SciELO - Scientific Electronic Library Online

vol.45 número1Micólogos argentinos pioneros en el estudio de la histoplasmosisRespuesta humoral y consecuencias reproductivas en ovejas desafiadas con Brucella ovis al final de la gestación índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados




  • No hay articulos citadosCitado por SciELO

Links relacionados


Revista argentina de microbiología

versión impresa ISSN 0325-7541

Rev. argent. microbiol. vol.45 no.1 Ciudad Autónoma de Buenos Aires mar. 2013



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

Renaud Berlemont1#, Olivier Spee1, Maud Delsaute1, Yannick Lara2, Jörg Schuldes3, Carola Simon3, Pablo Power1#*, Rolf Daniel3, Moreno Galleni1

1Laboratory of Biological Macromolecules and
2Laboratory of Cyanobacteria, Centre for Protein Engineering, University of Liège, Institut de Chimie B6a, Liège, Sart-Tilman, Belgium;
3Department of Genomics and Applied Microbiology, Institute of Microbiology and Genetics Georg-August-University Göttingen Grisebachstrasse 8, D-37077 Göttingen, Germany.

*Correspondence. E-mail:

# Current address: Dept. of Earth System Science and Dept. of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA.
#* Corresponding author current address: Departamento de Microbiología, Inmunología y Biotecnología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. Junín 956 (1113) Ciudad Autónoma de Buenos Aires, Argentina.


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.

Key words: Hormone-sensitive lipase family; Activity-driven metagenomics; Metagenomic library; abH4.04; Lysinibacillus sphaericus.


Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas. 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.

Palabras clave: Familia de lipasas sensibles a hormonas; Metagenomica funcional; Libreria metagenomica; abH4.04; Lysinibacillus sphaericus.


Metagenomics comprises a series of methodological approaches for accessing the genetic information (the "metagenome") from microbial consortia living in different habitats including uncultured microorganisms that collectively account for more than 95 % of the total, without the need of previous isolation techniques (15, 21). Methodologically, metagenomics is based on the extraction of total DNA from the sample, digestion and cloning of resulting fragments and transformation of ligation mixture into cultivable hosts like Escherichia coli (10, 17). The genetic content of the metagenome is finally stored in a metagenomic library from which different target genes or activities can be screened.

This technology paved the way for elucidating the functions of microbial communities, genomic analyses of uncultured soil microorganisms and to search for new genes coding for various proteins from unbiased gene pools.

Since evolution and natural selection have been occurring in the environment for billions of years, the metagenomic approach allows the isolation of enzymes that harbor tailor-made properties, which fit the physicochemical conditions of the habitats studied. Under this perspective, new enzymes with special biophysical features have been isolated from metagenomic libraries that were built from various environments such as temperate soils (18), hot springs (5), sandy ecosystems (29), oceanic waters (33), and cold environments (4). Among these enzymes, several esterases (EC. and lipases (EC. have been isolated (7-9, 19, 20, 23-25, 29, 30, 32).

Lipolytic enzymes represent an important part of the industrial enzymes used for the production of various food products and fine chemicals (27). The importance of their use relies on the possibility to obtain enantiomerically pure molecules starting from mixtures containing several isomers.

Since many substrates for lipolytic enzymes are poorly soluble in water, replacement of aqueous buffers by organic solvent-containing solutions reinforced the interest in organic solvent-tolerant enzymes (26, 37). Moreover, the possibility to perform enzyme-catalyzed reactions in organic solvents avoids microbial contaminations, modifies the substrate specificity and changes the thermodynamic equilibrium favoring synthesis over hydrolysis.

Triacylglycerols are natural substrates of esterases and lipases and are poorly soluble in aqueous buffer solutions. Some of these enzymes are active even in the presence of organic solvents, and are therefore referred to as organic solvent-tolerant (OST) enzymes, being valuable tools for food and fine chemical industry because of their ability to remain active under harsh conditions.

Lipolytic enzymes catalyze both the hydrolysis and the synthesis of ester bonds found in many molecules such as acylglycerols. Esterases prefer short-chain substrates (below 10 carbon chain), whereas lipases are able to hydrolyze either longchain acylglycerols (beyond 10 carbon chain). Both esterases and lipases from the hormone-sensitive lipase-like family (HSL) share a highly conserved motif (G-X-S-X-G) containing the essential active serine. Additional aspartate or glutamate, and histidine residues are also involved in the formation of a conserved catalytic triad. Also, lipolytic enzymes in the HSL family contain the conserved sequence H-G-G-(G/A) in their oxyanion hole (1).

In this report, we describe the construction of a small-insert metagenomic library derived from a temperate forest soil sample. The library was screened for genes coding for lipolytic enzymes by an activity-based screening system. A novel lipase/ esterase encoding gene was discovered, sequenced and analyzed. The enzyme (RBest1) was purified and its substrate specificity and tolerance to various organic solvents was evaluated.


DNA extraction and metagenomic library construction

In November 2005, 50 g of soil sample were collected in the area of the Göttingen beech forest (51°33' N - 9°57' E), near the Georg-August University in Göttingen (Germany). DNA was extracted using the direct approach described by Zhou et al. (41). Purified environmental DNA was partially digested using Bsp143I (Fermentas; St.Leon-Rot, Germany) and resulting fragments were resolved by overnight ultracentrifugation in a sucrose density gradient (10-40 %) at 27,000 × g and 4 °C. Fractions containing DNA fragments with molecular sizes between 3 and 5 kb were used for cloning at the BamHI site of the pCR2.1-TOPO vector (InvitrogenTM) and metagenomic library RB1 was constructed after transformation in competent Escherichia coli RR1 cells (an E. coli HB101 derived strain, Resulting recombinant cells were selected on Luria Bertani (LB) agar plates containing 100 µg/ml ampicillin, 30 µM IPTG , and 50 µg/ml X-gal (Fermentas).

Screening of lipolytic activity on metagenomic library RB1

In order to isolate clones producing lipolytic enzymes in the metagenomic library RB1, recombinant E. coli cells were spread on emulsified spirit blue agar medium (BDDifco; Franklin-Lake NJ, USA), supplemented with 100 µg/ml ampicillin, 50 µg/ml kanamycin, and 1 % emulsified tributyrin (Sigma; Bornem, Belgium). Colonies producing a blue and clear hydrolytic halo were selected as potential esterase/ lipase producers. Plasmids isolated from positive clones were retransformed into competent E. coli DH 10B cells (Invitrogen) for confirmation of the lipolytic phenotype.

Positive clones were cultivated overnight at 37 °C in LB medium. Subsequently, cells were harvested by centrifugation, resuspended in 20 mM Tris-HC l (pH 8.0), and disrupted by sonication (3 cycles of 30 s; amplitude: 10-12 µm). After eliminating cell debris by centrifugation, organic solvents were added (DM SO, benzene, toluene and xylene, 25 % v/v final concentration) and the mixture was incubated at 37 °C for 30 min under agitation. Finally, 50 µl of the protein suspension were loaded on apirit blue agar medium containing 1 % (v/v) emulsified tributyrin at pH 7.5. Hydrolysis was observed by formation of a blue hydrolytic halo after 48 h incubation at room temperature.

DNA sequencing and sequence analysis

DNA sequencing was performed at the GIGA sequencing platform of the University of Liège (Belgium) using universal M13 reverse and forward primers. Additional primers were used to fully sequence the insert from the OST-esterase/lipase producing clones (RBest1int1: 5'-CTC TCG TTG CCC GCT GGT CT -3'; RBest1int1rv: 5'-AGA CCA GCG GGC AAC GAG AG -3'). The amino acid sequence of the putative lipolytic enzyme (RBest1) was analyzed with BLA STp (, and phylogenetic analysis conducted by MUSCLE (11) and Geneious Pro 4.8.2 software ( for performing the multiple sequence alignment, and MEGA 4.0 for building the phylogenetic tree, using 5,000 replicates in a bootstrap test (40). Alignment was used as template to construct a similarity table using Protdist software from the PH YLIP package (13).

RBest1 production and purification

RBest1 expression was achieved from the transformed clone containing the environmental DNA fragment that includes the RBest1-encoding gene (pRBest1). Bacterial cells were cultivated overnight in 1 L LB medium at 18 °C under agitation. Soluble proteins from the cytoplasmic fraction were recovered after cell lysis by sonication and centrifugation at 20,000 × g for 40 min and 4 °C.

The RBest1 esterase was purified from the cytoplasmic fraction in a two-step strategy using an AKTA Prime Plus device (GE Healthcare, Uppsala, Sweden). The first purification step was carried out by cation exchange chromatography on a HiLoad S-Sepharose HP column (GE Healthcare), previously equilibrated with 20 mM acetate buffer pH 6.5 (buffer A). Proteins were eluted using a linear gradient of NaCl (0 - 250 mM) in buffer A. Collected fractions were analyzed by testing the activity on p-nitrophenyl butyrate (pNP B), and active fractions were further analyzed by 15 % SDS-PAGE gels. The RBest1-containing fractions were pooled and dialyzed overnight against 10 l of 20 mM Tris-HC l, pH 8.0 (buffer B). The dialyzed protein fraction was loaded on a 5-ml HiTrap Q-Sepharose HP column (GE Healthcare) equilibrated in buffer B and eluted using a linear gradient of NaCl (0 - 250 mM). Fractions containing the active esterase were analyzed as described above. Protein concentration and purity were determined by BCA protein quantification assay (Pierce, Rockford, IL, USA) using bovine serum albumin as standard, and by densitometry analysis using the ImageJ software ( on 15 % SDS-PAGE gels, respectively.

RBest1 substrate specificity assay

To test the in vivo substrate specificity, recombinant E. coli RR1clone expressing RBest1 was spread on spirit blue agar containing 1 % of different emulsified triacylglycerols: tributyrin (C4), tricaprylin (C8), tricaprin (C10) and triolein (C18) (all purchased from Sigma). Hydrolysis was observed by the formation of a clear blue halo after 48 h incubation at room temperature. To test the in vitro substrate specificity, cytoplasmic fractions were prepared as described above and used to measure the enzymatic hydrolytic activity against various nitrophenylesters (pNPE ): pNPA, acetate (C2); pNP B, butyrate (C4); pNPC, caprylate (C8 ); pNPD, decanoate (C10 ); and pNP S, stearate (C18) (all purchased from Sigma). The standard reaction mixture consisted of 1.25 mM pNPE in 1 ml of 20 mM Tris-HC l, pH 8.0, and the appropriate amount of cell extract capable of releasing at least 0.2 µM of p-nitrophenol per minute. When measured at pH higher than 7.5, the release of p-nitrophenol was measured at 405 nm (e405nm pNP = 16,500 / for 10 min on a Specord 50 spectrophotometer (Analytik Jena, Jena, Germany). One unit (U) of enzymatic activity corresponds to the release of 1 µmol p-nitrophenol per minute.

pH and thermal dependence of RBest1 activity

The pH dependence of RBest1 activity was determined by following the esterase activity at 346 nm (e346nm pNP = 4,800 / (34). Reactions were followed for 3 min in a 20 mM pHadjusted polybuffer solution (citrate-phosphate-CHE S-CAP S) ranging from pH 3.5 to pH 9.5.

The thermal dependence of the activity was determined as described before and at its optimal pH (6.5) using a thermostatized Specord 50 spectrophotometer. The enzyme was preincubated for 5 min at the appropriate temperature before measuring the activity. In order to test the enzymatic thermal stability, stocks of proteins were incubated at temperatures ranging from 30 to 60 °C for up to 6 h. The remaining activity at 40 °C was compared to the initial activity.

Kinetic parameters of RBest1

Kinetic parameters for pNP B hydrolysis were determined under initial rate conditions using a nonlinear regression analysis of the Michaelis-Menten equation. Hydrolysis was measured at 40 °C using pNP B as substrate at final concentrations ranging from 0 to 200 µM in a 20 mM Tris-HC l pH 8.0.

Tolerance to organic solvents and salts

The influence of organic solvents on the RBest1 esterase activity was carried out as previously described with minor modifications (35). Briefly, organic solvents were added to a final concentration of 25 % (v/v) to the protein solution (in 20 mM Tris-HC l pH 8.0) and incubated at 37 °C under rotational agitation for 2 h. Remaining activity was tested under standard conditions using water as control. Tested organic solvents were N,N'-dimethyl-formamide (DMF), dimethyl-sulfoxide (DMSO), acetonitrile, p-xylene, toluene, pentane, benzene and n-hexane. Among the parameters that characterize organic solvents, the log of the partition coefficient (log Ko/w) is the most commonly used parameter giving the best correlation with enzyme activity and/or stability (28); log Ko/w is the distribution of a specific molecule in a two-phase equimolar mixture of water and octanol: the higher the log Ko/w, the more hydrophobic the molecule is.

The impact of organic solvents on substrate specificity was investigated by incubating the enzyme in the presence of various solvents. Remaining activity on pNPA, pNP B and pNPC was compared to the activity measured for pNP B hydrolysis in 20 mM Tris-HC l (pH 8.0) at 40 °C.

The influence of different salts (NaCl, KCl, CaCl2, MgCl2, CoCl2, NiCl2 and MnCl2) in the esterase activity was studied in 20 mM Tris-HC l pH 8.0 at 40 °C.


Screening for esterase/lipase activity from metagenomic library RB1

A small-insert metagenomic library RB1 was built from a temperate forest soil sample. The library was constructed in pCR2.1-TOPO vector and consisted of ca. 70,000 recombinant E. coli bacterial colonies harboring an average insert size of 3.1 kb. The library was screened for lipase/esterase activity, and three clones were found to express a positive phenotype on agar plates containing 1 % emulsified tributyrin. Crude cell extracts were used to test for organic solvent tolerance and allowed the isolation of one recombinant clone, named E. coli/pRBest1, exhibiting detectable activity on tributyrin in the presence of DM SO, benzene, toluene and p-xylene (data not shown).

Sequence analysis

The DNA insert cloned in recombinant plasmid from esterase/lipase-producing E. coli/pRBest1 was sequenced, deposited under GenBank accession number FJ157327 (ACH 99848 for deduced aminoacid sequence). A single open reading frame (ORF) of 861 bp was located in the insert, which encodes a 30.7 kDa protein named RBest1.

RBest1 protein shares amino acid sequence similarity (ca. 50 %) to many putative a/b hydrolases, including esterases, peptidases, and other uncharacterized proteins. In addition, RBest1 exhibits all the conserved signatures for the Hormone Sensitive Lipase (HSL)-like family corresponding to the abH4.04 according to the LED classification (1): a catalytic triad including S148 (the active site serine, included in the highly conserved G-E-S148-A-G pentapeptide), D243 and H275, and a conserved H64-G65 sequence that is part of the oxyanion hole (Figure 1).

RBest1 also possesses a unique 11-amino acid insertion at position 99 to 109, not exhibited by other enzymes related to RBest1. Based on the EstE1 structure (PD B: 2C7B), the RBest1's closest metagenome-derived esterase whose structure has been solved (19 % identity, 36 % similarity) (6), this insertion is located at the surface of the protein, in opposition to the active site, between the fourth b-sheet and the third a-helix of the protein (Figure 1). This loop contains an unusually high ratio of charged residues (2 glutamic acids, 1 aspartic acid, 3 lysines), which could strongly interact with polar solvents.

Interestingly, RBest1 is also similar (22 % identity, 36 % similarity) to the lipase from Lysinibacillus sphaericus (formerly Bacillus sphaericus) 205y (BS-Lip), the model for the abH04.04 subgroup as described on the Lipase Engineering Database ( Both RBest1 and BS-Lip show an unusual conserved active serine embedded in the pentapeptide G-E-S-A-G. Indeed, the lipases from Bacillus species are known to possess a particular active serine-containing pentapeptide (A-X-S-X-G) (35). However, there is lack of information on the enzymes from the abH04.04 subgroup, since BS-Lip is the sole previously characterized enzyme and therefore comparison between enzymes is difficult.

Phylogenetic analysis on the abH04 family (including RBest1) using the Neighbor Joining (NJ) method highlighted the existence of two groups or subfamilies (Figure 2). The first group (Group A) is composed by reference members of subfamilies abH04.01, abH04.02, and abH04.03 and sequence similarities with RBest1 range from 15.5 % (Moraxella sp.) to 24.2 % (EstE1 of an uncultured archaeon).

Figure 2
. Neighbor Joining tree of the RBest1 sequence and related sequences. Bootstrap values were calculated for MP /ME / NJ method (see text).

Group A is divided in two sub clusters. The first one is composed by sequences from the Acinetobacter lipase family (abH04.02). In the second cluster, sequences from Moraxella lipase 2-like family (abH04.01) and eukaryotic N-deacetylase family (abH04.03) are grouped.

The second group (Group B, abH04.04) is composed by RBest1 and other 31 sequences, with similarities ranging from 29.5 % to 41.2 %. The best similarity score (41.2 %) was obtained for the sequence from Gemmata obscuriglobus UQM 2246 (ZP_02736946). Group B clusters 15 single branched sequences and seven clusters of two or three sequences.

Both Groups A and B share conserved HSL lipase signature as described above. Nevertheless, sequences from Group B share the following specific consensus H64-G65-G66, Y95, D120, G146-X147-S148-A149-G150, L153, G240-X-X-D243, and H275 (based on the RBest1 numbering), whereas sequences from Group A show an alternative consensus, H80-G81-G82, Y112, P116, P121, G152-X153-S154-X155-G256, S231, L253-X254-D255 and H281 (based on the EstE1 numbering). Other methods such as Minimum Evolution (ME ) yielded similar results, indicating that the phylogenetic analysis is consistent (data not shown).

Although accurate phylogenetic analysis highlights the vast diversity of previously annotated lipase-related sequences, it is assumed that the major part of the environmental enzyme diversity still remains untapped.

RBest1 production and purification

RBest1 production was performed using the pRBest1 plasmid isolated from the metagenomic library. A purification protocol using two different chromatographic steps was set up and allowed the purification of 1.4 mg of pure esterase (ca. 28 kDa) from the cytoplasmic fraction obtained from 1 L culture.

Biochemical characterization of RBest1

Although RBest1 was isolated on tributyrincontaining medium, its ability to hydrolyze other substrates was further investigated. Substrate specificity was studied both in vivo and in vitro, using either triacylglycerol emulsions (1 % of tributyrin, tricaprylin, tricaprin and triolein) or chromogenic nitrophenyl esters (pNPE ), respectively. For in vivo analysis, formation of a blue halo around the colonies in emulsified spirit blue agar medium after 48 h incubation was only observed for substrate tributyrin. To test in vitro substrate specificity, the highest activity was recorded with pNP B (C4: 300 mU/mg) followed by pNPA (C2: ca. 25 mU/mg). The relative activity of pNPC (C8), pNPD (C10) and pNP S (C18) as substrates was also determined (< 1 % of that recorded with pNPB).

Regarding the overall substrate specificity, RBest1 enzyme showed higher specificity for C4 substrates, confirming that RBest1 is an esterase rather than a lipase. On the other hand, BS-Lip displays the highest activity towards C8, C10 and C12 substrates (39).

The effect of pH on the activity was investigated using purified RBest1 and pNP B as substrate in different pH-adjusted buffers. Maximum activity was measured in the range of pH 6.5 and remained significant when pH increased (Figure 3). No activity was recorded at pH values lower than 5.5.

Figure 3
. Influence of pH on the RBest1 activity. The maximum measured activity at pH 6.5 was taken as 100 %. Enzyme activity was determined under standard conditions. Presented results are the mean values obtained from triplicate experiments.

Thermal dependence of the RBest1 activity was determined under standard conditions. The activity increased from 20 to 45 °C, being the latter the apparent maximum activity, and then quickly decreased (Figure 4A).

The thermal stability of RBest1 esterase was investigated by incubating the esterase at different temperatures for up to 6 h. Residual activity on pNP B at each temperature was determined. The enzyme remained significantly active when incubated at temperatures below 40 °C for up to 6 h. Nevertheless, when incubated at higher temperatures, rapid inactivation was observed (Figure 4B).

Kinetic parameters Km and kcat for pNPB hydrolysis were deduced to be 0.020 ± 0.002 mM and 88.1 ± 8.0 /min, respectively. The catalytic efficiency (kcat/Km) was calculated as 4,400 /mM.min.

Organic Solvent and Salt Tolerance

Protein stability and activity in the presence of organic solvents were tested in order to determine the OST behavior of RBest1.

Remaining activity against several pNPE was investigated after incubation for 30 min in the presence of organic solvents (Figure 5A). All the tested solvents appeared to affect the RBest1 activity. Non-miscible solvents (log Ko/w> 0) improved the pNPE hydrolysis whereas miscible solvents (log Ko/w < 0) had different effects. Inactivation occurred in the presence of acetonitrile (0 % remaining activity) whereas DM SO showed a slight activation of the hydrolytic activity. DMF appeared to moderately decrease the pNPE hydrolysis catalyzed by RBest1.

Figure 5
. (A) RBest1 Organic Solvent Tolerance, activity against pNP B in the presence of various organic solvents after incubation at 40 °C for 0 to 2 h under rotational agitation. Depicted results are the mean value obtained from triplicate experiments and relative to the activity measured in the buffer-containing control. (B) Influence of organic solvents on the RBest1 activity on pNPA, pNP B and pNPC. The enzyme was preincubated for 30 min at 40 °C under agitation in presence of organic solvent (25 %). Enzyme activity was determined under standard conditions. The measured activity for pNP B hydrolysis in aqueous buffer was taken as 100 %. Presented results are the mean values obtained from triplicate experiments.

After longer incubations (2 h) in the presence of 25 % benzene at 37 °C under agitation, activity against pNP B appeared to be stabilized in the previously observed enhanced state (Figure 5B). On the other hand, when the enzyme was incubated under the same conditions in the presence of DM SO, the esterase activity apparently became stabilized to the control state beyond 2 h. Incubation in the presence of p-xylene or DMF for a longer period seemed to increase the solvent effect; whereas the activity towards the first further increased, the latter reinforced the inactivation process (Figure 5B).

For both RBest1 and BS-Lip enzymes, it is noteworthy that organic solvents like benzene, toluene and xylene increase the stability and the activity of both crude extracts and purified enzymes (22, 35, 39). On the other hand, while acetonitrile has a deleterious effect on both enzymes, DMSO and DMF affect more slightly their hydrolytic activity.

The RBest1 hydrolytic activity on pNP B was stable over a wide range of NaCl concentrations; while the enzyme was not significantly affected by 1 M NaCl, the activity rate decreased to 50 % when NaCl concentration increased to 5 M (Figure 6). Several other salts were tested, showing that they were able to more severely affect the RBest1 activity on pNPB. In addition, divalent cations such as Ca2+, Mg2+, Co2+ and Ni2+ had a deleterious effect on the RBest1 activity (Figure 6).

Figure 6
. Effect of various salts on the RBest1 activity, measured at 40 °C under standard conditions. The optimal hydrolysis condition was defined as 100 % activity; presented values are means of triplicate experiments.

This halophilic behavior seems to correlate with the previously reported for many OST enzymes. Indeed, molecular adaptation to organic solvents converges to some extent with halophilic adaptation since increasing salt concentrations causes reduction of water activity (2, 36).

Bacteria growing in organic solvent-containing media circumvent the solvent's toxic effects by degradation and transformation of solvents or by active efflux of solvents (16). Lysinibacillus sphaericus is a common environmental organism which produces an insecticidal toxin similar to that produced by Bacillus thuringiensis (12). In fact, L. sphaericus 205y, producing the BS-Lip lipase, was isolated for its ability to grow in the presence of benzene, toluene, and p-xylenecontaining media (22).

Nevertheless, although both RBest1 and BS-Lip are OST enzymes sharing significant similarities and displaying the same range of tolerance against organic solvents, no clear evidence for their phylogenetic association could be established.

Lysinibacillus sphaericus 205y was isolated out of a presumably contaminated soil sample from Malaysia (22), while RBest1 is derived from a pristine German soil sample. In addition, the sample used for the metagenomic library construction had not been exposed to any organic solvent or to high salt concentration. Nonetheless, enzymes remaining active in low water-containing environments were observed in many physiological conditions including frozen ecosystems. Noteworthy, it is reported that Bacillus dormant spores are resistant to harsh conditions including desiccation for long periods of time (3, 38). Their reactivation involved many factors and the production of esterase/lipase was correlated with spore germination (3, 14, 31). However, functional analyses of lipase/esterase involved in spore germination still remain incomplete, and additional information is required to better understand their biophysical features.

As described here, the metagenomic approach gives the possibility to isolate enzymes displaying specific properties. The RBest1 ability to remain active in the presence of various organic solvents and the possibility to modulate its substrate specificity in the presence of organic solvents makes RBest1 a versatile new enzyme with potential biotechnological applications or in bioremediation processes.

Nevertheless, a better understanding of the microbial environmental diversity requires not only important efforts on DNA sequencing but also protein analysis in order to elucidate their physiological function. A major effort on the functional analysis of the metagenomederived enzymes remains to be done to better understand the role of individual genes and proteins in the environment. Indeed, attempts to elucidate the physiological function of proteins derived from metagenomes sometimes lead to unexpected results.

Acknowledgments: this project was partially supported by a grant from the First-Postdoc Program, Grant no. 916984 from the Région Wallonne (Belgium), and by a grant from FNRS (FRFC, 2.4561.07) to MG. PP is a member of the Carrera del Investigador Científico (CIC ) from CONICET, Argentina, and was a recipient of a Postdoctoral fellowship from the Belgian Science Policy Office (BEL SPO ).


1. Arpigny JL, Jaeger KE. Bacterial lipolytic enzymes: classification and properties. Biochem J 1999; 343 Pt 1: 177-83.         [ Links ]

2. Bell G, Janssen AEM, Halling PJ. Water activity fails to predict critical hydration level for enzyme activity in polar organic solvents: Interconversion of water concentrations and activities. Enz Microb Technol 1997; 20: 471-7.         [ Links ]

3. Bergman NH, Anderson EC, Swenson EE, Niemeyer MM, Miyoshi AD, Hanna PC. Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation. J Bacteriol 2006; 188: 6092-100.         [ Links ]

4. Berlemont R, Delsaute M, Pipers D, D'Amico S, Feller G, Galleni M, Power P. Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples. ISME J 2009; 3: 1070-81.         [ Links ]

5. Bhaya D, Grossman AR, Steunou AS, Khuri N, Cohan FM, Hamamura N, Melendrez MC, Bateson MM, Ward DM, Heidelberg JF. Population level functional diversity in a microbial community revealed by comparative genomic and metagenomic analyses. ISME J 2007; 1: 703-13.         [ Links ]

6. Byun JS, Rhee JK, Kim DU, Oh JW, Cho HS. Crystallization and preliminary X-ray crystallographic analysis of EstE1, a new and thermostable esterase cloned from a metagenomic library. Acta Crystallogr Sect F Struct Biol Cryst Commun 2006; 62: 145-7.         [ Links ]

7. Cieslinski H, Bialkowskaa A, Tkaczuk K, Dlugolecka A, Kur J, Turkiewicz M. Identification and molecular modeling of a novel lipase from an Antarctic soil metagenomic library. Pol J Microbiol 2009; 58: 199-204.         [ Links ]

8. Couto GH, Glogauer A, Faoro H, Chubatsu LS, Souza EM, Pedrosa FO. Isolation of a novel lipase from a metagenomic library derived from mangrove sediment from the south Brazilian coast. Genet Mol Res 9: 514-23.         [ Links ]

9. Chu X, He H, Guo C, Sun B. Identification of two novel esterases from a marine metagenomic library derived from South China Sea. Appl Microbiol Biotechnol 2008; 80: 615-25.         [ Links ]

10. Daniel R. The soil metagenome--a rich resource for the discovery of novel natural products. Curr Opin Biotechnol 2004; 15: 199-204.         [ Links ]

11. Edgar RC. MUSCLE : multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32: 1792-7.         [ Links ]

12. el-Bendary MA. Bacillus thuringiensis and Bacillus sphaericus biopesticides production. J Basic Microbiol 2006; 46: 158-70.         [ Links ]

13. Felsenstein J. An alternating least squares approach to inferring phylogenies from pairwise distances. Syst Biol 1997; 46: 101-11.         [ Links ]

14. Ferencko L, Cote MA, Rotman B. Esterase activity as a novel parameter of spore germination in Bacillus anthracis. Biochem Biophys Res Commun 2004; 319: 854-8.         [ Links ]

15. Frostegard A, Courtois S, Ramisse V, Clerc S, Bernillon D, Le Gall F, Jeannin P, Nesme X, Simonet P. Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol 1999; 65: 5409-20.         [ Links ]

16. Gupta A, Khare SK. Enzymes from solvent-tolerant microbes: useful biocatalysts for non-aqueous enzymology. Crit Rev Biotechnol 2009; 29: 44-54.         [ Links ]

17. Handelsman J, Liles M, Mann D, Riesenfeld C, Goodman RM. Cloning the metagenome: culture-independent access to the diversity and functions of the uncultivated microbial world. In: Wren B, Dorrell N, editors. Methods in Microbiology - Functional Microbial Genomics, 1st edition. New York, Academic Press, 2002, p. 241-55.         [ Links ]

18. Henne A, Daniel R, Schmitz RA, Gottschalk G. Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl Environ Microbiol 1999; 65: 3901-7.         [ Links ]

19. Henne A, Schmitz RA, Bomeke M, Gottschalk G, Daniel R. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl Environ Microbiol 2000; 66: 3113-6.         [ Links ]

20. Hong KS, Lim HK, Chung EJ, Park EJ, Lee MH, Kim JC, Choi GJ, Cho KY, Lee SW. Selection and characterization of forest soil metagenome genes encoding lipolytic enzymes. J Microbiol Biotechnol 2007; 17: 1655-60.         [ Links ]

21. Hugenholtz P, Goebel BM, Pace NR. Impact of cultureindependent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 1998; 180: 4765-74.         [ Links ]

22. Hun CJ, Rahman RN, Salleh AB, Basri M. A newly isolated organic solvent tolerant Bacillus sphaeriucs 205y producing organic solvent-stable lipase. Biochem Eng J 2003; 15: 147-51.         [ Links ]

23. Jeon JH, Kim JT, Kim YJ, Kim HK, Lee HS, Kang SG, Kim SJ, Lee JH. Cloning and characterization of a new coldactive lipase from a deep-sea sediment metagenome. Appl Microbiol Biotechnol 2009; 81: 865-74.         [ Links ]

24. Kim YH, Kwon EJ, Kim SK, Jeong YS, Kim J, Yun HD , Kim H. Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library. Biochem Biophys Res Commun 393: 45-9.         [ Links ]

25. Kim YJ, Choi GS, Kim SB, Yoon GS, Kim YS, Ryu YW. Screening and characterization of a novel esterase from a metagenomic library. Protein Expr Purif 2006; 45: 315-23.         [ Links ]

26. Klibanov AM. Improving enzymes by using them in organic solvents. Nature 2001; 409: 241-6.         [ Links ]

27. Koeller KM, Wong CH. Enzymes for chemical synthesis. Nature 2001; 409: 232-40.         [ Links ]

28. Laane C, Boeren S, Vos K, Veeger C. Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 1987; 30: 81-7.         [ Links ]

29. Lee MH, Lee CH, Oh TK, Song JK, Yoon JH. Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases. Appl Environ Microbiol 2006; 72: 7406-9.         [ Links ]

30. Lee SW, Won K, Lim HK, Kim JC, Choi GJ, Cho KY. Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl Microbiol Biotechnol 2004; 65: 720-6.         [ Links ]

31. Masayama A, Kuwana R, Takamatsu H, Hemmi H, Yoshimura T, Watabe K, Moriyama R. A novel lipolytic enzyme, YcsK (LipC), located in the spore coat of Bacillus subtilis, is involved in spore germination. J Bacteriol 2007; 189: 2369-75.         [ Links ]

32. Meilleur C, Hupe JF, Juteau P, Shareck F. Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library. J Ind Microbiol Biotechnol 2009; 36: 853-61.         [ Links ]

33. M oreira D, Rodriguez-Valera F, Lopez-Garcia P. Analysis of a genome fragment of a deep-sea uncultivated Group II euryarchaeote containing 16S rDNA, a spectinomycinlike operon and several energy metabolism genes. Environ Microbiol 2004; 6: 959-69.         [ Links ]

34. O tero C, Fernandez-Perez M, Hermoso JA, Ripoll MM . Activation in the family of Candida rugosa isolipases by polyethylene glycol. J Mol Cat B: Enzymatic 2005; 32: 225-9.         [ Links ]

35. Rahman RN, Chin JH, Salleh AB, Basri M. Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y. Mol Genet Genomics 2003; 269: 252-60.         [ Links ]

36. Sellek GA, Chaudhuri JB. Biocatalysis in organic media using enzymes from extremophiles. Enzyme Microb Technol 1999; 25: 471-82.         [ Links ]

37. Serdakowski AL, Dordick JS. Enzyme activation for organic solvents made easy. Trends Biotechnol 2008; 26: 48-54.         [ Links ]

38. Setlow P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 2006; 101: 514-25.         [ Links ]

39. Sulong MR, Abdul Rahman RN, Salleh AB, Basri M. A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene. Protein Expr Purif 2006; 49: 190-5.         [ Links ]

40. T amura K, Dudley J, Nei M, Kumar S. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA ) software version 4.0. Mol Biol Evol 2007; 24: 1596-9.         [ Links ]

41. Zhou J, Bruns MA, Tiedje JM. DNA recovery from soils of diverse composition. Appl Environ Microbiol 1996; 62: 316-22.         [ Links ]

Recibido: 10/7/2012
Aceptado: 7/12/2012