ranging from
50 mV to 190 mV with a supply of oxygen in the anode, but it declines gradually to zero in absence of aeration with time and
depletion of nutrients.
2019 - Profiling of filamentous bacteria in activated sludge by 16s RNA ampli...WALEBUBLÉ
Abstract: In this study, filamentous bacteria in the activated sludge of a WWTP were investigated throughout a one-year period using high-throughput short-read (Illumina) and full-length (PacBio) 16S rRNA gene amplicon sequencing. The results showed that a total of 28 filamentous bacteria genera
were identified using Illumina sequencing. Also, we found 25 species using PacBio sequencing, belonging to Curvibacter, Mycobacterium, Haliscomenobacter, Defluvicoccus, Sphaerotilus, Thiothrix, Leptothrix, Gordonia and Tetrasphaera genera. Active Volatile Suspended Solids (AVSS) were
calculated from ATP data contained in living microorganisms, this parameter represents the living biomass concentration, and the food/microorganisms ratio (F/M ratio) was calculated using AVSS instead of MLVSS. To assess the contribution of the F/M ratio to the variability observed in the filamentous bacteria structure we carried out distance-based linear models (DISTLM) and distancebased redundancy analysis (dbRDA).
The document contains slides from a course on microbial phylogenomics taught by Jonathan Eisen at UC Davis in winter 2016. The slides discuss various topics relating to metagenomics including the environmental genome shotgun sequencing of the Sargasso Sea, methods for binning sequences from metagenomic data like aligning to reference genomes or assembly, and examples of projects that used shotgun sequencing like the Wolbachia and glassy-winged sharpshooter projects. It also discusses challenges with assembly for metagenomic data due to variations in coverage and the DeLong lab's early work characterizing uncultured marine microbes.
Wurzbacher, Grimmett, Bärlocher, 2016. Metabarcoding fungal diversity Ivan Grimmett
This document summarizes a study that characterized fungal communities on coarse particulate organic matter (CPOM) like leaves and fine particulate organic matter (FPOM) in a stream in Nova Scotia, Canada using metabarcoding. Maple leaves were exposed in the stream for 4 weeks and 4 FPOM size fractions were collected over the same period by filtration. Pyrosequencing of the samples identified a total of 821 fungal operational taxonomic units, with 726 exclusive to particle samples and 47 to leaf samples. The study aimed to shed light on fungal processing of organic matter in streams and broaden understanding of freshwater fungal diversity.
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This document discusses Ectocarpus, a filamentous brown algae, as a model marine organism. Ectocarpus is found in temperate coastal regions worldwide and is used to study brown algal biology, including life cycle regulation, sex determination, morphogenesis, ecology, and stress response. Its genome was sequenced, revealing over 16,000 genes and repeated sequences that may play a role in silencing transposable elements. The sequencing also found an integrated DNA virus sequence that is silenced in some Ectocarpus strains. Ectocarpus has adapted well to survive in the intertidal zone through complex photosynthesis genes and enzymes that help with stress responses.
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Highly diverse biological system of solar salterns with different salinities, often provide high densities of mycofloral populations, makes the salterns excellent model systems for both its diverse and activity. In this study, diversity of halophilic fungi in six stations which includes reservoir, evaporator and crystallizer pond of both Marakkanam and Tuticorin saltpans in relation to environmental parameters were carried out for a period of two years. 95 species of halophilic fungi from water and sediment samples belongs to 41 genera were recorded in both saltpans. Aspergillus and Penicillium species were recorded as dominant, vast differences in growth of each isolate at different salt concentrations in the ponds were observed. This paper also elucidated the slight fluctuations in physico-chemical parameter among the ponds with respect to seasonal variations were also recorded.
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This study investigated the potential costs of acclimatization to warmer temperatures in the reef coral Acropora millepora and its symbiotic algae. Field and laboratory experiments found that A. millepora colonies with thermally tolerant symbiont type D grew more slowly than those with sensitive type C2 under normal temperatures. Growth was severely reduced for over 18 months after a bleaching event for all colony types. The stresses of heat damage and symbiont changes may compound to significantly compromise the growth and resilience of reef-building corals.
1. BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Diversity of hydrolases from hydrothermal vent sediments
of the Levante Bay, Vulcano Island (Aeolian archipelago)
identified by activity-based metagenomics and biochemical
characterization of new esterases and an arabinopyranosidase
Antonio Placido1
& Tran Hai2
& Manuel Ferrer3
& Tatyana N. Chernikova2
&
Marco Distaso2
& Dale Armstrong2
& Alexander F. Yakunin4
& Stepan V. Toshchakov5
&
Michail M. Yakimov6
& Ilya V. Kublanov7
& Olga V. Golyshina2
& Graziano Pesole1
&
Luigi R. Ceci1
& Peter N. Golyshin2
Received: 13 March 2015 /Revised: 12 June 2015 /Accepted: 20 July 2015
# The Author(s) 2015. This article is published with open access at Springerlink.com
Abstract A metagenomic fosmid expression library
established from environmental DNA (eDNA) from the shal-
low hot vent sediment sample collected from the Levante Bay,
Vulcano Island (Aeolian archipelago) was established in
Escherichia coli. Using activity-based screening assays, we
have assessed 9600 fosmid clones corresponding to approxi-
mately 350 Mbp of the cloned eDNA, for the lipases/ester-
ases/lactamases, haloalkane and haloacid dehalogenases, and
glycoside hydrolases. Thirty-four positive fosmid clones were
selected from the total of 120 positive hits and sequenced to
yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts
were attributed to the members of ten bacterial phyla, includ-
ing Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes,
Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae,
Armatimonadetes, and Planctomycetes. Of ca. 200 proteins
with high biotechnological potential identified therein, we
have characterized in detail three distinct α/β-hydrolases
(LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new
α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes
revealed distinct substrate specificities tested against 43 struc-
turally diverse esters and 4 p-nitrophenol carboxyl esters. Of
16 different glycosides tested, the GLV12_5 hydrolysed only
p-nitrophenol-α-(L)-arabinopyranose with a high specific ac-
tivity of about 2.7 kU/mg protein. Most of the α/β-hydrolases
were thermophilic and revealed a high tolerance to, and high
activities in the presence of, numerous heavy metal ions.
Among them, the LIPESV12_24 was the best temperature-
adapted, retaining its activity after 40 min of incubation at
90 °C. Furthermore, enzymes were active in organic solvents
(e.g., >30 % methanol). Both LIPESV12_24 and
LIPESV12_26 had the GXSXG pentapeptides and the cata-
lytic triads Ser-Asp-His typical to the representatives of
carboxylesterases of EC 3.1.1.1.
Keywords Vulcano Island . Fosmids . Metagenomic library .
Screening . Hydrolase . Lipase . Esterase .
Arabinopyranosidase
Antonio Placido and Tran Hai contributed equally to this work.
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-015-6873-x) contains supplementary material,
which is available to authorized users.
* Tran Hai
dr_tranhai@yahoo.de
1
Institute of Biomembranes and Bioenergetics (CNR), Via Amendola
165/A, 70126 Bari, Italy
2
School of Biological Sciences, Bangor University,
Bangor, Gwynedd LL57 2UW, UK
3
Institute of Catalysis, Consejo Superior de Investigaciones
Científicas (CSIC), 28049 Madrid, Spain
4
Department of Chemical Engineering and Applied Chemistry,
University of Toronto, Toronto, Ontario M5S 3E5, Canada
5
Immanuel Kant Baltic Federal University,
236040 Kaliningrad, Russia
6
Institute for Coastal Marine Environment CNR, 98122 Messina, Italy
7
S.N. Winogradsky Institute of Microbiology, Russian Academy of
Sciences, 117312 Moscow, Russia
Appl Microbiol Biotechnol
DOI 10.1007/s00253-015-6873-x
2. Introduction
The genomic and metabolic diversity of prokaryotic domains
of life is an extraordinary source for the development of inno-
vative bio-based products of a high application value (Erickson
et al. 2012; Buschke et al. 2013; Cragg and Newman 2013; He
et al. 2014; Yu 2014). Marine environments contain an excep-
tional biodiversity generated and supported by a diversified
range of special substrates and extreme conditions, such as high
and low temperatures, extreme pH values, elevated salinities,
pressure, and even irradiation. The microbes capable of growth
under such extreme physico-chemical and nutritional condi-
tions, the extremophiles, are of a special interest for biotech-
nology (Glöckner and Joint 2010; Zhang and Kim 2010; Levin
and Sibuet 2012). Among extreme marine habitats, the hydro-
thermal vents outflowing in volcanic areas are inhabited by
thermophilic and hyperthermophilic microorganisms (Zusuki
et al. 2004). The latter represent a source of novel
thermoresistant enzymes with many outstanding properties
(Sellek and Chaudhuri 1999; Bruins et al. 2001; Atomi 2005;
Kubalov et al. 2009; Wemheuer et al. 2013). The enzymes from
extremophilic microorganisms, the extremozymes, become in-
creasingly attractive for modern biotechnology, especially in
bio-conversion of waste and renewable substrates for produc-
ing biochemicals, and alternative energy (Nogi and Kato 1999;
Ferrer et al. 2005; Poli et al. 2010; Blunt et al. 2014; Elleuche
et al. 2014; López-López et al., 2014; Grawe et al. 2015). Since
extremozymes also exhibit high resistances to solvents, deter-
gents, and high pressure (Alcaide et al. 2014), they became the
catalysts of choice in different industrial bio-processes
(Egorova and Antranikian 2005). In particular, the hydrolytic
enzymes such as carboxylesterases (EC 3.1.1.1), lipases (EC
3.1.1.3), and cellulases (EC 3.2.1.4) are attractive biocatalysts
for a number of commercial applications in food, laundry, phar-
maceutical, and other chemical industries (Bornscheuer 2002;
Frazzetto 2003, Panda and Gowrishankar 2005; Kennedy et al.
2011).
The Levante Bay is situated on the northeastern side of the
Vulcano Island and has an active hot gas vent field at a depth
of less than 1 m (Frazzetta et al. 1984). The above sampling
site selection therefore fulfils the following important criteria:
physical and chemical stability, salinity, elevated temperature,
and variety of electron donors and acceptors, determining di-
verse microbial metabolisms, which makes it a valuable site
for metagenomic enzyme study. Moreover, it was shown ear-
lier that some sites of the Vulcano Island hosted dozens of new
genera of cultured bacteria and archaea (White et al. 2008). In
this work, we report the results of our initial activity-based
survey of the metagenomic expression library generated from
the environmental DNA (eDNA) extracted from the above
site. We characterized three carboxyesterases and one glycosyl
hydrolase with respect to their substrate specificities and bio-
chemical properties, and presented the details on the two novel
thermostable and solvent-resistant carboxylesterases (E.C.
3.1.1.1) from the family α/β-hydrolase-6, which also exhibit-
ed a robust activity in buffers containing polar organic sol-
vents and metal ions.
Materials and methods
Site description and sample collection
The sediments (each of approx. 300 g wet weight) were col-
lected at the Levante Bay (Vulcano Island, Aeolian archipel-
ago) (38.4162° N, 14.9603° E) on October 2, 2012, at the
depth of water column of about 0.5–2.0 m; the sediment depth
was 0–0.3 m.
Bacterial strains and vectors used in this work
The Escherichia coli strains applied in this research for the
construction of a metagenomic fosmid library as well as for
cloning and protein expression, and the primer pairs for clon-
ing the most significant hydrolases are shown in Table 1.
Luria-Bertani (LB) liquid broth and LB agar media were used
to grow E. coli. Antibiotics applied in media were chloram-
phenicol (12.5 mg/l) for fosmids and ampicillin (100 mg/l) for
pET-46c Ek/LIC expression vectors cloned in E. coli.
Extraction of eDNA from the Vulcano sediment samples
and generation of the fosmid library
Extraction of eDNA from 10 g of the sediments was done
using the protocol of Meta-G-Nome DNA Isolation Kit
(Epicentre Biotechnologies; WI, USA). The quality and sizes
of the extracted DNA were evaluated using agarose electro-
phoresis, and the DNA concentration was estimated with
Quant-iT dsDNA Assay Kit (Invitrogen). DNA fragments of
30–40 kb after the end repair were recovered using electro-
phoresis in a low-melting-point agarose gel, extracted using
the Gelase (Epicentre Biotechnologies; WI, USA) and ligated
to the linearized fosmid vector pCC2FOS according to the
protocol of the manufacturer (CopyControl™ Fosmid Library
Production Kit, Epicentre). After the in vitro packaging into
the phage lambda (MaxPlax™ Lambda Packaging Extract,
Epicentre), the transfected phage T1-resistant EPI300™-T1R
E. coli cells were spread on LB agar medium containing
12.5 μg/ml chloramphenicol and incubated at 37 °C overnight
to determine the titre of the phage particles. The resulting
library dubbed V12 had a total titre of 40,000 clones. The
ten randomly chosen fosmid clones were analysed using NotI
and/or BamHI endonuclease digestion of purified fosmids to
evaluate the size of the cloned eDNA. For longer-term stor-
age, the whole library was plated onto the same solid medium,
and after an overnight growth, colonies were collected from
Appl Microbiol Biotechnol
3. the agar surface by using liquid LB medium containing 20 %
(v/v) sterile glycerol and the aliquots were stored at −80 °C.
Screening the metagenomic library V12
Single fosmid clones obtained by plating the pooled library
from the above step on LB agar were arrayed in 384-microtitre
plates containing LB medium supplemented with chloram-
phenicol. The cells were grown at 37 °C overnight and then
directly used for screening assays. Replica plates were also
produced and stored at −80 °C in the LB broth with chloram-
phenicol (12.5 μg/ml) and 20 % (v/v) glycerol for next using.
Agar-based esterase/lipase activity screening
Every six master 384-well microtitre plates were printed on
the surface of a large (22.5 cm × 22.5 cm) square Petri dish
containing LB medium supplemented with chloramphenicol
(12.5 mg/l), copy control fosmid induction solution (Epicentre
Biotechnologies; Madison, USA) (2 ml/l), and 0.5–1.0 % (v/v)
tributyrin emulsified with gum arabic (2:1, v/v) by sonication.
The replicated clones were grown for 18–40 h at 37 °C. The
active lipolytic enzymes hydrolysed tributyrin and formed
transparent zones around the colonies. The active esterase hits
were verified by using Fast Blue RR and α-naphthyl acetate
solution, as described previously (Khalameyzer et al. 1999).
Screening for cellulase activities
The cellulolytic active hits were identified by using low-
density carboxymethyl cellulose (CMC) with a final
concentration of 0.5 % weight to volume (w/v). After 48 h
of growth at 37 °C, the colonies were thoroughly removed
from the agar plates by small volumes of 0.1 % (w/v) Congo
Red (CR) solution in 20 % (v/v) ethanol. Then, the agar plates
were submerged in the new CR solution and stained for
20 min with shaking. The unbound CR was removed from
the plates by washing with 1 M NaCl two times, each for
30 min. The cellulose-active clones formed transparent zones
around the colonies. The activity confirmation in selected
clones was done in ø 9-cm Petri dishes.
Screening for other glycoside hydrolase activities
The glycosidase activity screening was carried out by using
chromogenic glycosides, such as 5-bromo-4-chloro-3-
indolyl-β-D-galactopyranoside (X-gal) for beta-
galactosidases and other substrate-non-specific glycosidases
(30 μg/ml), in which the actively expressed enzymes hydro-
lyse the glycosidic bonds and turned the colourless substrate
into insoluble indigo-like blue precipitates. The rescreening
was done in mini agar plates (ø 6 cm) by using the fluorescent
aglicon of 4-methylumbelliferyl beta-(D)-glycoside.
Screening for haloalkane dehalogenases and haloacid
dehalogenases
Clones were replicated into 96-well microtitre plates contain-
ing per well 200 μl of LB broth with chloramphenicol and the
induction solution (as above), and grown overnight (18 h) at
37 °C with shaking at 220 rpm. Then, 20 μl of reaction cock-
tail of haloacids and haloalkanoic acids (chloroacetic acid,
Table 1 Bacterial strains and vectors used for this research
Strain, vector, or primers Relevant markers and characteristics Reference
Escherichia coli EPI300 The cells contain an inducible mutant trfA and tonA genotype:
F-mcrA Δ(mrr-hsdRMS-mcrBC) φ80dlacZΔM15 ΔlacX74 recA1
endA1 araD139 Δ(ara, leu)7697 galU galK Γ- rpsL nupG trfA tonA
Epicentre, Madison, WI, USA
E. coli NovaBlue endA1 hsdR17 (rK12
−
mK12
+
) supE44 thi-1 recA1 gyrA96 relA1 lac F
′[proA+
B+
lacIq
ZΔM15::Tn10] (TetR
)
Novagen, Merck, Germany
E. coli BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo
ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5
Biolab, CA, USA
pCC2FOS-vector A copy control fosmid kit (Cat. No. CCFOS059) Epicentre, USA
pET46c/Lic Ligase-independent cloning vector kit Ek/LIC cloning kit of Novagen
(Cat. No. 71335)
Novagen, Merck, Germany
E. coli NovaBlue Transformant strain harbouring LIPESV12_24 This study
E. coli BL21(DE3) Protein expression strain harbouring LIPESV12_24 This study
E. coli NovaBlue Transformant strain harbouring LIPESV12_26 This study
E. coli BL21(DE3) Protein expression strain harbouring LIPESV12_26 This study
E. coli NovaBlue Transformant strain harbouring ABO_1197 This study
E. coli BL21(DE3) Protein expression strain harbouring ABO_1197 This study
E. coli NovaBlue Transformant strain harbouring ABO_1251 This study
E. coli BL21(DE3) Protein expression strain harbouring ABO_1251 This study
Appl Microbiol Biotechnol
4. bromoacetic acid, 1,3-dichloropropanol, 1,2-
dibromopropanol, 3-bromo-2-methylpropionic acid) at a con-
centration of 0.1 mM each in 10 mM N-(2-
hydroxyethyl)piperazine-N′-(3-propanesulphonic acid)
(EPPS) buffer pH 8.0, containing 20 % (v/v) of
dimethylsulphoxide, was added. Sealed plates were incubated
at 37 °C with shaking at 180 rpm for further 30 min. Then,
5 μl of 0.5 mM phenol red in the EPPS buffer was added to the
vials and positive hits were detected by the production of a
yellow colour due to the liberation of inorganic haloacids into
the medium. The activity was re-confirmed on agar plates.
Overnight-grown colonies were covered with a soft (0.6 %)
agar in the EPPS buffer containing the substrate cocktail (4 %
w/v). After a short incubation (30 min) at 37 °C, the hits were
verified by adding the pH indicators.
Extraction of fosmid DNA for sequencing
The fosmid DNA extraction was done by using the Large-
Construct Kit (QIAGEN, Hilden). The host chromosomal
DNA contamination in the samples was reduced by using
ATP-dependent exonuclease of Epicentre (Cambio Ltd., Cam-
bridge, UK). The purity of the fosmid DNA and the assess-
ment of the approximate size of the cloned fragment were
done by NotI and/or BamHI endonuclease digestion and frag-
ments’ visualization after agarose gel electrophoresis.
DNA sequencing, insert assembly, and annotation
The Sanger reads for terminal nucleotide stretches of each
purified fosmid were done by Macrogen Ltd. (Amsterdam,
The Netherlands). Then, the pools of the individual fosmids
were sequenced using the Illumina MiSeq platform. For the
preparation of libraries for next-generation sequencing, we
pooled five fosmids in a minicentrifuge tube in equimolar
ratios. DNA fragmentation was conducted with Bioruptor
UCD-200 sonicator (Diagenode, Belgium) using parameters
adjusted to obtain 800–1000-bp fragments. The fragment li-
braries have been prepared by NebNext Ultra DNA Library
preparation kit (New England Biolabs, USA) according to the
manufacturer’s instructions. Then, the multiplexed libraries
were sequenced on MiSeq Sequencing System (Illumina,
San Diego, USA) with a 2 × 150-bp sequencing kit.
Obtained paired end reads were subjected to stringent qual-
ity filtering and trimming by CLC Genomics Workbench 6.5
(CLCbio, Denmark), and removal of reads associated to E. coli
K12 genomic DNA and pCC2FOS vector DNA was per-
formed by the Deconseq software package (Schmieder and
Edwards 2011). Reads remaining after the filtering step were
used for de novo assembly with a CLC assembler and further
scaffolding by SSPACE software (Boetzer et al. 2011) and in
silico filling of gaps using GapFiller (Boetzer and Pirovano,
2012). Assembled contigs were checked for quality using
mapping of all the reads back to contigs and analysis of uni-
formity of coverage and distribution of broken read pairs
along the contig. Completeness of insert sequences of a contig
was confirmed by occurrence of pCC2FOS polylinker se-
quences. To identify fosmids in the pool, we initially per-
formed Sanger sequencing of the termini of each fosmid using
standard pCC2FOS sequencing primers (Epicentre, UK) and
aligned resulting reads with all contigs obtained for a pool
using a local BLAST algorithm.
Gene prediction and primary functional annotation were
done using the RAST annotation pipeline (Aziz et al. 2008;
Overbeek et al. 2014) and the MetaGeneMark annotation soft-
ware (http://opal.biology.gatech.edu). The taxonomy of the
hosts was analysed also by using BLAST2GO software
(Conesa et al. 2005). Amino acid alignment was done by
using ClustalW2 as well as HMMER tools (http://hmmer.
janelia.org, Finn et al. 2011), and the phyla and orders were
predicted using an E value < e−20
as a cut-off. The multiple
protein alignments were conducted also by using the
MUSCLE application (Edgar 2004) and ClustalW in BioEdit
software (Hall 1999) with default settings. The neighbour-
joining and maximum likelihood trees were constructed in
MEGA v.6.06 (Tamura et al. 2013) using the settings for the
Poisson model and homogenous patterning between lineages.
The bootstrapping was performed with 1000 replicates, if not
indicated otherwise. The 3D structure prediction for
LIPESV12_24 and LIPESV12_26 was analysed using
Phyr_2 (http://www.sbg.bio.ic.ac.uk/phyre2) and Pymol
(www.pymol.org).
Gene cloning and protein purification
For characterization of enzymes predicted in the positive
fosmid clones, we have chosen three carboxylesterases and
one glycosyl hydrolase. Their genes were amplified by PCR
using MyTaq™ Red DNA polymerase (Bioline, London, UK)
and the custom oligonucleotide primer pairs: LIPESV12-9-FP
and RP; LIPESV12_24-FP and RP; LIPESV12_26-FP and
RP; ABO_1197-FP and RP; ABO_1251-FP and RP; and
GLV12_5-FP and RP. The oligonucleotide sequences with
pET-46c Ek/LIC adaptors are given in Table 2. The corre-
sponding positive fosmid was used as a template to amplify
the target genes.
Cloning and expression of selected genes in E. coli
The PCR primer pairs (Table 2) with the nucleotide adapters
used in this research were designed following a ligation-
independent cloning protocol of Novagen (Darmstadt,
Germany). The reactions were done in a Techne TC-5000
cycler (CA, USA) using the following programme: 1 cycle
of 95 °C/3 min following 35 cycles of 95 °C for 30s, 55 °C
for 30s, and 72 °C for 1 min per 1000 nucleotides, followed by
Appl Microbiol Biotechnol
5. one extension at 72 °C for 5 min. The purified PCR products
were then purified, treated with an endonuclease, annealed to
His-tag harbouring the pET-46c Ek/LIC vector, and trans-
formed into E. coli NovaBlue according to the protocol of the
manufacturer (Novagen, Darmstadt, Germany). The DNA in-
serts in the resulting plasmids were verified by sequencing
services of Macrogen Ltd. (Amsterdam, The Netherlands).
The expression of hydrolases in E. coli BL-21 (DE3) was
carried out in two steps: at first, the inocula were grown in LB
medium supplemented with 100 μg/l ampicillin in an incuba-
tor at 37 °C, with shaking at 220 rpm to the OD600 of 0.8–0.9.
The cultures were then transferred to an incubator at 18 °C and
induced by adding isopropyl-β-D-galactopyranoside (IPTG)
at a final concentration of 0.5 mM. The cells were grown
overnight under the above conditions and then harvested by
centrifugation (5000g for 30 min at 4 °C). The recombinant
proteins were purified by affinity chromatography on Ni-NTA
His-Bind columns (Novagen) and gel filtration using 10-kDa
cut-off centrifugal filter units (Merck KGaA) according to the
protocol of the manufacturers. The size and purities of the his-
tagged protein preparations have been analysed by sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) using 10 % v/v precast gels (Expedeon).
Hydrolytic activity assay
The general assay methods were reported previously (Gupta
et al. 2002). In our study, the hydrolytic activities of the
LIPESV12 α/β-hydrolases were (if not mentioned otherwise)
estimated colorimetrically in 500 μl of 50 mM potassium
phosphate (K,P) buffer (pH 7.0), containing 0.1 % Triton
X-100, substrate, and enzyme as described recently (Pérez
et al. 2012; Tchigvintsev et al. 2014). Briefly, the substrates
para-nitrophenyl (p-NP)-carboxylic acid esters were added
from 10 mM stock solution in a buffer containing
acetonitrile:dimethyl sulphoxide (1:1 of v/v) to get a final con-
centration of 1 mM. The enzyme (1.8 μg) for reaction (native)
or for reference (overheated) was added from stock solution
after 1 min of preincubation of the reaction vials at an appro-
priate temperature as requested. The reactions (if not men-
tioned otherwise) were incubated in an Eppendorf
thermomixer comfort with a mixing frequency of 500 rpm.
After 10 min of incubation, if not indicated otherwise, the
reactions were stopped by adding 500 μl of cold K,P-buffer
(50 mM, pH 8.0) containing 10 mM EDTA (pH 8.0). The
stopping solution did change the pH in the reaction vial, at
the values tested, to slightly alkaline (pH 7.2–8.0) for main-
taining the released p-NP in the phenolate form. The absorp-
tion was measured on a spectrophotometer of model
JENWAY 6300 (Progen, UK), if not described otherwise, at
410 nm in the temperature range from 10 to 50 °C or at
380 nm (55–80 °C) with respect to hypsochromic shift and
blunting peak formation of the overheated p-NP. The blank
samples with all reaction components and with inactivated
enzymes were run in parallel. Before reading the absorbance
of each enzyme reaction, the absorbance of the blank was set
to 0, in order to omit the background rates caused by random
hydrolysis of the p-NP-ester substrates during incubation.
Then, the concentration of enzyme products was calculated
using simple linear regression equation (Microsoft Excel) giv-
en on each individual standard curve of p-NP (Sigma-Aldrich,
UK, PA grade) for each test series. The substrate profiling
assays for other hydrolytic activities against esters other than
p-NP esters were tested in 96-well plates using 43 structurally
diverse esters (read at 540 nm) as described recently (Alcaide
et al. 2015). Briefly, assay reactions (in triplicate) were con-
ducted at 30 °C measuring the absorbance during a total time
of 30 min each 1 min in a Synergy HT Multi-Mode Microplate
Reader (BioTek, Bad Friedrichshall, Germany). Reaction
mixture started by adding 1.3 μg protein stock solution to an
Table 2 Primers were designed
and used in this study Primersa
Oligosequences of direction 5′ to 3′
LIPESV12-9-FP GACGACGACAAGATGCGGTACCTGAATGAAGTG
LIPESV12-9-RP GAGGAGAAGCCCGGTTATTTAAAAAAAGACTTC
LIPESV12_24-FP GACGACGACAAGATGACCATCACCACCAGCGAAAG
LIPESV12_24-RP GAGGAGAAGCCCGGTTAACTTGAGGCGGGCGGGG
LIPESV12_26-FP GACGACGACAAGATGCCGCACCCCACCATCCAGAC
LIPESV12_26-RP GAGGAGAAGCCCGGTTACGATTTGCTGGAAGAGAC
ABO_1197-FP GACGACGACAAGATGCAACTGAAACACCTTTTTC
ABO_1197-RP GAGGAGAAGCCCGGTTAGGGGCGAACTTCGCGCCAGC
ABO_1251-FP GACGACGACAAGATGATGACAGCAATAATTCGTC
ABO_1251-RP GAGGAGAAGCCCGGTTAAACCACCGGGATGATGTC
GLV12_5-FP GACGACGACAAGATGCCTGTGAAGAACGTCCTTC
GLV12_5-RP GAGGAGAAGCCCGGTTACCGGAAATCCAGTTCGTAC
a
The oligomers were designed with adapter after Ek/LIC Cloning Kit instruction of Novagen (Merck, Germany)
and were purchased from Eurofins (Eurofins Genomics, Ebersberg, Germany)
Appl Microbiol Biotechnol
6. assay mixture containing 4 μl of ester stock solution
(200 mg/ml in acetonitrile), to a final concentration of
4 mg/ml, in 196 μl of 5 mM EPPS buffer, pH 8.0, containing
0.45 mM phenol red (ε at 540 nm = 8500 M−1
cm−1
).
Glycosidase activity for GLV12_5 was assayed using 15
different p-NP sugar derivatives that included α/β-glucose,
α/β-galactose, α-maltose, β-cellobiose, α/β-
arabinopyranose, α/β-arabinofuranose, α/β-xylose, β-man-
nose, α-rhamnose, and α-fucose in 96-well plates as previ-
ously described (Alcaide et al. 2015). Briefly, assay reactions
were conducted at 30 °C by adding 1 μg enzyme and 10 μl of
substrate from a stock solution (10 mg/ml freshly prepared in
45 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
(HEPES) buffer, pH 7.0) in 190 μl of 45 mM HEPES buffer,
pH 7.0, to get a final volume of each vial of 200 μl, and the
final substrate concentration was 500 μg/ml. Absorbance was
read in triplicate assays at 405 nm in a Synergy HT Multi-
Mode Microplate Reader (BioTek, Bad Friedrichshall, Ger-
many) to follow the extent of the hydrolysis (εp-NP at
405 nm = 16,325 M−1
cm−1
) during 30 min. All values were
corrected with non-enzymatic transformation as blank.
The p-NP-butyrate (C4) is visually more stable relating to
diverse temperatures and pH values of buffers as well as in the
presence of metal ions or organic solvents in comparison to p-
NP-acetate (C2). Therefore, we used p-NP-C4 in this study as
a universal substrate for biochemical enzyme characterization.
Thermal stability was determined after incubation of the en-
zyme for 5, 20, 30, and 40 min at temperatures 55, 60, 70, 80,
and 90 °C with shaking at 500 rpm. The enzyme solutions
were cooled down on ice, and the activity was estimated fol-
lowing the standard protocol as described in the BMaterials
and methods^. The half-life for LIPESV12_24 was measured
by incubating the enzyme at 55 °C, and samples were taken
after 0.5, 1, 2, 3, 4, and 24 h. Then, the retaining activities
were assayed using 1 mM p-NP-C4 as substrate.
The optimal pH for the enzyme activity was determined in
the range of pH 5.5–9.0 at 30 °C in 20 mM of different buffer
systems including sodium citrate (pH 5.5), potassium phos-
phate (pH 7.0), HEPES (pH 7.5), Tris-HCl (pH 8.0), and Tris-
HCl (pH 9.0), with incubation time of 10 min. The substrate
for the assays was 1 mM p-NP-C4. The reference variant with
inactivated enzymes was applied and used also as blank for
absorbance reading. The enzyme reactions were stopped by
adding the buffer with higher pH values for maintaining p-NP
released in a phenolate form as described above. The concen-
tration of released p-NP was determined using an individual
standard curve for each experiment as described above.
The effects of different bivalent cations (Cu2+
, Mn2+
,
Mg2+
, Zn2+
, Co2+
, and Ca2+
) each at 1 mM, and organic sol-
vents (acetonitrile, methanol, ethanol, isopropanol, 1,2-
propanediol, and dibutyl phosphate) in a range of concentra-
tions from 5 to 80 % (v/v) on the activity of the LIPESV12
proteins were also evaluated. All the enzyme tests were
performed in triplicate at 30 °C/10 min using 1 mM p-NP-
C4 as substrate as mentioned above. The average values with
standard deviations were applied.
In all cases, the specific activity of enzymes were given in
units per milligram of protein, where one unit (U) of activity
was defined as the amount of enzyme required to transform
1 μmol of substrate in 1 min under the assay conditions.
Chemicals
All chemicals used for enzymatic tests were of PA grade,
which have been purchased from Sigma-Aldrich Chemical
Co. (St. Louis, MO, USA). The kits applied in this study with
individual manufacturers have been noticed in the text.
Accession numbers
The genes cloned in this study for LIPESV12_9,
LIPESV12_24, LIPESTV12_26, and GLV12_5 are deposited
in the EMBL/GenBank/DDBJ databases under the accession
numbers KR919661, KP861227, KP861228, and KR919662,
respectively.
Results
V12 fosmid library generation and screening
the industrial relevant enzymes
As estimated by the library titration and insert restriction anal-
ysis of a set of randomly chosen fosmids, the V12 library
contained approx. 40,000 clones with cloned DNA fragments
between 30 and 40 kbp in size, which implies the V12 library
size corresponds to approximately 1.4 Gbp of the cloned
eDNA.
Using agar plates containing one of the following sub-
strates, tributyrin, carboxylmethylcellulose, mixture of
haloacids and haloalkanoic acids, or X-gal, we screened
9600 clones and identified 120 positive hits. Among them,
50 hits were positive for the hydrolysis of tributyrin showing
carboxylesterase/lipase activities, 36 hits for the hydrolysis of
X-gal showing β-glycosyl hydrolase (galactosidase) activi-
ties, 10 hits for the hydrolysis of carboxylmethylcellulose
(CMC) showing cellulase activity, and 24 hits revealed
dehalogenase activities.
Among the 120 hits, a set of 40 most active clones, which
revealed larger halo zones on agar plates containing tributyrin
(lipases/esterases (LIPES) relating hits) and cellulases
(CMCases) or strong chromogenic signals of glycosidase
(GLY) and dehalogenases (HAD), were chosen for further
analysis (some representative hits are presented on Fig. 1).
The termini of purified fosmids were Sanger-sequenced by
using standard pCC2-FOS forward and reverse primers. The
Appl Microbiol Biotechnol
7. Sanger read analysis allowed us to filter redundant hits or
those with high similarity with already-characterized proteins
or with absolute nucleotide identities with known genomes. In
that manner, six hits including four HADs, one LIPES, and
one GH were discarded from further analysis.
DNA sequence analysis of hits
The total size of the eDNA corresponding to the selected 34
hits was about 1360 kbp. By the annotation of the sequences,
we have identified about 200 open reading frames (ORFs)
encoding relevant hydrolytic enzymes of interest. In fact, most
of them showed only partial domain similarities to hydrolases
from non-redundant databases. We have chosen 60 ORFs
encoding esterases/lipases (26 hits) (EC 3.1.1.-) and β-
lactamases (9 hits) (EC 3.5.2.-), and 5 ORFs encoding
haloalkane and haloacid dehalogenases (HAD) (EC 3.8.1.-),
C-N hydrolases (5 hits) (EC 3.5.-.), and glycosyl hydrolases
(EC 3.2.1.-) (15 hits) (Supplementary Table S1). Among the
annotated ORFs, the β-lactamases, as well as some
thioesterases and the C-N hydrolase genes, were deduced
from the nucleotide sequences of the hits exhibiting tributyrin
hydrolytic activities as described in the BMaterials and
methods^ section.
Phylogenetic analysis of hydrolytic enzymes
From BLAST analysis carried out using the selected 60 ORFs
as query sequences, we have identified the proteins with the
highest identity and putative producing organisms. The 60
hydrolases were affiliated within a broad spectrum of bacterial
phyla to include Proteobacteria (E value ranging from
1.00e−35
to 0), Bacteroidetes (5.00e−48
to 0), Acidobacteria
(1.00e− 1 2 8
–0), Firmicutes (2.00e− 2 2
–5.00e− 1 2 7
),
Verrucomicrobia (5.00e−22
–9.00e−114
), Chloroflexi (0),
Spirochaetes (1.00e−99
), Thermotogae (1.00e−164
),
Armatimonadetes (1.00e−50
), and Planctomycetes
(1.00e−156
). Among only the lipolytic and dehalogenase hits,
18 associated to known and three to unknown bacterial orders.
For more details about the nearest microbial taxa from which
cloned DNA fragments were derived, see Tables S1 and S2 in
the Electronic supplementary material (ESM). The protein
families within each hydrolase group, namely, the
LIPESV12_series for lipases/esterases (EC 3.1.1.-), the
BLAV12_series for β-lactamases (EC 3.5.2.-), the C-
NV12_series for nitrilases and other C-N hydrolases (EC
3.5.-.-), the HADV12_proteins of haloalkane and haloacid
dehalogenases (EC 3.8.1.-), and the GLV12_series for glyco-
syl hydrolases (EC 3.2.1.-), have been established using inter-
active sequence similarity searching and HMMER software
and is presented in Supplementary Table 2. The clan-specific
phylogenetic positions of the lipases/esterases (LIPESV12_),
9 beta-lactamases (BLAV12_), and 5 C-N hydrolases (C-
NV12_), in lipolytic positive hits have been also highlighted
in Figs. S1, S2, and S3 of the ESM, respectively. The relation-
ship between five putative HADV12_proteins and their
nearest counterparts is presented in Fig. S4. As the
HADV12_3 and HADV12_4 were related to α/β-hydrolase
superfamily including trehalose phosphatases, the other
HADV12_proteins clustered nearly to their counterparts from
Desulfarculus baarsii and Microbulbifer agariliticus
(Fig. S4). The phylogenetic analysis for 15 GLV12_proteins
is also illustrated in Supplementary Fig. S5. The phyloge-
netic position of GLV12_5 enzyme, one of the glycosyl
hydrolases identified with the largest halo zone on CMC
(Fig. 1) with 37 other closely clustered proteins. As a
result of the phylogenetic analysis, we have chosen a set
of significant new hydrolases, particularly three LIPESV12
(Fig. 2) and one GLV12 enzymes, for further biochemical
characterization.
Biochemical characterization of His-tagged proteins
Substrate specificity of LIPESV12_enzymes
After verifying the nucleotide sequences by the Sanger
readers, we purified three 6His-tagged LIPESV12 proteins
(LIPESV12-9, LIPESV12_24, LIPESV12_26) and one gly-
cosyl hydrolase (GLV12_5) to homogeneity using Ni-NTA
affinity chromatography and ultrafiltration with 10 kDa cut-
off. The relative molecular masses of the purified protein prep-
arations were confirmed by SDS-PAGE and are illustrated in
Fig. S6. For the biochemical characterization, we applied dif-
ferent p-NP esters such as p-NP-C2, p-NP-C4, p-NP-laurate
(C12), and p-NP-palmitate (C16). The standard enzyme tests
have been done as described in the BMaterials and methods^.
As references, we have chosen two carboxyesterases,
ABO_1177 and ABO_1521, which were characterized recent-
ly (Tchviginsev et al. 2014). All of the LIPESV12 hydrolases
under standard assay conditions showed higher activities
against the p-NP-acetate in comparison to the referent ABO
enzymes, which preferred the p-NP-C4 as optimal substrate
(Table 3). The hydrolytic activities of LIPESV12 enzymes
against long-chain fatty acid p-NP-esters were significantly
lower, even not detectable for LIPESV12-9 and ABO_1251
even after 50-min incubations. After 50 min of reaction under
the conditions described in the BMaterials and methods^, the
other three, LIPESV12_24, LIPESV12_26, and ABO_1197,
were significantly activated also in the hydrolysis of the long-
chain ester p-NP-C16 and released approx. 128.0, 554.0, and
12.0 μmol p-NP per mg protein, respectively. Further, to study
the substrate specificities of the LIPESV12 enzymes with re-
spect to other esters with diverse chain lengths and stereo-
configurations in more detail, we performed the substrate fin-
gerprinting, with the results presented below.
Appl Microbiol Biotechnol
8. Substrate fingerprinting for the representative LIPESV12
enzymes
A total of 43 ester-like chemicals, other than p-NP esters,
were used to evaluate the substrate ranges and specific ac-
tivities (U/mg) of the three LIPESV12 enzymes at pH 8.0
and at 30 °C (Fig. 3). Under our assay conditions, the three
ester hydrolases hydrolysed short-to-medium-chain-length
tri-acyl-glycerides and alkyl esters, but with different ranges
of reactivities and orders of preference. Thus, substrate fin-
gerprints revealed that V12_26 (33 positive substrates) ex-
hibited the widest substrate range. Among the three en-
zymes, V12_26 did show more lipase character, as com-
pared to V12_24 and V12-9; this was demonstrated by its
higher capacity to hydrolyse methyl and ethyl hexanoate
and octanoate and ethyl decanoate. Halogenated esters were
also hydrolysed by V12-9 and V12_26, with the latter being
the only one acting against halogenated esters with double
bonds (i.e. the alkenyl halogenated ester methyl 2-bromo-2-
butenoate). Under our assay conditions, all three ester hy-
drolases were also found to be enantio-selective to different
degrees and preferences for at least eight chiral esters, in-
cluding methyl-(±)-mandelate, methyl-(±)-lactate, (±)-
menthylacetate, (±)-neomenthyl acetate, (±)-glycidyl 4-
nitrobenzoate, (±)-pantolactone, and (±)-methyl (S)-3-
hydroxybutyrate. Three hydrolases further utilized tri-O-
acetyl-glucan and two the carbohydrate ester α-D-glucose
pentaacetate (Fig. 3).
LIPES
CMC-aseGLY
HAD
hits
hit
hit
hits
LIPES
CMC-aseGLY
HAD
hits
hit
hit
hits
Fig. 1 Agar-based activity
screens of lipolytic enzymes
(LIPES), haloalkane
dehalogenases (HAD),
glycosidases (GLY), and
cellulases (CMC-ase) from the
Vulcano fosmid library. The halo
zones produced by hits are
indicated. Among them, the HAD
hits, as mentioned in the
BMaterials and methods^, have
been rescreened from microtitre
plate-based activity screening
LIPESV12 24
WP 013259677.1 abhydrolase Desulfarculus baarsii
WP 006420035.1 esterase delta proteobacterium NaphS2
WP 028322184.1 hyp. protein Desulfobacterium anilini
WP 027358265.1 hyp. protein Desulforegula conservatrix
WP 027982158.1 hyp. protein delta proteobact. PSCGC 5342
WP 013032277.1 alpha/beta hydrolase Nitrosococcus halophilus
WP 007414066.1 alpha/beta hydrolase Pedosphaera parvula
LIPESV12 26
WP 012844996.1 alpha/beta hydrolase Rhodothermus marinus
WP 022835384.1 alpha/beta hydrolase Salisaeta longa
WP 013061539.1 alpha/beta hydrolase Salinibacter ruber
LIPESV12-9
WP 002775684.1 abhydrolase Leptonema illini
WP 004767837.1 phospholipase Leptospira kirschneri
WP 000816747.1 abhydrolase Leptospira interrogans
WP 020772540.1 phospholipase Leptospira alstoni
WP 002998695.1 phospholipase Leptospira weilii99
27
100
98
100
100
97
87
63
100
99
96
100
49
76
0.2
Fig. 2 Neighbour-joining
phylogenetic tree of the
carboxylesterases LIPESV12-9,
LIPESV12_24, and LIPESV12_
26 and their closely clustered
enzymes. The multiple protein
alignment was conducted using
the MUSCLE application (Edgar,
2004) and BioEdit software (Hall,
1999) with default settings. The
phylogenetic neighbour-joining
trees were constructed using
MEGA v.6.06 (Tamura et al.,
2013) as described in the
BMaterials and methods^ with
100 bootstrap replicates. The
scale bar reflects the number of
substitutions per position
Appl Microbiol Biotechnol
9. Influence of temperature on stability and activity
of LIPESV12_24
Since the V12 library was generated from a hydrothermal
site, we expected LIPESV12 enzymes to exhibit some
habitat-specific features. Indeed, not all of the purified
enzymes exhibited their thermophilic nature. The maximal
activity for activity of LIPESV12_24 was at about 70–
80 °C (∼2800 and ∼3980 U/mg), while LIPESV12_26
reached its maximum activity at 50 °C (∼1312 U/mg). A
summary of the enzyme activity vs temperature profile is
shown in Table 4.
The thermostability profiles of LIPESV12_24 are shown in
Fig. 4. Noteworthy, the protein manifested a robust residual
activity at temperatures ranging from 55 to 90 °C. After 5 and
10 min of incubation at 60–70 °C, the activity was highest and
decreased after 40 min of incubation. After a short incubation at
90 °C (for 5 min), the enzyme revealed a lower yet significant
activity (∼ 203 U/mg) pointing at its thermostability. The en-
zyme became inactive only after a longer incubation time
(40 min, 90 °C). The half-life of LIPESV12_24 at 55 °C was
also estimated by incubating the enzyme at the temperature and
withdrawing aliquots for measurement after 0.5, 1, 2, 3, 4, and
24 h by the manner described in the BMaterials and methods^.
Table 3 Substrate specificities, as maximum activities, of the LIPESV12 enzymes in comparison to ABO esterases as references
Enzymes/substratea
p-NP-C2 p-NP-C4 p-NP-C12b
p-NP-C16b
LIPESV12_9 1145.5 ± 5.3 334.4 ± 2.3 0 0
LIPESV12_24 960.0 ± 0.5 392.4 ± 0.6 42.1 ± 1.2 127.6 ± 0.6
LIPESV12_26 1053.4 ± 1.7 806.0 ± 0.5 314.4 ± 2.7 553.5 ± 1.7
ABO_1197 61.3 ± 6.6 254.7 ± 1.8 110.4 ± 7.3 10.2 ± 12.6
ABO_1251 650.1 ± 4.5 2100.0 ± 0.3 57.4 ± 5.8 0
a
Enzyme reactions were carried out as described in the BMaterials and methods^. The p-NP-esters were added from 10 mM stock solution of each to get
an end concentration of 1 mM. The enzyme assays were set up in triplicate
b
The incubation time prolonged to 50 min. The specific activities corresponding to units per milligram of protein with standard deviation (±SD) rounded
to decimal were given. Protein concentration here and hereafter was estimated by using the standard Bradford reagent kit B-6916 from Sigma
Fig. 3 Substrate profiles of the enzymes with a set of 43 structurally
diverse compounds. The specific activities were calculated in triplicate,
and average values with standard deviation are shown. The specific
esterase/lipase activity of each enzyme (in units/mg protein) against a
set of structurally diverse esters was measured after incubation at 30 °C
each minute within 30 min using 1.3 μg protein at pH 8.0 in 5 mM EPPS
buffer and 4 mg/ml esters. Of the total 43 compounds, the LIPESV12-9
(solid bar) hydrolysed 17 esters, LIPESV12_24 (grey bar) hydrolysed 15
esters, and LIPESV12_26 (open bar) hydrolysed 33 esters. Some of the
activity values (for the cases of ethyl myristate, ethyl benzoate, methyl
decanoate, methyl oleate, (R)-(+)-glycidol, (S)-(-)-glycidol, and gama-
valerolactone) were negligible and we rounded to 0
Appl Microbiol Biotechnol
10. The LIPESV12_24 exhibited about 60 and 40 % its maximum
activity (100 % activity corresponded to 610 ± 0.5 U/mg) after
3 and 4 h of incubation. Therefore, its half-life was estimated as
being approx. 3–3.5 h.
Effects of pH and bivalent cations on the activity
of enzymes
We have stopped the enzyme reaction by adding cold alkaline
buffer containing EDTA (pH 8.0) in order to quench the re-
leased p-NP into para-nitrophenylate form allowing the col-
orimetric measurement of the latter at a standard wavelength
of 410 nm. These measurements were usefully applied to the
pH activity optimum determination. The pH range was
established at 5.5, 7.0, 7.5, 8.0, and 9.0 by applying citrate,
phosphate, HEPES, and Tris-HCl buffers as described in the
BMaterials and methods^. Both the LIPESV12_24 and
LIPESV12_26 enzymes altered significantly the activities at
acidic pH values: under slightly acidic conditions (pH 5.5), the
activity of LIPESV12_24 decreased for about 25 % while the
LIPESV12_26 was fully inactivated (remained <1 %) in com-
parison to their activities at pH 7.0. Both enzymes were par-
ticularly active at slightly alkaline (pH 8.0) and neutral (7.0)
pH values (Fig. 5). The inhibitory effect was observed also for
the LIPESV12_26, even at pH 8.0. On the contrary, the
LIPESV12_24 retained about 75 % of its activity at pH 9.0
in comparison to its highest values (Fig. 5).
The enzyme kinetics may also be influenced by cofactors
such as metal ions. Volcanic environments are characterized
by the presence of metal ions at elevated concentrations. We
measured the influence of bivalent cations such as Cu2+
, Co2+
,
Zn2+
, Mn2+
, Mg2+
, and Ca2+
on the activity of purified
LIPESV12_24 during short-term (5 min) and long-term
(50 min) incubations. After 5 min of the exposure, the enzyme
activity was increased in comparison to the control. Reaction
mixtures containing calcium, cobalt, and magnesium salts,
each of 1 mM, showed almost a twofold increase in the activ-
ities as compared to the control (Fig. 6). Long incubation of
the enzyme in metal ions in most cases seemed to be not result
the increase of enzyme activities. In opposite, some inhibitory
effects of copper, zinc, and manganese were observed after a
long-term incubation (data not shown). The common effects
of the divalent metal ions on the activities of the
LIPESV12_24 and LIPESV12_26 enzymes are clearly con-
sistent with the metal-rich conditions in the environment of
origin of the source microorganisms.
Effect of organic solvents
We analysed the activity of both enzymes using p-NP-butyrate
in the buffer supplemented at different concentrations with 1,
2-propanediol, isopropanol, ethanol, methanol, acetonitrile,
and dibutyl phosphate, the main component of degradation
products of tributyl phosphate (TBP), the extractant used in
the nuclear fuel reprocessing known as the PUREX process
(Table 5). By adding to 5 % (v/v) dibutyl phosphate in the
potassium phosphate buffer (pH 7.0), the specific activities
of both, LIPESV12_24 and LIPESV12_26, enzymes were
strongly stimulated (∼2600 and ∼955 U/mg, respectively).
Interestingly, both LIPESV12_enzymes were activated by
acetonitrile with a final concentration of 30 % (v/v) (∼1746
Table 4 Temperature profiles of the V12 α/β-hydrolases
Activity (%)b
T (°C)a
LIPESV12_24 LIPESV12_26
10 62.0 ± 0.5 20.0 ± 0.5
20 57.0 ± 0.5 72.0 ± 0.5
30 100c
100e
37 406.0 ± 0.5 116.0 ± 1.0
45 451 ± 0.5 200.0 ± 1.0
50 517 ± 0.5 258.0 ± 0.1
60 100d
87.0 ± 0.5e
70 127.0 ± 0.5 n.d
80 173.0 ± 0.5 n.d
a
Enzyme reactions were carried out in triplicate as described in the
BMaterials and methods^ by using p-NP-butyrate (1 mM) as substrate
b
The measurements of absorbance in the range 10–50 °C were conducted
at the wavelength 410 nm and at 60–80 °C—at 380 nm, since the heated
p-NP revealed a hypsochromic shift as mentioned in the BMaterials and
methods^, and the results with ±SD are given, where 100 % activity of
c
359.0 ± 0.5, d
2219.0 ± 0.5, and e
524 ± 1.0 U/mg of proteins
Fig. 4 The thermostability of LIPESV12_24 as a function of incubation
time. Since the LIPESV12_24 showed a broad temperature profile and
actively hydrolysed the substrates at high temperatures (see Table 3), we
checked its thermostability at different incubation times (5, 10, 30, and
40 min) and temperatures of 55, 60, 70, 80, and 90 °C. Residual activity
was detected using standard test at 30 °C as described in the BMaterials
and methods^ section. The activity values given on the top of each column
were corresponded also to incubation time: after 5 (solid bar), 20 (light-
grey), 30 (dashed grey), and 40 (open bar) min using 1 mM p-NP-
butyrate
Appl Microbiol Biotechnol
11. and 954.0 U/mg, respectively). Although to a lesser extent
than in dibutyl phosphate and acetonitrile, LIPESV12_ pro-
teins revealed a high specific activity in different alcohols,
such as 30 % of 1,2-propanediol (∼1039 U/mg for
LIPESV12_24 and ∼592 U/mg for LIPESV12_26) and
10 % ethanol (∼500 and ∼542 U/mg, respectively) (Table 5).
We have tested also ethanol of concentration up to 30 % (v/v),
but no activation effects have been observed (data not shown).
The LIPESV12_24 enzyme displayed the following activities
in methanol: ∼685, ∼945, and ∼769 U/mg in buffer supple-
mented with 10, 20, and 30 % (vol/vol), respectively. At
higher concentrations of methanol, the LIPESV12_24 still
revealed a significant activity: 126 U/mg at 40 % and
127 U/mg at 50 % (v/v). The similar profile showed also the
LIPESV12_26, which in the presence of 30 % (v/v) methanol
had activity of ∼914 U/mg (Table 5). Both LIPESV12 en-
zymes were also active in 20 % (v/v) isopropanol and devel-
oped significant catalytic activities of ∼732 and ∼341 U/mg,
respectively. The obtained data indicated the metagenome-
derived new α/β-hydrolases were capable of retaining their
activities in different polar protic and aprotic solvents.
Moreover, we have determined also some kinetic parame-
ters such as Michaelis constant (Km) for the LIPESV12 α/β-
hydrolases. The concentrations of substrates, either p-NP-C3
or p-NP-C4, varied between 1 and 40 mM. Under the standard
assay conditions, the Km for p-NP-C4 was 100 μM for
LIPESV12_26 and 10 μM for LIPESV12_24. For the
ABO_1197 and ABO_1251, the activity varied as per sub-
strate between 780 μM (p-NP-C3) and 5 μM (p-NP-C4), re-
spectively, as reported recently (Tchigvintsev et al. 2014).
Structure prediction analysis for two new hydrolases:
LIPESV12_24 and LIPESV12_26
Both HMMER (Supplementary Table S2) and BLAST indi-
cated that the LIPESV12_26 has a high identity (70–81 %)
with the proteins WP_012844996.1 and WP_014067971.1 of
Rhodothermus marinus, while LIPESV12_24 exhibited a sig-
nificantly lower identity (50 %) with α/β-hydrolases of
D. baarsii, delta-proteobacterium NaphS2 (42 %),
Desulfatiglans aniline (39 %), and Desulforegula
conservatrix (38 %). Meanwhile, the maximum identities to
the LIPESV12-9 were found to predict α/β-hydrolase homo-
logues from Leptonema illini (WP_002775684.1) of 47 % and
from Leptospira alstoni (WP_020772540.1) of 42 %. A more
detailed phylogenetic analysis for the above findings is pre-
sented in Fig. 2.
The putative catalytic triads and the reactive centres of the
LIPESV12_24 and LIPESV12_26 with 272 and 275 amino
acids, respectively, were analysed by using the Phyre2 and
PyMol software available in the abovementioned tools.
The α/β fold of different lipases and esterases contains the
typical GXSXG. This motif together with a conserved histi-
dine and aspartate (or glutamate) generates the catalytic triad
of α/β-hydrolases (Ollis et al. 1992, Jaeger et al. 1994), which
is also present in both V12 hydrolases (see Figs. S7A and B).
The possible catalytic triads for LIPESV12_24 and
LIPESV12_26 were identified with amino acids S107,
D217, and H245, and S94, D218, and H245, respectively.
The distances between H245/D217 and H245/D218 in the
amino acid sequences of LIPESV12_24 and LIPESV12_26
were 28 and 27 amino acid residues, respectively, similar to
the H and D/E distance of about 29 residues reported previ-
ously for carboxylic ester hydrolases (Ollis et al. 1992, Jaeger
et al. 1994). Based on the three-dimensional model, a shorter
distance (3.1 Å) between His245 and Asp217 in the V12_24
in comparison to the distance between His245 and Asp218 in
its counterpart reached 5.9 Å (Fig. S7A and B). The
80.5
398.6
432.4
673.6
504.5
5.0
509.4
385.2
275.9
18.6
0
100
200
300
400
500
600
700
5.5 7.0 7.5 8.0 9.0
U/mgprotein
pH
LIPESV12_24
LIPESV12_26
Fig. 5 Hydrolytic activities of LIPESV12_24 and LIPESV12_26.
LIPESV12_24 against p-NP-butyrate at different pH values. A wide pH
spectrum of the enzyme activities was demonstrated. The data indicates
that the enzymes were neutrophilic or slightly alkaline
0
200
400
600
800
1000
378
889
527
960
591
673
788
520
674
495
850
804
560
650
U/mgprotein
Metal ions (1mM)
Fig. 6 Influences of bivalent metal ions: Cu+2
, Co+2
, Zn+2
, Mn+2
, Mg+2
,
and Ca+2
on the activity of LIPESV12_24 (solid bar) and LIPESV12_26
(grey bar). The reaction mixture in each vial was preincubated at 30 °C in
the presence of 1 mM bivalent cation for 5 min. Then, the substrate
(1 mM of p-NP-butyrate) was added and activity was measured under
standard conditions as described in the BMaterials and methods^. Both
enzymes were activated by the addition of cations: Co2+
, Ca2+
, and Mn2+
stimulated activities twofold to threefold towards the substrate in
comparison to the control variants. After a longer incubation period
(50 min), activities decreased, and in the case of Cu2+
and Mn+2
, the
activity of LIPESV12_24 dropped from ∼527 and ∼788 U/mg (5 min
of incubation) to ∼21 and ∼73 U/mg (50 min of incubation), respectively
Appl Microbiol Biotechnol
12. polypeptides contain eight α-helix turns and six typical β-
sheets, which are also predicted for enzymes of the α/β-hy-
drolase_6 superfamily.
Characterization of GLV12_5
The glycosyl hydrolase selected for this study, GLV12_5, was
derived from a fosmid exhibiting the strongest CMC hydroly-
sis halo (seen as the largest clearance zone in Fig. 1). The
homology as well as the phylogenetic relationship of the
GLV12_5 was analysed and presented also in the HMMER
electronic supplementary table (Table S2) and in Fig. 7. The
new glucosyl hydrolase was clustered with some
uncharacterized proteins and also some known cellulases
and glycosyl hydrolases from Opitutaceae bacterium species
(Fig. 7). The PCR product of the gene was cloned using the
primer pair GLV12_5-FP and RP with pET46 Ek/LIC
adapters, as described in the BMaterials and methods^
(Table 2). Substrate specificity of the GH enzyme revealed
activity towards just one of 16 sugar esters tested, namely, p-
NP-α-arabinopyranose (see Supplementary Table S3). Under
the standard assay conditions, the maximal activity was at
35 °C and pH 5.5 (∼2683 ± 26 U/mg). Therefore, the enzyme
should be considered as α-arabinopyranosidase with a quite
restricted substrate profile.
Discussion
The volcanic and geothermal sites are popular subjects for the
enzyme biodiversity prospecting, as they harbour microbial
communities often composed of extremophiles (Handelsman
et al. 1998; Ferrer et al. 2007; Simon and Daniel 2011;
Wemheuer et al. 2013; Alcaide et al. 2014). In the present
study, we provided a first report on the metagenomic
prospecting of hydrolytic enzymes from the shallow hydro-
thermal vents of the Vulcano Island, Italy, a renowned hot spot
of biodiversity of extremophilic microorganisms.
Our results demonstrated that using enzyme activity
screening methods, we have successfully obtained numerous
hydrolytic enzymes, including lipases and esterases,
lactamases/nitrilases, dehalogenases, and glycosyl hydrolases.
From DNA sequence analysis of positive fosmid hits, we have
predicted and annotated about 200 genes with either full
length or with domain homologies to the non-redundant pro-
tein databases. From the annotated ORFs, 60 complete pro-
teins deduced have been analysed in more detail. Within this
small set of peptide sequences, by performing homology anal-
ysis using HMMER and BLAST tools with a low E value cut-
off filter, ten known bacterial phyla were predicted as potential
hosts of the cloned DNA. The analysis indicated a high taxo-
nomic complexity in the microbial community of the Levante
Bay in the Vulcano Island, the enzymatic diversity of which is
far from being fully explored. As expected, all affiliated orders
were related to marine bacteria, mostly thermophiles from the
phyla Proteobacteria, Bacteroidetes, and Acidobacteria (see
also Table S1). The organisms donating the eDNA fragments
in positive clones were restricted to the organisms typically
present in thermophilic, as well as in metal ion- and sulphur-
rich milieus. The enzymes studied here in detail,
LIPESV12_24, LIPESV12_26, and also LIPESV12-9 and
GLV12_5, have been derived from the yet uncultured organ-
isms of the orders Desulfarculales and Incertae sedis from
order II of Bacteroidetes, and from the uncharacterized protein
of some Opitutaceae species (Fig. 2), respectively. The phy-
logenetic position and homology of both LIPESV12 enzymes
with their counterparts from non-redundant protein databases
suggested they belong to the α/β-hydrolase_6 (ABHD6) su-
perfamily. The highest similarity, as well as the closest clus-
tering of LIPESV12_24 to the homologous proteins from ma-
rine bacteria of the order Desulfarculales, and of
LIPESV12_26 to the R. marinus, and, at the same time, their
distant phylogenetic placement one from another (see Fig. 2,
Fig. S1, and Table S2), suggests their distinct origin despite a
somewhat similar function. Both the LIPESV12 proteins pos-
sessed the typical positions of catalytic centres for the
Table 5 Hydrolytic activity of
LIPESV12_24 and LIPESV12_
26 in solvents
KP buffer (50 mM)
supplemented with
Solvent added (%, v/v) Spec. act. in U/mg (protein)a
LIPESV12_24 LIPESV12_26
KP buffer None 360.0 ± 0.1 524.0 ± 0.5
Dibutyl phosphate 5 2600.0 ± 0.5 955.0 ± 0.5
Acetonitrile 30 1746.0 ± 0.5 954.0 ± 0.1
1,2-Propanediol 30 1039.0 ± 0.5 592.0 ± 1.0
Methanol 30 769.0 ± 0.3 914.0 ± 1.0
Isopropanol 20 732.0 ± 0.1 341.0 ± 1.0
Ethanol 10 500.0 ± 0.5 542.0 ± 0.1
a
Enzyme reactions were carried out as described in the notes of Tables 3 and 4. The enzyme assay was set up in
triplicate, and the results ± SD are given in the table
Appl Microbiol Biotechnol
13. carboxylesterases with the conserved triads Ser-His-Asp. The
identified numbers of helix turns and beta-sheets indicated the
similarity of both enzymes to the carboxylic lipase/esterase
group, consistent with the data obtained in the primary poly-
peptide sequence analysis. But the predicted 3D structures of
the LIPESV12 proteins were clearly different from the canon-
ical fold of α/β-hydrolases from other extremozymes such as
carboxylic ester hydrolases from hyperthermophiles
(Levisson et al. 2009) and the referent proteins in this re-
search: ABO_1197 and ABO_1251 from Alcanivorax
bokumensis (see also Tchigvinsev et al. 2014). The nearest
homologues of LIPESV12_24 are the α/β-hydrolase
YP_003808833.1 from D. baarsii DSM 2075 (50 % sequence
identity), which has been annotated as a lipase (cd00741) with
a preference to long-chain esters, and several α/β-hydrolases
from the family 6 (ABHD6), which are active in hydrolysis of
long-acyl-chain glycerol esters such as arachidonic glycerol
ester. In the structural models of LIPESV12_24 and
LIPESV12_26, the active sites are located in a deep hydro-
phobic groove and include the putative catalytic triad residues
Ser107/94, His245/245, and Asp217/218, respectively. In
both active sites, the catalytic serine residues (Ser107 and
Ser94) are located on a sharp turn of the protein backbone
structure (the nucleophilic elbow), whereas the interacting
residues of both catalytic triads (except for Asp218 in
LIPESV12_26) are positioned within hydrogen bonding. This
suggests that both proteins use the classical Ser-His-Asp cat-
alytic triad mechanism with serine acting as the nucleophile,
histidine as the general acid-base, and aspartate helping to
neutralize the charge forming on histidine during the catalysis.
These active sites appear to be capable of binding acyl esters
with short acyl chain lengths. The structural models also sug-
gest that LIPESV12_26 might have a more open active site
that is in line with a broader substrate range of this enzyme
tr|W0JC82|W0JC82 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 22980 PE 3 SV 1
tr|E0SRZ8|E0SRZ8 IGNAA Uncharacterized protein OS Ignisphaera aggregans (strain DSM 17230
tr|A0A052I9Y4|A0A052I9Y4 9BORD Putative N-acetyltransferase YedL OS Bordetella hinzii 8-296-03
sp|P54583|GUN1 ACIC1 Endoglucanase E1 Acidothermus cellulolyticus ATCC 43068 Active 203323
sp|P58599|GUN1 RALSO Endoglucanase Ralstonia solanacearum Active 247359
tr|W0IXA7|W0IXA7 9BACT Glycosyl hydrolase family 5 OS Opitutaceae bacterium TAV5 GN OPIT5 01025 PE 3 SV 1
tr|W0J1Z4|W0J1Z4 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 11855 PE 4 SV 1
tr|W0J4X8|W0J4X8 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 19170 PE 4 SV 1
tr|W0IXI8|W0IXI8 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 02055 PE 3 SV 1
tr|I6B4B5|I6B4B5 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV1 GN OpiT1DRAFT 00687 PE 4 SV 1
tr|I6B3U1|I6B3U1 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV1 GN OpiT1DRAFT 00510 PE 4 SV 1
tr|W0IXA2|W0IXA2 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 01000 PE 4 SV 1
tr|I6B267|I6B267 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV1 GN OpiT1DRAFT 05935 PE 4 SV 1
tr|I6AVT0|I6AVT0 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV1 GN OpiT1DRAFT 03625 PE 4 SV 1
tr|I6AYE8|I6AYE8 9BACT Cellulase (Glycosyl hydrolase family 5) OS Opitutaceae bacterium TAV1 GN OpiT1DRAFT 04576 PE 3 SV 1
tr|W0IYP6|W0IYP6 9BACT Glycosyl hydrolase family 5 OS Opitutaceae bacterium TAV5 GN OPIT5 04140 PE 3 SV 1
tr|W0J6Y8|W0J6Y8 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 21635 PE 4 SV 1
tr|I6AVN9|I6AVN9 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV1 GN OpiT1DRAFT 03584 PE 4 SV 1
GLV12_5
tr|W0J103|W0J103 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 19240 PE 4 SV 1
tr|R7L6D6|R7L6D6 9BACT Uncharacterized protein OS Coraliomargarita sp. CAG:312 GN BN601 00088 PE 4 SV 1
tr|W0JBF2|W0JBF2 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 04550 PE 3 SV 1
tr|W0IZF8|W0IZF8 9BACT Uncharacterized protein OS Opitutaceae bacterium TAV5 GN OPIT5 07565 PE 4 SV 1
tr|D7DPA2|D7DPA2 METV0 Uncharacterized protein OS Methylotenera versatilis (strain 301) GN M301 0762 PE 3 SV 1
tr|A6WVW1|A6WVW1 OCHA4 Uncharacterized protein OS Ochrobactrum anthropi (strain ATCC 49188 / DSM 6882 / NCTC 12168)
tr|M5JQQ0|M5JQQ0 9RHIZ Uncharacterized protein OS Ochrobactrum intermedium M86 GN D584 06917 PE 4 SV 1
tr|A0A095V4V0|A0A095V4V0 9RHIZ Glycoside hydrolase OS Rhizobium sp. YS-1r GN JL39 16855 PE 4 SV 1
tr|A0A085FUB9|A0A085FUB9 9BRAD Endo-beta-mannanase OS Bosea sp. LC85 GN FG93 00824 PE 4 SV 1
tr|A6UHN5|A6UHN5 SINMW Uncharacterized protein OS Sinorhizobium medicae (strain WSM419) GN Smed 4360 PE 4 SV 1
tr|F6E7B1|F6E7B1 SINMK Glycoside hydrolase family 5 OS Sinorhizobium meliloti (strain AK83) GN Sinme 4405 PE 4 SV 1
tr|W8IKV6|W8IKV6 ENSAD Uncharacterized protein OS Ensifer adhaerens OV14 GN OV14 a0372 PE 4 SV 1
tr|A0A069HRT9|A0A069HRT9 ENSAD Glycoside hydrolase OS Ensifer adhaerens GN FA04 15010 PE 4 SV 1
tr|A0A072CN10|A0A072CN10 9RHIZ Uncharacterized protein OS Sinorhizobium americanum CCGM7 GN SAMCCGM7 c1696 PE 4 SV 1
tr|C3KLR4|C3KLR4 RHISN Uncharacterized protein OS Rhizobium sp. (strain NGR234) GN NGR b19030 PE 4 SV 1
tr|I3X2D3|I3X2D3 RHIFR Uncharacterized protein OS Sinorhizobium fredii USDA 257 GN USDA257 c14490 PE 4 SV 1
GH5
tr|Q56306|Q56306 THEMA Beta-galactosidase (lacZ) OS Thermotoga maritima PE 4 SV 1
sp|O69315|BGAL THETH Beta-galactosidase OS Thermus thermophilus Active 141312
sp|P94804|BGAL HALL2 Beta-galactosidase BgaH OS Haloferax lucentense (DSM 14919) Active 142311
GH42
82
46
73
99
34
48
32
25
16
22
46
22
38
100
50
68
96
43
15
55
84
4
77
41
19
61
9
25
19
4
1
4
2
5
7
0.5
Fig. 7 Phylogenetic position of GLV12_5. The conserved functional
domains of each protein were determined using Pfam (http://pfam.xfam.
org/search) (Finn et al. 2011). The obtained BLAST data sets of the
lowest E values were aligned in MEGA6 (Tamura et al. 2013) using
MUSCLE (Edgar 2004). The tree was constructed by using the
neighbour-joining method (Saitou & Nei 1987) as described in the
BMaterials and methods^
Appl Microbiol Biotechnol
14. compared to LIPESV12_24. Indeed, in our assays,
LIPESV12_26 revealed a wider substrate specificity (hydro-
lysed 33 from 43 esters tested) compared to LIPESV12-9 (7
from 43) and LIPESV12_24 (15 from 43) (Fig. S7A and
Fig. S7B). This was confirmed in experiments using p-NP-
C12 and p-NP-C18 as substrates showing the capacity to hy-
drolyse longer alkyl esters. In contrast to the ABO proteins
and LIPESV12-9, after a long incubation, LIPESV12_24 and
LIPESV12_26 were able to hydrolyse significant amounts of
long-chain fatty acids, including p-NP-C12 and p-NP-C16.
To this end, the LIPESV12_enzymes are therefore quali-
fied as carboxylesterase EC 3.1.1.1 with wider substrate spec-
ificities. The protein engineering and evolution research for
these target proteins basing either on naïve error-prone or on
direct mutagenesis with more specific databases and tools,
such as 3DM (Kourist et al. 2010), have also been planned
for our future research. Furthermore, from a biochemical point
of view, the enzymes, especially the LIPESV12_24, pointed at
a thermophilic nature of their hosts, retaining the activity even
after a short incubation at temperatures near the water boiling
point.
The enzymes were tolerant to, and active with, different
divalent cations such as calcium, cobalt and magnesium, cop-
per, zinc, and manganese. The three latter metals exhibited
inhibition on LIPESV12_24 only after a long incubation.
The activity of several metal ions is positively influenced at
high temperatures possibly due to ionic bond formation, sta-
bilizing the protein molecules.
The pH values have a considerable influence on enzyme
kinetics due to the influence on the formation or breaking of
electrostatic, hydrogen, and other bonds (Li et al. 2005). The
pH profile analysis for the enzymes showed their higher ac-
tivities in the neutral and slightly alkaline ranges. While the
LIPESV12_24 was still active at pH 5.5, the LIPESV12_24 at
that pH had almost no activity. This may be explained either
by a high buffering capacity of the seawater and thus the
selection for neutrophils or by a possible intracellular locali-
zation of enzymes.
The use of organic solvents in the industrial biocatalysis
offers advantages as compared to the aqueous media due to a
higher solubility of hydrophobic substrates, reduced water
activity altering the hydrolytic equilibrium, decreased risk of
microbial contamination, and others (Sellek and Chaudhuri
1999). Both hydrolases, LIPESV12_24 and LIPESV12_26,
could serve as potential candidates for industrial applications,
since they kept their stability and activity in organic solvents,
even at high concentrations of isopropanol and methanol.
The phylogenetic analysis showed that the representative
of GLV12 proteins identified in this study, the GLV12_5,
clustered with GH42 and GH5 glycosyl hydrolase groups,
which embraced the most cellulose-degrading enzymes. Inter-
estingly, the purified recombinant GLV12_5 protein was high-
ly substrate specific, which revealed activity towards just one
of the 16 p-NP-glycosides tested, namely the p-NP-α-
arabinopyranose. This enzyme should be considered as α-
arabinopyranosidase (EC 3.2.1_x) with a restricted substrate
profile.
The obtained data suggest a high biodiversity of hydrolases
in the hydrothermal vents of the Vulcano Island, pointing at a
high potential of these environments for mining and exploita-
tion of new industrially relevant enzymes.
Acknowledgments This work was funded by the EU FP7 MicroB3
Research Project (OCEAN-2011-287589) to OVG, HT, and PNG. PNG
and OVG acknowledge the Royal Society-Russia Exchange Grant
IE130218. SVT thanks the Russian Federal Targeted Program for R and
D for support (Grant No. 14.587.21.0007). IVK and AP are thankful to
the Russian Foundation of Basic Research (Grant No. 13-04-02157) and
Consiglio Nazionale delle Ricerche (Project, Medicina Personalizzata),
respectively. We thank EU Horizon 2020 Program for the support of the
Project INMARE H2020-BG-2014-2634486. MF thanks grants
BIO2011-25012, PIA102014-1 and BIO2014-54494-R from the Spanish
Ministry of Economy and Competitiveness. The authors gratefully ac-
knowledge the financial support provided by the European Regional De-
velopment Fund (ERDF).
Conflict of interest All authors have declared that they have no conflict
of interest. On behalf of all authors, I hereby certify that this article
contains the original data from our research activities and is for the first
time submitted for publication.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a link
to the Creative Commons license, and indicate if changes were made.
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