Lipases: it's not just pancreatic lipase!

Lipases are water-soluble enzymes that hydrolyze water-insoluble lipid molecules, such as triglycerides, phospholipids, and galactolipids. They are ubiquitous in nature and are present in humans, animals, insects, plants, fungi, and microorganisms. While we commonly consider pancreatic lipase, this review provides an overview of several lipases that are important for the digestion and metabolism of lipids in veterinary species. All of these enzymes have specific functions but share a common α/ß-hydrolase fold and a catalytic triad where substrate hydrolysis occurs. The pancreatic lipase gene family is one of the best characterized lipase gene families and consists of 7 mammalian subfamilies: pancreatic lipase, pancreatic lipase related proteins 1 and 2, hepatic lipase, lipoprotein lipase, endothelial lipase, and phosphatidylserine phospholipase A1. Other mammalian lipases that play integral roles in lipid digestion include carboxyl ester lipase and gastric lipase. Although most enzymes have preferred substrate specificity, much overlap occurs across the plethora of lipases because of the similarities in their structures. This has major implications for the development and clinical utilization of diagnostic assays. These implications are further explored in our companion Currents in One Health article by Lim et al in the August 2022 issue of the Journal of American Veterinary Medical Association, which focuses on pancreatic lipase assays for the diagnosis of pancreatitis.

Detection and diversity of the mannosylerythritol lipid (MEL) gene cluster and lipase A and B genes of Moesziomyces antarcticus isolated from terrestrial sites chronically contaminated with crude oil in Trinidad.

BACKGROUND: Mannosylerythritol lipids (MELs) belong to the class of glycolipid biosurfactants and are produced by members of the Ustilago and Moesziomyces genera. Production of MELs is regulated by a biosynthetic gene cluster (MEL BGC). Extracellular lipase activity is also associated with MEL production. Most microbial glycolipid-producers are isolated from oil-contaminated environments. MEL-producing yeast that are capable of metabolizing crude oil are understudied, and there is very limited data on indigenous strains from tropical climates. Analysis of the MEL BGC and lipase genes in Trinidad M. antarcticus strains, using a gene-targeted approach, revealed a correlation between their intrinsic capability to degrade crude oil and their adaptation to survive in a chronically polluted terrestrial environment. RESULTS: M. antarcticus was isolated from naturally-occurring crude oil seeps and an asphaltic mud volcano in Trinidad; these are habitats that have not been previously reported for this species. Genus identification was confirmed by the large-subunit (LSU) and the small-subunit (SSU) sequence comparisons and species identification was confirmed by ITS sequence comparisons and phylogenetic inference. The essential genes (Emt1, Mac1, Mac2, Mmf1) of the MEL BGC were detected with gene-specific primers. Emt1p, Mac1p and Mmf1p sequence analyses confirmed that the Trinidad strains harboured novel synonymous amino acid (aa) substitutions and structural comparisons revealed different regions of disorder, specifically for the Emt1p sequence. Functionality of each protein sequence was confirmed through motif mining and mutation prediction. Phylogenetic relatedness was inferred for Emt1p, Mac1p and Mmf1p sequences. The Trinidad strains clustered with other M. antarcticus sequences, however, the representative Trinidad M. antarcticus sequences consistently formed a separate, highly supported branch for each protein. Similar phylogenetic placement was indicated for LipA and LipB nucleotide and protein sequences. The Trinidad strains also demonstrated lipolytic activity in culture, with an ability to utilize different carbon sources. Comparative evolution of MEL BGC and LipA gene suggested early and late duplication events, depending on the gene, followed by a number of speciation events within Ustilaginaceae. M. antarcticus and M. aphidis were separated from all other members of Ustilaginaceae and two gene homologues were detected, one for each species. CONCLUSIONS: Sequence analyses was based on a novel gene-targeted approach to analyze the essential genes of the MEL BGC and LipA and LipB genes of M. antarcticus strains from Trinidad. The findings indicated that these strains accumulated nucleotide mutations to a threshold level that did not affect the function of specific proteins encoded by the MEL BGC and LipA and LipB genes. The biosurfactant and lipase enzymes secreted by these Trinidad M. antarcticus strains facilitated their survival in oil-contaminated terrestrial environments. These findings suggest that the Trinidad strains should be explored as promising candidates for the commercial production of MEL biosurfactants and lipase enzymes.

Candida lipases: a review on biochemical, molecular and pathogenic aspects. / [Lipasas de especies Candida: una revisión sobre aspectos bioquímicos, moleculares y patogénicos.]

In the last half century there was a significant increase in the incidence of fungal infections being likely to become a global health priority. The sophisticated degree of host-Candida interaction is the product of different virulence strategies used by the fungus to invade the tissues and the various defense mechanisms that it develops to control it. There is a significant amount of literature that indicates that this opportunistic commensal fungus has components that can be considered virulence factors related to the stage of the infectious process. Among the virulence factors of this fungus can be mentioned the adherence to cell surfaces, the formation of biofilms and the production of hydrolytic enzymes. The most studied hydrolases secreted by C. albicans are aspartyl proteinases, phospholipases and esterases, while lipases have been the least studied. These enzymes would have the function to facilitate active penetration into the cells, participating in the digestion and synthesis of lipid esters for their nutrition and contributing to the invasion of the tissue by hydrolyzing the lipid components of the host cell membranes. There is also bibliographic evidence that these enzymes are capable to damage cells and molecules of the immune system to avoid the antimicrobial activity.Taking into account the foregoing, this review provides an updated description of biochemical and molecular characteristics of the lipases secreted by Candida, its role as a virulence factor and its potential for the development of new antifungal drugs.

En el último medio siglo se produjo un aumento significativo en la incidencia de infecciones fúngicas siendo probable que se conviertan en una prioridad de salud global. El sofisticado grado de interacción hospedador-Candida es producto de diferentes estrategias de virulencia que utiliza el hongo para invadir los tejidos y de los diversos mecanismos de defensa que este último desarrolla para controlarlo. Existe bibliografía que indica que este hongo comensal oportunista posee componentes que pueden ser considerados factores de virulencia asociados a la etapa del proceso infeccioso. Dentro de los factores de virulencia de este hongo pueden mencionarse la adherencia a las superficies celulares, la formación de biofilms y la producción de enzimas hidrolíticas. Las hidrolasas secretadas por C. albicans más estudiadas son las aspartil proteinasas, las fosfolipasas y las esterasas, mientras que las lipasas han sido las menos exploradas. Estas enzimas tendrían como función facilitar la penetración activa en las células, participar en la digestión y síntesis de ésteres de lípidos para su nutrición y contribuir a la invasión del tejido al hidrolizar los componentes lipídicos de las membranas celulares del hospedador. También hay evidencia bibliográfica que indica que estas enzimas son capaces de dañar células y moléculas del sistema inmune para evitar la actividad antimicrobiana. Teniendo en cuenta lo precedente, esta revisión, proporciona una actualizada descripción de las características bioquímicas y moleculares de las lipasas secretadas por el hongo Candida, su rol como factor de virulencia y su potencial para el desarrollo de nuevos fármacos antifúngicos.

Comparative genomic analysis of the Lipase3 gene family in five plant species reveals distinct evolutionary origins.

Lipases are physiologically important and ubiquitous enzymes that share a conserved domain and are classified into eight different families based on their amino acid sequences and fundamental biological properties. The Lipase3 family of lipases was reported to possess a canonical fold typical of α/ß hydrolases and a typical catalytic triad, suggesting a distinct evolutionary origin for this family. Genes in the Lipase3 family do not have the same functions, but maintain the conserved Lipase3 domain. There have been extensive studies of Lipase3 structures and functions, but little is known about their evolutionary histories. In this study, all lipases within five plant species were identified, and their phylogenetic relationships and genetic properties were analyzed and used to group them into distinct evolutionary families. Each identified lipase family contained at least one dicot and monocot Lipase3 protein, indicating that the gene family was established before the split of dicots and monocots. Similar intron/exon numbers and predicted protein sequence lengths were found within individual groups. Twenty-four tandem Lipase3 gene duplications were identified, implying that the distinctive function of Lipase3 genes appears to be a consequence of translocation and neofunctionalization after gene duplication. The functional genes EDS1, PAD4, and SAG101 that are reportedly involved in pathogen response were all located in the same group. The nucleotide diversity (Dxy) and the ratio of nonsynonymous to synonymous nucleotide substitutions rates (Ka/Ks) of the three genes were significantly greater than the average across the genomes. We further observed evidence for selection maintaining diversity on three genes in the Toll-Interleukin-1 receptor type of nucleotide binding/leucine-rich repeat immune receptor (TIR-NBS LRR) immunity-response signaling pathway, indicating that they could be vulnerable to pathogen effectors.

GDSL lipases modulate immunity through lipid homeostasis in rice.

Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of OsGLIP1 and OsGLIP2 was suppressed by pathogen infection and salicylic acid (SA) treatment. OsGLIP1 was mainly expressed in leaf and leaf sheath, while OsGLIP2 showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of OsGLIP1 and OsGLIP2 increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in OsGLIP1 and OsGLIP2 overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER) membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of OsGLIP gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity.

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

BACKGROUND: Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. RESULTS: In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. CONCLUSIONS: The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Multiple resistance to pirimiphos-methyl and bifenthrin in Tribolium castaneum involves the activity of lipases, esterases, and laccase2.

Several recent studies have elucidated the molecular mechanisms that confer insecticide resistance on insect pests. However, little is known about multiple resistance in red flour beetle (Tribolium castaneum) at molecular level. The multiple resistance is characterized as resistance to different classes of insecticides that have different target sites, and is mediated by several enzymatic systems. In this study, we investigated the biochemical and molecular mechanisms involved in multiple resistance of T. castaneum to bifenthrin (pyrethroid [Pyr]) and pirimiphos-methyl (organophosphate [Org]). We used artificial selection, biochemical and in silico approaches including structural computational biology. After five generations of artificial selection in the presence of bifenthrin (F5Pyr) or pirimiphos-methyl (F5Org), we found high levels of multiple resistance. The hierarchical enzymatic cluster revealed a pool of esterases (E), lipases (LIPs) and laccase2 (LAC2) potentially contributing to the resistance in different ways throughout development, after one or more generations in the presence of insecticides. The enzyme-insecticide interaction network indicated that E2, E3, LIP3, and LAC2 are enzymes potentially required for multiple resistance phenotype. Kinetic analysis of esterases from F5Pyr and F5Org showed that pirimiphos-methyl and specially bifenthrin promote enzyme inhibition, indicating that esterases mediate resistance by sequestering bifenthrin and pirimiphos-methyl. Our computational data were in accordance with kinetic results, indicating that bifenthrin has higher affinity at the active site of esterase than pirimiphos-methyl. We also report the capability of these insecticides to modify the development in T. castaneum. Our study provide insights into the biochemical mechanisms employed by T. castaneum to acquire multiple resistance.

A novel thermophilic and halophilic esterase from Janibacter sp. R02, the first member of a new lipase family (Family XVII).

Janibacter sp. strain R02 (BNM 560) was isolated in our laboratory from an Antarctic soil sample. A remarkable trait of the strain was its high lipolytic activity, detected in Rhodamine-olive oil supplemented plates. Supernatants of Janibacter sp. R02 displayed superb activity on transesterification of acyl glycerols, thus being a good candidate for lipase prospection. Considering the lack of information concerning lipases of the genus Janibacter, we focused on the identification, cloning, expression and characterization of the extracellular lipases of this strain. By means of sequence alignment and clustering of consensus nucleotide sequences, a DNA fragment of 1272bp was amplified, cloned and expressed in E. coli. The resulting recombinant enzyme, named LipJ2, showed preference for short to medium chain-length substrates, and displayed maximum activity at 80°C and pH 8-9, being strongly activated by a mixture of Na+ and K+. The enzyme presented an outstanding stability regarding both pH and temperature. Bioinformatics analysis of the amino acid sequence of LipJ2 revealed the presence of a consensus catalytic triad and a canonical pentapeptide. However, two additional rare motifs were found in LipJ2: an SXXL ß-lactamase motif and two putative Y-type oxyanion holes (YAP). Although some of the previous features could allow assigning LipJ2 to the bacterial lipase families VIII or X, the phylogenetic analysis showed that LipJ2 clusters apart from other members of known lipase families, indicating that the newly isolated Janibacter esterase LipJ2 would be the first characterized member of a new family of bacterial lipases.

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/ß-hydrolase_1, α/ß-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/ß-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform for the selection of candidate lipase genes for further detailed functional study.

Enzyme classification using complex dynamic hemithioacetal systems.

A complex dynamic hemithioacetal system was generated for the evaluation of lipase reactivities in organic media. In combination with pattern recognition methodology, twelve different lipases were successfully classified into four distinct groups following their reaction selectivities and reactivities. A probe lipase was further categorized using the training matrix with predicted reactivity.

Hydrolytic enzymes expressivity in different parts of the Rapana digestive system.

The relevance of comprehensive studies of the Rapana vital functions is determined by its considerab­le negative impact on the ecosystem of the Black Sea. The aim of the work was to find out the polymorphism and activity of the main hydrolases in the different parts of the digestive system of Rapana. Hydrolases (proteases, amylases, esterases, lipases and phosphatases) in glandular structures of the Rapana digestive system were studied by electrophoresis. It was found that different sets of hydrolytic enzymes are functioning in certain parts of the Rapana digestive tract. The gland of Leiblein and hepatopancreas played the most important role in the digestion of food components. The salivary glands had the significant influence on proteolysis.

The genotypic diversity and lipase production of some thermophilic bacilli from different genera

Abstract Thermophilic 32 isolates and 20 reference bacilli were subjected to Rep-PCR and ITS-PCR fingerprinting for determination of their genotypic diversity, before screening lipase activities. By these methods, all the isolates and references could easily be differentiated up to subspecies level from each other. In screening assay, 11 isolates and 7 references were found to be lipase producing. Their extracellular lipase activities were measured quantitatively by incubating in both tributyrin and olive oil broths at 60 °C and pH 7.0. During the 24, 48 and 72-h period of incubation, the changes in the lipase activities, culture absorbance, wet weight of biomass and pH were all measured. The activity was determined by using pNPB in 50 mM phosphate buffer at pH 7.0 at 60 °C. The lipase production of the isolates in olive oil broths varied between 0.008 and 0.052, whereas these values were found to be 0.002-0.019 (U/mL) in the case of tyributyrin. For comparison, an index was established by dividing the lipase activities to cell biomass (U/mg). The maximum thermostable lipase production was achieved by the isolates F84a, F84b, and G. thermodenitrificans DSM 465T (0.009, 0.008 and 0.008 U/mg) within olive oil broth, whereas G. stearothermophilus A113 displayed the highest lipase activity than its type strain in tyributyrin. Therefore, as some of these isolates displayed higher activities in comparison to references, new lipase producing bacilli were determined by presenting their genotypic diversity with DNA fingerprinting techniques.

Identification and characterization of lipases from Malassezia restricta, a causative agent of dandruff.

Dandruff, a skin disorder affecting 50% of the world population, is linked with proliferation of lipophilic yeasts of the genus Malassezia (particularly Malassezia globosa and M. restricta). Most Malassezia species show a unique lipid dependency and require external lipids for growth. Genome mining of the incomplete M. restricta genome led to the identification of eight lipase sequences. Sequences representing the class 3 and LIP lipase families were used to clone the lipases MrLip1, MrLip2 and MrLip3, recombinantly expressed in Pichia pastoris, and tested for their activity using mono-, di- and triacylglycerol substrates. Hydrolysis by the M. restricta lipase MrLip1 and MrLip2 (family class 3) was limited to the mono- and diacylglycerol, while MrLip3 (family LIP) hydrolyzed all three substrates. This result confirms that Malassezia family LIP lipases are responsible for the hydrolysis of triacylglycerols, the main component of human sebum. Furthermore, the information regarding lipases from M. restricta presented here might aid in the search for anti-dandruff agents.

Isolation of a novel alkaline-stable lipase from a metagenomic library and its specific application for milkfat flavor production.

BACKGROUND: Lipolytic enzymes are commonly used to produce desired flavors in lipolyzed milkfat (LMF) manufacturing processes. However, the choice of enzyme is critical because it determines the final profile of fatty acids released and the consequent flavor of the product. We previously constructed a metagenomic library from marine sediments, to explore the novel enzymes which have unique properties useful in flavor-enhancing LMF. RESULTS: A novel lipase Est_p6 was isolated from a metagenomic library and was expressed highly in E.coli. Bioinformatic analysis indicated that Est_p6 belongs to lipolytic enzyme family IV, the molecular weight of purified Est_p6 was estimated at 36 kDa by SDS-PAGE. The hydrolytic activity of the enzyme was stable under alkaline condition and the optimal temperature was 50°C. It had a high specific activity (2500 U/mg) toward pNP butyrate (pNP-C4), with K(m) and V(max) values of 1.148 mM and 3497 µmol∙min⁻¹âˆ™mg⁻¹, respectively. The enzyme activity was enhanced by DTT and was not significantly inhibited by PMSF, EDTA or SDS. This enzyme also showed high hydrolysis specificity for myristate (C14) and palmitate (C16). It seems that Est_p6 has safety for commercial LMF flavor production and food manufacturing processes. CONCLUSIONS: The ocean is a vast and largely unexplored resource for enzymes. According the outstanding alkaline-stability of Est_p6 and it produced myristic acid and palmitic acid more efficiently than other free fatty acids in lipolyzed milkfat. This novel lipase may be used to impart a distinctive and desirable flavor and odor in milkfat flavor production.

Isolation and characterization of a thermostable esterase from a metagenomic library.

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser192-Glu313-His412 with Ser92 in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K(m) and k(cat) of 0.33 mM and 36.21 s⁻¹, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.

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/ß 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.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

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 a/ß 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.(AU)

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.(AU)

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

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 a/ß 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.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.

ESTHER, the database of the α/ß-hydrolase fold superfamily of proteins: tools to explore diversity of functions.

The ESTHER database, which is freely available via a web server (http://bioweb.ensam.inra.fr/esther) and is widely used, is dedicated to proteins with an α/ß-hydrolase fold, and it currently contains >30 000 manually curated proteins. Herein, we report those substantial changes towards improvement that we have made to improve ESTHER during the past 8 years since our 2004 update. In particular, we generated 87 new families and increased the coverage of the UniProt Knowledgebase (UniProtKB). We also renewed the ESTHER website and added new visualization tools, such as the Overall Table and the Family Tree. We also address two topics of particular interest to the ESTHER users. First, we explain how the different enzyme classifications (bacterial lipases, peptidases, carboxylesterases) used by different communities of users are combined in ESTHER. Second, we discuss how variations of core architecture or in predicted active site residues result in a more precise clustering of families, and whether this strategy provides trustable hints to identify enzyme-like proteins with no catalytic activity.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification. / Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.

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/ß 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.