No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Identification and characterization of a novel salt-tolerant esterase from a Tibetan glacier metagenomic library
Biotechnology Progress
Abstract
A salt-tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p-nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three-dimensional model of H9Est revealed that S200, D294 and H324 formed the H9Est catalytic triad. Circular Dichroism (CD) spectra and molecular dynamic (MD) simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat-resistant features. This article is protected by copyright. All rights reserved.
© 2015 American Institute of Chemical Engineers.
... and lipases (EC 3.1.1.3) are carboxylic ester hydrolases that catalyze the cleavage and formation of ester bonds 3,6 . A wide range of different esterases exist that differ in their substrate specificity, their protein structure, and their biological function. ...
The esterases catalyzing the hydrolysis of acyl esters are highly desired in industrial applications. An extracellular esterase producing, mesophilic and alkaliphilic Gram positive bacterial isolate obtained from human ear wax (sebum) was identified as Bacillus safensis strain. The B. safensis strain produced relatively higher amount of esterase after 36 h at 37o C under shaking conditions when coconut oil (1.75 %, v/ v) and gelatin (0.6 %, w/v) were used in the Mineral-Based broth as carbon and nitrogen source, respectively. The esterase production by B. safensis strain increased by ~208 % by consecutive optimization of physico�chemical parameters. The esterase was partially purified by acetone precipitation. Enzyme showed greater affinity towards a relatively short C-chain ester p-NPA as substrate. The Km and Vmax values of the esterase were found to be 0.127 mM and 8.82 U/ml/min, respectively. The esterase was purified up to 2.8 fold by acetone precipitation (20-40 % saturation). The esterase was found to be a heptameric protein of 49 kDa (under native-PAGE) comprising seven units of 4 7 kDa as revealed by SDS-PAGE.
... It retained over 90% of its maximum activity at 20, 25, 35, and 40 °C. In contrast, the catalytic activities of many other mesophilic metagenomederived esterases and cutinases decrease dramatically below 30 °C [47,55,65]. The relative activity of CEstKAD01 at 45 and 50 °C was 79.31 ± 2.45 and 65.12 ± 1.33, respectively. ...
Plastic pollution has threatened biodiversity and human health by shrinking habitats, reducing food quality, and limiting the activities of organisms. Therefore, global interest in discovering novel enzymes capable of degrading plastics has increased considerably. Within this context, the functional metagenomic approach, which allows for unlocking the functional potential of uncultivable microbial biodiversity, was used to discover a plastic-degrading enzyme. First, metagenomic libraries derived from microplastic-associated microbiota were screened for esterases capable of degrading both tributyrin and polycaprolactone. Clone KAD01 produced esterase highly active against p-nitrophenyl esters (C2–C16). The gene corresponding to the enzyme activity showed moderate identity (≤ 55.94%) to any known esterases/cutinases. The gene was extracellularly expressed with a 6× histidine tag in E. coli BL21(DE3), extracellularly. Titer of the enzyme (CEstKAD01) was raised from 21.32 to 35.17 U/mL by the statistical optimization of expression conditions and media components. CEstKAD01 was most active at pH 7.0 and 30 °C. It was noteworthy stable over a wide pH (6.0–10.0) and temperature (20–50 °C). The enzyme was active and stable in elevated NaCl concentrations up to 12% (w/v). Pre-incubation of CEstKAD01 with Mg²⁺, Mn²⁺, and Ca²⁺ increased the enzyme activity. CEstKAD01 displayed an excellent tolerance against various chemicals and solvents. It was determined that 1 mg of the enzyme caused the release of 5.39 ± 0.18 mM fatty acids from 1 g apple cutin in 120 min. Km and Vmax values of CEstKAD01 against p-nitrophenyl butyrate were calculated to be 1.48 mM and 20.37 µmol/min, respectively. The enzyme caused 6.94 ± 0.55, 8.71 ± 0.56, 7.47 ± 0.47, and 9.22 ± 0.18% of weight loss in polystyrene, high-density polyethylene, low-density polyethylene, and polyvinyl chloride after 30-day incubation. The scanning electron microscopy (SEM) analysis indicated the formation of holes and pits on the plastic surfaces supporting the degradation. In addition, the change in chemical structure in plastics treated with the enzyme was determined by Fourier Transform Infrared Spectroscopy (FTIR) analysis. Finally, the degradation products were found to have no genotoxic potential. To our knowledge, no cutinolytic esterase with the potential to degrade polystyrene (PS), high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polyvinyl chloride (PVC) has been identified from metagenomes derived from microplastic-associated microbiota.
... In addition to aquatic sediments, other types of samples with metagenomic exploitation potential come from arid/semiarid and mountain soils, for example in the Ladakh region [70] and the Apharwat mountain [41,44], respectively, both in the northwestern Himalayas that have distinct geo-climatic characteristics like extremely cold and dry weather, high altitude and glacial and permafrost soils. Relevant examples of this are the Karuola glacier in Tibetan Plateau [45] and the Kolyma Lowland permafrost in north-eastern Siberia [47], which are extremely hostile environments and inhabited by unique microbial communities. ...
Natural resources are considered a promising source of microorganisms responsible for producing biocatalysts with great relevance in several industrial areas. However, a significant fraction of the environmental microorganisms remains unknown or unexploited due to the limitations associated with their cultivation in the laboratory through classical techniques. Metagenomics has emerged as an innovative and strategic approach to explore these unculturable microorganisms through the analysis of DNA extracted from environmental samples. In this review, a detailed discussion is presented on the application of metagenomics to unravel the biotechnological potential of natural resources for the discovery of promising biocatalysts. An extensive bibliographic survey was carried out between 2010 and 2021, covering diverse metagenomic studies using soil and/or water samples from different types and locations. The review comprises, for the first time, an overview of the worldwide metagenomic studies performed in soil and water and provides a complete and global vision of the enzyme diversity associated with each specific environment.
... Cultivation-independent metagenomic approach circumvents the culture of microorganisms and extracts total microbial DNA directly from an environmental sample to construct a library, following by functional screening for desired enzyme [13]. Through this approach, numerous esterases with biotechnological importance have been successfully identified from the metagenomic libraries from different environmental samples, such as soil [14][15][16], lake and ocean [17,18], compost [19], hot spring [20], glacier [21,22], and desert [23]. Therefore, there is great interest dedicating metagenomic-based searches for novel enzymes with greater industrial applicability from different sources. ...
An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0–10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure–function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.
... Prediction of protein structures is a fundamental approach to highlight conformational aspects of molecules of biotechnological interest, for example, elucidating structural features related to environmental adaptation, such as warm or cold-adapted mechanisms that confer thermostability in extremophilic enzymes [233,234], or specifying enzymatic action useful for biotechnological applications [235]. The prediction of protein structures follows two main strategies: (1) comparative approaches, based on homology modeling [236] or protein threading techniques [237,238], which predict new structures by modeling sequences from unknown structures using solved structures from homolog sequences as templates, or by recognizing common protein folds in protein sequences that lack homolog sequences; and (2) ab initio approaches [239], based on intrinsic chemical and physical characteristics of amino acid sequences rather than previously solved structures. ...
The sea represents a major source of biodiversity. It exhibits many different ecosystems in a huge variety of environmental conditions where marine organisms have evolved with extensive diversification of structures and functions, making the marine environment a treasure trove of molecules with potential for biotechnological applications and innovation in many different areas. Rapid progress of the omics sciences has revealed novel opportunities to advance the knowledge of biological systems, paving the way for an unprecedented revolution in the field and expanding marine research from model organisms to an increasing number of marine species. Multi-level approaches based on molecular investigations at genomic, metagenomic, transcriptomic, metatranscriptomic, proteomic, and metabolomic levels are essential to discover marine resources and further explore key molecular processes involved in their production and action. As a consequence, omics approaches, accompanied by the associated bioinformatic resources and computational tools for molecular analyses and modeling, are boosting the rapid advancement of biotechnologies. In this review, we provide an overview of the most relevant bioinformatic resources and major approaches, highlighting perspectives and bottlenecks for an appropriate exploitation of these opportunities for biotechnology applications from marine resources.
... The comparison of the effect of metal ions (1 mM) on the activity of hAGEst and other enzymes in the literature is summarized in Table 4. The enzymes to compare are selected according to their origin or salt tolerance properties: a metal-resistant esterase from Red Sea brine pool (Mohamed et al. 2013), a salt-tolerant esterase from Tibetan glacier (De Santi et al. 2015), a halophilic esterase from Antarctic soil sample (Castilla et al. 2017), and a salt-tolerant esterase from deep-sea sediment (Zhang et al. 2017). The activity of hAGEst is higher than other enzymes for many of the metal ions. ...
The aim of this study was to isolate a novel esterase from a hypersaline lake by sequence-based metagenomics. The metagenomic DNA was isolated from the enriched hypersaline lake sediment. Degenerate primers targeting the conserved regions of lipolytic enzymes of halophilic microorganisms were used for polymerase chain reaction (PCR) and a whole gene was identified by genome walking. The gene was composed of 783 bp, which corresponds to 260 amino acids with a molecular weight of 28.2 kDa. The deduced amino acid sequence best matched with the esterase from Halomonas gudaonensis with an identity of 91%. Recombinantly expressed enzyme exhibited maximum activity towards pNP-hexanoate with a kcat value of 12.30 s⁻¹. The optimum pH and temperature of the enzyme were found as 9 and 30 °C, respectively. The effects of NaCl, solvents, metal ions, detergents and enzyme inhibitors were also studied. In conclusion, a novel enzyme, named as hypersaline lake “Acıgöl” esterase (hAGEst), was identified by sequence-based metagenomics. The high expression level, the ability to maintain activity at cold temperatures and tolerance to DMSO and metal ions are the most outstanding properties of the hAGEst.
A moderately halotolerant serine protease was previously isolated from Bacillus subtilis from salted, fermented food. Eight mutation sites on the protein surface were selected for protein engineering based on sequence and structural comparisons with moderately halotolerant proteases and homologous non‐halotolerant proteases. The newly constructed multiple mutants with substituted Asp and Arg residues were compared with the recombinant wild type (rApr) and the previously constructed mAla‐8 substituted with Ala to analyze the contribution of protein surface charge to the salt adaptation of the protease. The three mutants showed >1.2‐fold greater halotolerance than rApr. In addition, the mutants showed a broader range of pH stability than rApr, retaining >80% of their maximum activity in the pH range 5.0–11. The mutants also retained >75% of their activity after incubation for 1 h at pH 8.0 and 55°C or at pH 11.5 and 25°C. The Asp and Arg residues exchanged by multiple substitution probably played a role in increasing protein surface hydration and solubility in high salt conditions. This study illustrated that increasing a high proportion of the negative or positive charge on the surface of the Bacillus serine protease stably improved the protein's salt adaptation.
Extremophiles are remarkable organisms that thrive in the harshest environments on Earth, such as hydrothermal vents, hypersaline lakes and pools, alkaline soda lakes, deserts, cold oceans, and volcanic areas. These organisms have developed several strategies to overcome environmental stress and nutrient limitations. Thus, they are among the best model organisms to study adaptive mechanisms that lead to stress tolerance. Genetic and structural information derived from extremophiles and extremozymes can be used for bioengineering other nontolerant enzymes. Furthermore, extremophiles can be a valuable resource for novel biotechnological and biomedical products due to their biosynthetic properties. However, understanding life under extreme conditions is challenging due to the difficulties of in vitro cultivation and observation since > 99% of organisms cannot be cultivated. Consequently, only a minor percentage of the potential extremophiles on Earth have been discovered and characterized. Herein, we present a review of culture-independent methods, sequence-based metagenomics (SBM), and single amplified genomes (SAGs) for studying enzymes from extremophiles, with a focus on prokaryotic (archaea and bacteria) microorganisms. Additionally, we provide a comprehensive list of extremozymes discovered via metagenomics and SAGs.
A esterase gene was characterized from a halophilic bacterium Chromohalobacter canadensis which was originally isolated from a salt well mine. Sequence analysis showed that the esterase, named as EstSHJ2, contained active site serine encompassed by a conserved pentapeptide motif (GSSMG). The EstSHJ2 was classified into a new lipase/esterase family by phylogenetic association analysis. Molecular weight of EstSHJ2 was 26 kDa and the preferred substrate was p-NP butyrate. The EstSHJ2 exhibited a maximum activity at 2.5 M NaCl concentration. Intriguingly, the optimum temperature, pH and stability of EstSHJ2 were related to NaCl concentration. At 2.5 M NaCl concentration, the optimum temperature and pH of EstSHJ2 were 65 ℃ and pH 9.0, and enzyme remained 81% active after 80 ℃ treatment for 2 h. Additionally, the EstSHJ2 showed strong tolerance to metal ions and organic solvents. Among these, 10 mM K+, Ca2+ , Mg2+ and 30% hexane, benzene, toluene has significantly improved activity of EstSHJ2. The EstSHJ2 was the first reported esterase from Chromohalobacter canadensis, and may carry considerable potential for industrial applications under extreme conditions.
Chiral lactic acids and their ester derivatives are crucial building blocks and intermediates for the synthesis of a great variety of valuable functional materials and pharmaceuticals. Before our study, the reports about the enantioselective preparation of pure L-lactic acid and its ester derivatives through direct hydrolysis of racemic substrate were quite rare. Herein, we heterologously expressed and functionally characterized one novel microbial esterase WDEst9 from Dactylosporangium aurantiacum, which exhibited high resistance to diverse metal ions, organic solvents, surfactants, NaCl and KCl. We further utilized WDEst9 as a green biocatalyst in the kinetic resolution of (±)-methyl lactate through direct hydrolysis and generated L-methyl lactate with high enantiomeric excess (e.e. >99%) and high yield (>86%) after process optimization. Notably, the enantioselectivity of WDEst9 was opposite than that of two previously reported esterases PHE14 and BSE01701 that can generate D-methyl lactate though kinetic resolution of (±)-methyl lactate. Microbial esterase WDEst9 is a promising green biocatalyst in the preparation of valuable chiral chemicals and opens the door for the identification of useful industrial enzymes and biocatalysts from the genus Dactylosporangium.
The two enantiomers of ethyl 3‐hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)‐3‐hydroxybutyrate. Herein, we also functionally characterized one novel salt‐tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)‐3‐hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio‐selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates.
In the transition to the post-petroleum economy, there is a growing demand for novel enzymes with high process performances to replace traditional chemistry with a more 'green' approach. To date, microorganisms encompass the richest source of industrial biocatalysts, but the Earth-living microbiota remains largely untapped by using traditional isolation and cultivation methods. Metagenomics, which is culture-independent, represents a powerful tool for discovering novel enzymes from unculturable microorganisms. Herein, we summarize the variety of approaches adopted for mining environmental DNA and, based on a systematic literature review, we provide a comprehensive list of 332 industrially relevant enzymes discovered from metagenomes within the last three years.
A novel marine microbial esterase PHE14 was cloned from the genome of Pseudomonas oryzihabitans HUP022 isolated from the deep sea of the western Pacific Ocean. Esterase PHE14 exhibited very good tolerance to most organic solvents, surfactants and metal ions tested, thus making it a good esterase candidate for organic synthesis that requires an organic solvent, surfactants or metal ions. Esterase PHE14 was utilized as a biocatalyst in the asymmetric synthesis of D-methyl lactate by enzymatic kinetic resolution. D-methyl lactate is a key chiral chemical. Contrary to some previous reports, the addition of an organic solvent and surfactants in the enzymatic reaction did not have a beneficial effect on the kinetic resolution catalyzed by esterase PHE14. Our study is the first report on the preparation of the enantiomerically enriched product D-methyl lactate by enzymatic kinetic resolution. The desired enantiomerically enriched product D-methyl lactate was obtained with a high enantiomeric excess of 99% and yield of 88.7% after process optimization. The deep sea microbial esterase PHE14 is a green biocatalyst with very good potential in asymmetric synthesis in industry and can replace the traditional organic synthesis that causes pollution to the environment.
Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.
The 97-megabase genomic sequence of the nematode Caenorhabditis elegans reveals over 19,000 genes. More than 40 percent of the predicted protein products find significant matches in other organisms.
There is a variety of repeated sequences, both local and dispersed. The distinctive distribution of some repeats and highly
conserved genes provides evidence for a regional organization of the chromosomes.
Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications.
Bacterial lipolytic enzymes were originally classified into eight different families defined by Arpigny and Jaeger (families I-VIII). Recently, the discovery of new lipolytic enzymes allowed for extending the original classification to fourteen families (I-XIV). We previously reported that G. thermodenitrificans EstGtA2 (access no. AEN92268) belonged to a novel group of bacterial lipolytic enzymes. Here we propose a 15(th) family (family XV) and suggest criteria for the assignation of protein sequences to the N' subfamily. Five selected salt bridges, hallmarks of the N' subfamily (E3/R54, E12/R37, E66/R140, D124/K178 and D205/R220) were disrupted in EstGtA2 using a combinatorial alanine-scanning approach. A set of 14 (R/K→A) mutants was produced, including five single, three double, three triple and three quadruple mutants. Despite a high tolerance to non-conservative mutations for folding, all the alanine substitutions were destabilizing (decreasing T m by 5 to 14°C). A particular combination of four substitutions exceeded this tolerance and prevents the correct folding of EstGtA2, leading to enzyme inactivation. Although other mutants remain active at low temperatures, the accumulation of more than two mutations had a dramatic impact on EstGtA2 activity at high temperatures suggesting an important role of these conserved salt bridge-forming residues in thermostability of lipolytic enzymes from the N' subfamily. We also identified a particular interloop salt bridge in EstGtA2 (D194/H222), located at position i -2 and i -4 residues from the catalytic Asp and His respectively which is conserved in other related bacterial lipolytic enzymes (families IV and XIII) with high tolerance to mutations and charge reversal. We investigated the role of residue identity at position 222 in controlling stability-pH dependence in EstGtA2. The introduction of a His to Arg mutation led to increase thermostability under alkaline pH. Our results suggest primary targets for optimization of EstGtA2 for specific biotechnological purposes.
in order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 α/β hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.
The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity and activities. Herein, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46' N, 2°59' W; 655 m altitude). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from the Lake Arreo suggests that its sediment contain moderately halophilic and cold-adapted Proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origin. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics as they exhibit maximal activity at alkaline pH (8.0-8.5), 16-40°C and are stimulated (1.5-2.2 times) by chloride ions (0.1-1.2 M) reflecting an adaption to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e. production of enantiomers and sugar esters) because these enzymes are salt-tolerant and are active at low temperatures and against a broad range of substrates. As example, the ability of a single protein to hydrolyze at similar extent triacylglycerols, (non) halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones and chiral epoxides was demonstrated.
A soil metagenomic library was constructed and two functionally diverse lipase genes, SMlipB and SMlipD, were screened by a function-driven approach and characterized. The optimal temperature for enzyme activity of SMlipB and SMlipD was 50°C and 30°C, respectively, and optimal pH was determined to be 7.0 and 9.0, respectively. Both enzymes exhibited broad substrate specificity and showed enhanced activity in the presence of SDS and Tween 20. The SMlipB enzyme was highly resistant to many organic solvents, especially isopropanol, ethanediol, DMSO, methanol and xylene, whereas SMlipD activity was inhibited in all the solvents except xylene. Sequence analysis revealed SMlipB consisted of an open reading frame of 1,212 bp and encoded for 404 amino acids. It contained the GXXGXD motifs, which are supposed to be involved in Ca(2+) binding in proteases and lipases, and an extreme C-terminal motif consisting of a negatively charged amino acid followed by four hydrophobic residues, essential for the secretion of metalloprotease, and belongs to lipase subfamily I.3. SMlipD contained 1,071 bp ORF and encoded for 357 amino acids. It contains Ca(2+) ion binding sites extending from amino acid 282 to 294 and two Cystein residues (218,308), proven necessary for forming a disulfide bridge and belongs to lipase subfamily1.2.
Psychrophiles inhabit more than 70% of Earth's surface which includes Ocean depths, polar and alpine regions. Oceans have a constant temperature of 4–5°C below a depth of 1,000 m irrespective of the latitude. Psychrophiles and oceans are one of the underutilizing resources which have great economical and environmental potential. As the organisms should withstand to strong negative effect of low temperatures they adapted modifications in their membrane components and produces anti-freeze proteins which protects from forming ice inside the cell. The enzymes from psychrophiles are cold active due to their unique structural, kinetic and activation parameters which differ from mesophiles and thermophiles. By having the properties like high specific activity at low temperatures and thermolabile nature they are having potential applications in various fields of biotechnology. characterized originated from the Antarctic terrestrial and the Antarctic sea water (Gerday et al., 1997 and Russell, 1998). Specific activity of cold enzymes from wild strains and some of their recombinant forms have been determined in organisms of Antarctic and arctic regions including alcohol dehydrogenase (Feller, 1994 (a,b)), α-amylase (Vazquez et al., 1995), Aspartate transcarbamylase (Feller, 1992), Ca+Zn+2 protease, (Villeret, 1997) citratesynthetase, (Gerike et al., 1997) β-lactamase (Feller, 1997), malate dehydrogenase, subtilisin (Narinx, 1997), triose phosphate isomerase (Alvarez, 1998), and xylanase (Reddy, 2003). Typically cold enzymes show higher specific activity than that of their mesophilic enzymes. By considering all unique properties of these enzymes people realize that there is a huge Biotechnological potential of enzymes from psychrophiles. So there is urgent need to exploit them for stimulating further advances in this field. In this review we focused on psychrophilic enzymes and their cold adaptations at structural and molecular level activity-parameters and their applications in various Industries. Microorganisms that have colonized the cold environments are referred to as psychrophiles or cold adapted. They are found in deep sea to mountain and Polar Regions as well (Morita, 1975). In the strictest o definition, psychrophiles do not grow above 20 C. Organisms growing o well at low temperatures and also grow above 20 C are referred as psychrotolerant or psychrotrophs (Russell, 1992;1997). Thus, to avoid the difficulty of dealing with the varying maximum growth temperature, Neidhardt et al. (1990), defined psychrophiles as organisms that grow at ~5°C or below up to 20°C to distinguish them from mesophiles which o grow best at 37 C. Based on their surroundings Psychrophiles are further divided in to Piezo-psychrophiles, lives in the ocean depths and sediments and they usually face extremely high pressures, Halo-psychrophiles which inhabit in high salt concentrations (Yayanos, 1999)
Background
Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome.
Results
A metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C β-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3–12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 μmol/min, respectively.
Conclusion
We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.
The PROCHECK suite of programs provides a detailed check on the stereochemistry of a protein structure. Its outputs comprise a number of plots in PostScript format and a comprehensive residue-by-residue listing. These give an assessment of the overall quality of the structure as compared with well refined structures of the same resolution and also highlight regions that may need further investigation. The PROCHECK programs are useful for assessing the quality not only of protein structures in the process of being solved but also of existing structures and of those being modelled on known structures.
The gene estF27, encoding a protein with feruloyl esterase activity, was cloned through functional screening from a soil metagenomic library and expressed in Escherichiacoli BL21 (DE3) with high solubility. Sequence analysis showed that estF27 encoded a protein of 291 amino acids with a predicted molecular mass of 31.16 kDa. According to the substrate specificity, EstF27 was classified as a type A feruloyl esterase. EstF27 displayed optimal activity at 40°C and pH 6.8. This enzyme was stable in a broad pH range of 5.0-10.0 over 24 h, and retained more than 50% of its activity after 96 or 120 h incubation in the presence of 3 M KCl or 5 M NaCl. The enzyme activity was slightly enhanced by the addition of Mg(2+) and Fe(3+) at a low concentration, and completely inhibited by Cu(2+). In the enzymatic hydrolysis of destarched wheat bran, EstF27 could release ferulic acid from it in the presence of xylanase from Thermomyces lanuginosus. Given its alkalitolerance, halotolerance and highly soluble expression, EstF27 is a promising candidate for industrial applications.
Comparative analysis of molecular sequence data is essential for reconstructing the evolutionary histories of species and inferring the nature and extent of selective forces shaping the evolution of genes and species. Here, we announce the release of Molecular Evolutionary Genetics Analysis version 5 (MEGA5), which is a user-friendly software for mining online databases, building sequence alignments and phylogenetic trees, and using methods of evolutionary bioinformatics in basic biology, biomedicine, and evolution. The newest addition in MEGA5 is a collection of maximum likelihood (ML) analyses for inferring evolutionary trees, selecting best-fit substitution models (nucleotide or amino acid), inferring ancestral states and sequences (along with probabilities), and estimating evolutionary rates site-by-site. In computer simulation analyses, ML tree inference algorithms in MEGA5 compared favorably with other software packages in terms of computational efficiency and the accuracy of the estimates of phylogenetic trees, substitution parameters, and rate variation among sites. The MEGA user interface has now been enhanced to be activity driven to make it easier for the use of both beginners and experienced scientists. This version of MEGA is intended for the Windows platform, and it has been configured for effective use on Mac OS X and Linux desktops. It is available free of charge from http://www.megasoftware.net.
Two esterase genes (designated as estAT1 and estAT11, respectively) were cloned by activity-based screening of a fosmid library constructed with seashore sediment sample of the Arctic. The sequence analysis of the genes revealed that these esterase genes encoded proteins of 303 and 312 amino acids, respectively, and showed 40-50% identities to members of the hormone-sensitive lipase (HSL) family retaining a catalytic triad with a conserved GDSAG sequence and an oxyanion hole (HGGG). The esterases genes were overexpressed in Escherichia coli by co-expressing GroEL-GroES chaperonine, and the recombinant proteins (rEstAT1 and rEstAT11) were purified to homogeneity. The purified EstAT1 and EstAT11 were active in a broad range of temperature from 20 to 40 degrees C with an optimum temperature at 30 degrees C. The activation energies of rEstAT1 and rEstAT11 to hydrolyze p-nitrophenyl esters of butyrate were determined to be 12.65 kcal/mol and 11.26 kcal/mol, respectively, indicating that they are cold-adapted esterases. The purified EstAT1 and EstAT11 could hydrolyze racemic ofloxacin esters, and further rEstAT11 hydrolyzed preferentially (S)-racemic ofloxacin butyl ester with an enantiomeric excess (ee(p)) value of 70.3%. This work represents an example that develops enzymes from the Arctic using metagenomic approach, potentially applicable to chiral resolution of heat-labile substrates.
A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
A simple, rapid method for bacterial lysis and direct extraction of DNA from soils with minimal shearing was developed to address the risk of chimera formation from small template DNA during subsequent PCR. The method was based on lysis with a high-salt extraction buffer (1.5 M NaCl) and extended heating (2 to 3 h) of the soil suspension in the presence of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide, and proteinase K. The extraction method required 6 h and was tested on eight soils differing in organic carbon, clay content, and pH, including ones from which DNA extraction is difficult. The DNA fragment size in crude extracts from all soils was > 23 kb. Preliminary trials indicated that DNA recovery from two soils seeded with gram-negative bacteria was 92 to 99%. When the method was tested on all eight unseeded soils, microscopic examination of indigenous bacteria in soil pellets before and after extraction showed variable cell lysis efficiency (26 to 92%). Crude DNA yields from the eight soils ranged from 2.5 to 26.9 micrograms of DNA g-1, and these were positively correlated with the organic carbon content in the soil (r = 0.73). DNA yields from gram-positive bacteria from pure cultures were two to six times higher when the high-salt-SDS-heat method was combined with mortar-and-pestle grinding and freeze-thawing, and most DNA recovered was of high molecular weight. Four methods for purifying crude DNA were also evaluated for percent recovery, fragment size, speed, enzyme restriction, PCR amplification, and DNA-DNA hybridization. In general, all methods produced DNA pure enough for PCR amplification. Since soil type and microbial community characteristics will influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods on the basis of experimental goals.
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
Psychrophilic, mesophilic, and thermophilic α-amylases have been studied as regards their conformational stability, heat inactivation,
irreversible unfolding, activation parameters of the reaction, properties of the enzyme in complex with a transition state
analog, and structural permeability. These data allowed us to propose an energy landscape for a family of extremophilic enzymes
based on the folding funnel model, integrating the main differences in conformational energy, cooperativity of protein unfolding,
and temperature dependence of the activity. In particular, the shape of the funnel bottom, which depicts the stability of
the native state ensemble, also accounts for the thermodynamic parameters of activation that characterize these extremophilic
enzymes, therefore providing a rational basis for stability-activity relationships in protein adaptation to extreme temperatures.
POPS (Parameter OPtimsed Surfaces) is a new method to calculate solvent accessible surface areas, which is based on an empirically
parameterisable analytical formula and fast to compute. Atomic and residue areas (the latter represented by a single sphere
centered on the Cα atom of amino acids and at the P atom of nucleotides) have been optimised versus accurate all-atom methods. The parameterisation
has been derived from a selected dataset of proteins and nucleic acids of different sizes and topologies. The residue based
approach POPS-R, has been devised as a useful tool for the analysis of large macromolecular assemblies like the ribosome and it is specially
suited for the refinement of low resolution structures. POPS-R also allows for estimates of the loss of free energy of solvation upon complex formation, which should be particularly useful
for the design of new protein–protein and protein–nucleic acid complexes. The program POPS is available at http://mathbio.nimr.mrc.ac.uk/~ffranca/POPS and at the mirror site http://www.cs.vu.nl/~ibivu/programs/popswww.
A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.
Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
A functional screen of a metagenomic library from “Upo” swamp sediment in Korea identified a gene EstL28, the product of which displayed lipolytic properties on a tributyrin-supplemented medium. The EstL28 sequence encodes a 290 amino acid protein (designated as EstL28), with a predicted molecular weight of 31.3 kDa. The encoded EstL28 protein exhibited the highest sequence similarity (45 %) to a hydrolase found in Streptococcus
sanguinis. Phylogenetic analysis indicated that EstL28 belongs to a currently uncharacterized family of esterases. Within the conserved α/β-hydrolase 6 domain, the EstL28 retains the catalytic triad Ser103–Asp248–His268 that is typical of esterases. The Ser103 residue in the catalytic triad is located in the consensus pentapeptide motif GXSXG. The purified EstL28 enzyme worked optimally at 35 °C and pH 8.5 and remained stable at temperatures lower than 20 °C. The catalytic activity of EstL28 was maximal with p-nitrophenyl butyrate, indicating that it was an esterase. This enzyme also exhibited stable activity in the presence of methanol, ethanol, isopropanol, and dimethyl sulfoxide. Therefore, the level of stability in organic solvents and cold temperature suggests that EstL28 has potential for many biotechnological applications.
A novel alkaliphilic esterase (EstJ) was identified from a soil metagenome of Jeju Island, Korea, using a 96-well plate-based functional assay for determination of pH dependence of activity. The amino acid sequence of EstJ showed low similarity (32-45 %) to putative α/β hydrolases derived from whole-genome sequencing studies. EstJ, although not belonging to any of the known families of bacterial lipolytic enzymes, however, it showed closest sequence identity to the family IV enzymes that are related to the mammalian hormone-sensitive lipases. The highly conserved motifs of family IV enzymes were found in EstJ, but the corresponding sequences of each motif in EstJ were unique; most particularly the -(F/Y)(F/Y/L)HGGG- motif was represented by -WMVSGG-. The purified EstJ was highly active from pH 8.5 to 10.5. More than 90 % of maximum activity was also retained over a wide pH range of 5.5-0.5 after prolonged incubation. EstJ was also moderately thermophilic with an optimum temperature of 55 °C. Therefore, EstJ is the first metagenome-derived bacterial family IV esterase possessing both highly alkaliphilic and moderately thermophilic properties.
A novel cold-adapted lipolytic enzyme gene, est97, was identified from a high Arctic intertidal zone sediment metagenomic library. The deduced amino acid sequence of Est97 showed low similarity with other lipolytic enzymes, the maximum being 30 % identity with a putative lipase from Vibrio caribbenthicus. Common features of lipolytic enzymes, such as the GXSXG sequence motif, were detected. The gene product was over-expressed in Escherichia coli and purified. The recombinant Est97 (rEst97) hydrolysed various ρ-nitrophenyl esters with the best substrate being ρ-nitrophenyl hexanoate (K (m) and k (cat) of 39 μM and 25.8 s(-1), respectively). This esterase activity of rEst97 was optimal at 35 °C and pH 7.5 and the enzyme was unstable at temperatures above 25 °C. The apparent melting temperature, as determined by differential scanning calorimetry was 39 °C, substantiating Est97 as a cold-adapted esterase. The crystal structure of rEst97 was determined by the single wavelength anomalous dispersion method to 1.6 Å resolution. The protein was found to have a typical α/β-hydrolase fold with Ser144-His226-Asp197 as the catalytic triad. A suggested, relatively short lid domain of rEst97 is composed of residues 80-114, which form an α-helix and a disordered loop. The cold adaptation features seem primarily related to a high number of methionine and glycine residues and flexible loops in the high-resolution structures.
Dried solid-state fermented solids (biocatalysts) produced by seven thermotolerant fungal strains were tested for lipase activity and stability in organic solvents. Two strains of Rhizopus sp. (19 and 43a) produced biocatalysts (L-19 and L-43a) that showed high lipase activities (74 and 72 U/g of dry matter, respectively) comparable to Lipozyme® RM IM (118 U/g DM). The use of the dipole moment of the organic solvents along with their classification based on the functional groups (non-polar, protic polar, aprotic polar) allowed the establishment of four different relative activity profiles for the seven biocatalysts evaluated. Compared to a biocatalyst not exposed to the organic solvent (100% relative activity), all biocatalysts showed a high relative activity (greater than 90%) in aprotic polar solvents (acetonitrile, acetone and ethyl acetate), whereas in protic polar solvents (ethanol and i-propanol) activity was reduced (lower than 40%). In addition, the incubation of biocatalysts L-19 and L-43a in i-amyl alcohol increased lipase activity in the synthesis of ethyl oleate 3.36 and 1.46 times, respectively. L-19 activity also increased after incubation in toluene (2.0 times), i-propanol (1.5 times) and acetonitrile (1.3 times) at temperatures from 30 to 50 °C. The results suggest that these biocatalysts can be used for a broad range of lipase reactions.
Lipase from Mucor miehei was covalently immobilised onto the graft copolymer poly(ethylene)-g.co-hydroxyethyl methacrylate (PE-g.co-HEMA), partially hydrolysed, via a spacer arm of 1,6-diaminohexane activated with glu-taraldehyde. To improve the lipolytic activity of the immobilised lipase (for the synthesis of isoamyl-caprylate, as a model), the effect of several additives was investigated. Polyethylene glycol (PEG), glutaraldehyde, organic solvents and buffers, were added during the immobilisation procedure and their effects are reported and compared with the behaviour of the lipolytic preparation without pre-treatment. An increase of 40 – 100% in the activity was obtained when small quantities of PEG 2000 and glutaraldehyde (used also as an activator of the spacer arm) were added. The activity had a maximum when the pH of the lipase attachment solution was 7.2 and buffered with phosphate. The effect of the aggregation level of biocatalyst particles on the amount of water retained, as well as the effect of the immobilisation on solid supports on the stability to organic solvents, is also reported. © 1998 Elsevier Science B.V. All rights reserved.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
Psychrophilic enzymes produced by cold-adapted microorganisms display a high catalytic efficiency and are most often, if not always, associated with high thermosensitivity. Using X-ray crystallography, these properties are beginning to become understood, and the rules governing their adaptation to cold appear to be relatively diverse. The application of these enzymes offers considerable potential to the biotechnology industry, for example, in the detergent and food industries, for the production of fine chemicals and in bioremediation processes.
A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1-50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1-40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.
A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909 bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.
To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50-57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.
The aims of this study were to access the bacterial diversity and isolate lipolytic genes using the metagenomic approach in activated sludge of a swine wastewater treatment facility. On the basis of BLASTN analysis of 16S rRNA gene clones, most of these communities (90%) were of uncultivated bacteria. The metagenomic library was constructed using a plasmid vector and DNA extracted directly from activated sludge samples. The average insert size was approximately 5.1 kb. A total of 12 unique and lipolytic clones were obtained using the tributyrin plate assay. The rate of discovering a lipolytic clone in this study was as high as 0.31%. Molecular analysis revealed that most of the 16 putative lipolytic enzymes showed 28-55% identity with non-redundant protein sequences in the database. Briefly, this study demonstrates that activated sludge is an ideal bioresource for isolating new lipolytic enzymes.
Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 degrees C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C(2) to C(12) and the preferred substrate was pNP-pentanoate showing a k(cat) = 26.2 +/- 0.1 s(-1), K(M) = 0.122 +/- 0.006 mM and a k(cat)/K(M) = 215 +/- 11 mM(-1) s(-1). The enzyme was strongly inhibited by Hg(2+), Zn(2+), Cu(2+), Fe(3+), Mn(2+) ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C(50) values of 1.98 M and 0.92 M, respectively. The region surrounding PsyHSL catalytic site showed an unexpected homology with the human HSL. Further, both enzymes are characterised by the presence of an extra N-terminal domain, which role in the human protein has been related to regulative function. To clarify the function of PsyHSL N-terminal domain, a 97 amino acids deleted version of the enzyme was produced in E. coli in soluble form, purified and characterised. This mutant was inactive towards all tested substrates, indicating the involvement of this region in the catalytic process.
Metagenomic cloning is a powerful tool for the discovery of novel genes and biocatalysts from environmental microorganisms. Based on activity screening of a marine sediment microbial metagenomic library, a total of 19 fosmid clones showing lipolytic activity were identified. After subcloning, 15 different lipolytic genes were obtained; their encoded proteins showed 32-68% amino acid identity with proteins in the database. Multiple sequence alignment and phylogenetic tree analysis demonstrated that most of these predicted proteins are new members of known families of bacterial lipolytic enzymes. However, two proteins, FLS18C and FLS18D, could not be assigned to any known family, thus probably representing a novel family of the bacterial lipolytic enzyme. The activity assay results indicated that most of these lipolytic enzymes showed optimum temperature for hydrolysis at 40-50 degrees C with p-nitrophenol butyrate as a substrate. The lipolytic gene fls18D was overexpressed, and the resulting protein FLS18D was characterized as an alkaline esterase. Furthermore, the whole sequence of fosmid pFL18 containing FLS18C and FLS18D was shotgun sequenced, and a total of 26 ORFs on it were analyzed and annotated.
A bacterial strain 5YN5-8(T) was isolated from peat layer on Yongneup in Korea. Cells of strain 5YN5-8(T) were strictly aerobic, Gram-negative, coccobacilli, non-spore forming, and non-motile. The isolate exhibited optimal growth at 28 degrees C, pH 7.0, and 0-1% NaCl. Results of 16S rRNA gene sequence analyses indicated a close relationship of this isolate to Acinetobacter calcoaceticus (97.8% similarity for strain DSM 30006(T)). It also exhibited 94.4-97.8% 16S rRNA gene sequence similarities to the validly published Acinetobacter species. The value for DNA-DNA hybridization between strain 5YN5-8(T) and other members of the genus Acinetobacter ranged from 16 to 28%. Predominant cellular fatty acids were C(18:1) omega9c, summed feature 4 containing C(15:0) iso 2-OH and/or C(16:1) omega7c, and C(16:0). The DNA G+C content was 43.9 mol%. Phylogenetic, phenotypic, and chemotaxonomic data accumulated in this study revealed that the isolate could be classified in a novel species of the genus Acinetobacter. The name Acinetobacter brisouii sp. nov. is proposed for the novel species, with 5YN5-8(T) (=KACC 11602(T) = DSM 18516(T)) as the type strain.
Using a metagenome library constructed from a bacterial associated with a marine sponge Hyrtios erecta, we identified a novel esterase that belongs to the SGNH hydrolase superfamily of esterases. The substrate specificity of EstHE1 was determined using p-nitrophenyl (pNP) ester (C2: acetate, C4: butylate, C6: caproate, C12: laurate, C16: palmitate). EstHE1 exhibited activity against C2 (5.6 U/mg), C4 (5.1 U/mg), and C6 (2.8 U/mg) substrates. The optimal temperature for EstHE1 esterase activity of the pNP acetate substrate was 40 degrees C, and EstHE1 retained 60% of its enzymatic activity in the 30-50 degrees C range. This esterase showed moderate thermostability, retaining 58% of its activity even after preincubation for 12 h at 40 degrees C. EstHE1 also maintained activity in high concentrations of NaCl, indicating that this esterase is salt-tolerant. Thus, EstHE1 has the thermal stability and salt tolerance necessary for use as an industrial enzyme.
The quest for a quick and easy detection of the neurotoxin levels in the environment has fostered the search for systems alternative to currently employed analytical methods such as spectrophotometer, gas-liquid chromatography, thin-layer chromatography, and more recently mass spectrometry. These drawbacks lead to intense research efforts to develop biosensor devices for the determination of these compounds. In this review, we present an overview of the actual development of research in neurotoxin detection by using enzymatic biosensors based on esterase activity, in particular cholinesterases, and carboxylesterases. Detection by enzymatic activity could be carried out measuring the hydrolysis products or the residual enzymatic activity after inhibition, using a transducer system that makes possible the correlation between the determined activity and the analyte concentration. Several transducer systems were adopted for the neurotoxins identification using esterases, including electrochemical, optical, conductimetric and piezoelectric procedures. The differences in the used transducer determine the final sensitivity and specificity of the biosensor. Moreover, a brief description of immobilization procedure, that is an important step in the biosensor development and could affect the final characteristic of biosensor (sensibility, stability, response time and reproducibility), was accomplished. Final considerations on advantages and problems, related to actual development of these technologies, and its prospective were discussed.
Metagenomics has accelerated the process of discovery of novel biocatalysts by enabling scientists to tap directly into the entire diversity of enzymes held within natural microbial populations. Their characterization has revealed a great deal of valuable information about enzymatic activity in terms of factors which influence their stability and activity under a wide range of conditions. Many of the biocatalysts have particular properties making them suitable for biotechnological applications. A diverse array of strategies has been developed to optimize each step of the process of generating and screening metagenomic libraries for novel biocatalysts. This review covers the diversity of metagenome-derived enzymes characterized to date, and the strategies currently being developed to optimize discovery of novel metagenomic biocatalysts.
Sequence comparison studies revealed that the drug resistance transporter of Streptomyces peucetius (DrrAB) and two nodulation gene products (NodIJ) of Rhizobium leguminosarum are homologous to proteins encoded by three sets of genes that comprise capsular polysaccharide export systems in gram-negative bacteria: KpsTM of Escherichia coli, BexABC of Haemophilus influenzae, and CtrDCB of Neisseria meningitidis. These five systems comprise a new subfamily within the family of ATP binding cassette (ABC)-type transporters. We have termed this subfamily the ABC-2 subfamily. For three of the systems comprising this subfamily (Drr, Nod, and Kps) only one integral membrane constituent has been identified, whereas for the other two systems (Bex and Ctr) two dis-similar integral membrane constituents have been found. This observation suggests that the transmembrane channels of ABC-2-type transporters can be formed of homo- or heterooligomers as is true of several other classes of transport systems.
A FORTRAN 77 computer program has been written to aid with macromolecular modeling and drug design. Called WHAT IF, it provides an intelligent and flexible environment for displaying, manipulating, and analyzing small molecules, proteins, nucleic acids, and their interactions. A relational protein structure database is incorporated to be queried. The program is suitable for most common crystallographic work. The menu-driven operation of WHAT IF, combined with the use of default values wherever user input is required, makes it very easy to use for a novice user while keeping full flexibility for more sophisticated studies. Although there are not too many unique features in WHAT IF, the fact that everything is integrated in one program makes it a unique tool for many purposes.
Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.
The crystal structure of a novel esterase from Streptomyces scabies, a causal agent of the potato scab disease, was solved at 2.1 A resolution. The tertiary fold of the enzyme is substantially different from that of the alpha/beta hydrolase family and unique among all known hydrolases. The active site contains a dyad of Ser 14 and His 283, closely resembling two of the three components of typical Ser-His-Asp(Glu) triads from other serine hydrolases. Proper orientation of the active site imidazol is maintained by a hydrogen bond between the N delta-H group and a main chain oxygen. Thus, the enzyme constitutes the first known natural variation of the chymotrypsin-like triad in which a carboxylic acid is replaced by a neutral hydrogen-bond acceptor.
We describe a comparative protein modelling method designed to find the most probable structure for a sequence given its alignment with related structures. The three-dimensional (3D) model is obtained by optimally satisfying spatial restraints derived from the alignment and expressed as probability density functions (pdfs) for the features restrained. For example, the probabilities for main-chain conformations of a modelled residue may be restrained by its residue type, main-chain conformation of an equivalent residue in a related protein, and the local similarity between the two sequences. Several such pdfs are obtained from the correlations between structural features in 17 families of homologous proteins which have been aligned on the basis of their 3D structures. The pdfs restrain C alpha-C alpha distances, main-chain N-O distances, main-chain and side-chain dihedral angles. A smoothing procedure is used in the derivation of these relationships to minimize the problem of a sparse database. The 3D model of a protein is obtained by optimization of the molecular pdf such that the model violates the input restraints as little as possible. The molecular pdf is derived as a combination of pdfs restraining individual spatial features of the whole molecule. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non-hydrogen atoms. The method is automated and is illustrated by the modelling of trypsin from two other serine proteinases.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.
Psychrophilic organisms have successfully colonized polar and alpine regions and are able to grow efficiently at sub-zero temperatures. At the enzymatic level, such organisms have to cope with the reduction of chemical reaction rates induced by low temperatures in order to maintain adequate metabolic fluxes. Thermal compensation in cold-adapted enzymes is reached through improved turnover number and catalytic efficiency. This optimization of the catalytic parameters can originate from a highly flexible structure which provides enhanced abilities to undergo conformational changes during catalysis. Thermal instability of cold-adapted enzymes is therefore regarded as a consequence of their conformational flexibility. A survey of the psychrophilic enzymes studied so far reveals only minor alterations of the primary structure when compared to mesophilic or thermophilic homologues. However, all known structural factors and weak interactions involved in protein stability are either reduced in number or modified in order to increase their flexibility.
EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at temperatures higher than 50°C. The Michaelis constant (K
m
) and V
max for p-nitrophenyl butyrate were 11 μM and 131.6 μM min−1 mg of protein-1, respectively. The enzyme was strongly inhibited by Hg2−, Ca2+, Mg2+, Fe2+, Cu2+, Zn2+, Mn2+, Co2+, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP).
Esterases (EC 3.1.1.x) represent a diverse group of hydrolases catalyzing the cleavage and formation of ester bonds and are widely distributed in animals, plants and microorganisms. Beside lipases, a considerable number of microbial carboxyl esterases have also been discovered and overexpressed. This review summarizes their properties and classification. Special emphasis is given on their application in organic synthesis for the resolution of racemates and prostereogenic compounds. In addition, recent results for altering their properties by directed evolution are presented.
The esterase-encoding gene, estA, was cloned from Acinetobacter lwoffii I6C-1 genomic DNA into Escherichia coli BL21(DE3) with plasmid vector pET-22b (pEM1). pEM1 has a 4.4-kb EcoRI insert that contained the complete estA gene. A 2.4-kb AvaI- SphI DNA fragment was subcloned (pEM3) and sequenced. estA gene encodes a protein of 366 amino acids (40,687 Da) with a pI of 9.17. The EstA signal peptide was 31 amino acids long, and the mature esterase sequence is 335 amino acids long (37.5 kDa). The conserved catalytic serine residue of EstA is in position 210. The EstA sequence was similar to that of the carboxylesterase from Acinetobacter calcoaceticus (75% identity, 85% similarity), Archaeoglobus fulgidus (37% identity, 59% similarity), and Mycobacterium tuberculosis (35% identity, 51% similarity). These enzymes contained the conserved motif G-X(1)-S-X(2)-G carrying the active-site serine of hydrolytic enzyme. The EstA activity in A. lwoffii I6C-1 remains constant throughout the stationary phase, and the activity in E. coil BL21 (DE3) with pEM1 was similar to A. lwoffii I6C-1.