The first description of a hormone‐sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealed Bjerkandera adusta BaEstB as a novel esterase

Abstract The heterologous expression and characterization of a Hormone‐Sensitive Lipases (HSL) esterase (BaEstB) from the Basidiomycete fungus Bjerkandera adusta is reported for the first time. According to structural analysis, amino acid similarities and conservation of particular motifs, it was established that this enzyme belongs to the (HSL) family. The cDNA sequence consisted of 969 nucleotides, while the gene comprised 1133, including three introns of 57, 50, and 57 nucleotides. Through three‐dimensional modeling and phylogenetic analysis, we conclude that BaEstB is an ortholog of the previously described RmEstB‐HSL from the phylogenetically distant fungus Rhizomucor miehei. The purified BaEstB was characterized in terms of its specificity for the hydrolysis of different acyl substrates confirming its low lipolytic activity and a noticeable esterase activity. The biochemical characterization of BaEstB, the DLS analysis and the kinetic parameters determination revealed this enzyme as a true esterase, preferentially found in a dimeric state, displaying activity under alkaline conditions and relative low temperature (pH = 10, 20°C). Our data suggest that BaEstB is more active on substrates with short acyl chains and bulky aromatic moieties. Phylogenetic data allow us to suggest that a number of fungal hypothetical proteins could belong to the HSL family.

Esterases and lipases have been isolated from microorganisms, plants, animals, and metagenomes (Fojan et al., 2000;Gopinath, Anbu, Lakshmipriya, & Hilda, 2013). They have also been produced in heterologous hosts upon bioinformatics screening of public databases of genomes (Barriuso, Prieto, & Martínez, 2013). There is still interest in the characterization of microbial lipolytic enzymes and esterases because of their high production yields and the easy genetic manipulation of the microorganisms compared with other organisms. Both enzymes share the α/β fold in their structure and most of them present the consensus sequence GXSXG as a signature motif. According to amino acid similarities and conservation of particular motifs, lipolytic enzymes (including esterases) are classified into C, H, X, and L blocks (Lenfant et al., 2013).
Block H includes Hormone-Sensitive Lipases (HSL), where the majority of the enzymes resulted to be esterases (Ali, Verger, & Abousalham, 2012;Huang et al., 2016;Li et al., 2015). In mammals, HSL have been proposed as esterases that hydrolyze diacylglycerol for the generation of ATP and cholesteryl esters precursors to deliver cholesterol for the synthesis of steroid hormones (Kraemer, 2007). In fungi, ergosterol is used instead of cholesterol to maintain fluidity, permeability and integrity of the plasma membrane and adequate function of membrane-bound proteins (Holick, 2003;Sun, Gao, Ling, & Lou, 2005).
Ergosteryl esters are found in lipid particles in the cytoplasm and when unesterified may be used as a source for ergosterol in membrane synthesis (Shobayashi et al., 2005;Zweytick, Athenstaedt, & Daum, 2000).
Bacterial lipolytic enzymes are classified in eight families, where proteins with high similarity to mammalian HSL belong to family IV (Arpigny & Jaeger, 1999). Based on massive sequence alignment and different conserved motifs, two subfamilies of bacterial HSL have been proposed, the GTSAG motif subfamily and the GDSAG motif subfamily, where the labeled amino acids are defined as arbitrary residues in the pentapeptide conserved motif GXSXG (Jeon et al., 2012;Li et al., 2014).
Lipolytic enzymes from fungi have been extensively characterized from Candida antarctica, C. rugosa, and from filamentous fungi as Aspergillus niger, Rhizopus orizae, Penicillium camembertii among others, that have been commercialized and used in dairy, oil, and fat industries (Anobom et al., 2014;Borrelli & Trono, 2015;Houde, Kademi, & Leblanc, 2004). However, only two fungal lipolytic enzymes from the HSL family (RmEstA and RmEstB) have been studied (Rm: Rhizomucor miehei, Est: Esterase). Both of them were identified from the mucoral thermophilic fungus R. miehei. Interestingly, RmEstA and RmEstB exhibit distinct substrate specificities: RmEstA shows high activity toward long-chain esters, whereas RmEstB favors hydrolysis of shortchain esters (Liu et al., 2013;Yan et al., 2014;Yang, Qin, Duan, Yan, & Jiang, 2015). In spite of these reports, there is still a lack of information of lipolytic enzymes from the HSL family in fungi and their role is uncertain.
Moreover, lipolytic activity was stimulated in the presence of humic acids (Belcarz, Ginalska, & Kornillowicz-Kowalska, 2005). However, the study did not show data of purified enzymes.
In this work, we report the characterization of the first HSL esterase reported from a Basidiomycete (BaEstB). The cDNA was obtained from a B. adusta library grown on crude oil. Blastp results showed high similarity with esterase family IV that includes HSL members.
Classification within the HSL family was confirmed through phylogenetic analysis and homology modeling of BaEstB. The esterase activity was confirmed by the preference of the enzyme for shorter chain substrates and the lack of activity on a rhodamine triglyceride assay.

| BaEstB modeling, structural alignment, threedimensional superposition and structure-based and sequence-based phylogenies
Upon PSI-BLAST (Position-Specific Iterated) analysis of 768 sequenced clones from a cDNA library form B. adusta, we identified a sequence with homology to the α/β hydrolase Esterase/Lipase superfamily. The Open Reading Frame (ORF) in this sequence was named as BaEstB (GenBank accession number KX580963). The complete amino acid sequence of the BaEstB was submitted to the I-TASSER server (Roy, Kucukural, & Zhang, 2010;Yang, Qin, et al., 2015;Zhang, 2008) without constraints in order to get a three-dimensional model. A second modeling round was performed using PDB (Protein Data Bank) 4WY8 and 4ZRS as templates, since these PDBs were the best templates found during the first modeling round. Structural alignments and threedimensional superpositions were obtained considering the PDBs identified by I-TASSER as close structural neighbors in order to identify the cap and catalytic domains, the catalytic triad, the conserved motifs, and other residues involved in the catalysis of BaEstB.
The model visualization and structural alignment, including the Root Mean Square Deviation (RMSD) values, were obtained in Visual Molecular Dynamic program (VMD) (Humphrey, Dalke, & Schulten, 1996).
A structure-based phylogenetic tree according with the RMSD derived from BaEstB's structural comparison with its closest structural analogs was prepared in VMD and visualized in Phylogeny.
fr server (http://www.phylogeny.fr/). A second phylogenetic tree (amino acid sequence-based reconstruction) was obtained on line in the same server in order to describe the relationships of BaEstB with related sequences obtained from the Blast results. Phylogeny. fr considers various bioinformatics algorithms to construct a robust phylogenetic tree from a set of sequences (Dereeper, Audic, Claverie, & Blanc, 2010;Dereeper et al., 2008); for the generation of phylogenetic trees, MUSCLE was used for the multiple alignments, Gblocks for the automatic alignment curation (in order to eliminate poorly aligned positions, not allowing smaller final blocks and less strict flanking positions), PhyML, for tree building and TreeDyn for tree drawing (Dereeper et al., 2008). The Maximum Likelihood method was used to estimate the phylogenetic tree; the branch support was assessed using the ALTr algorithm and the Jones-Thornton-Taylor (JTT) model was used to estimate distances for amino acids (Dereeper et al., 2008).
The parameters used during the MUSCLE alignment were those recommended by the Phylogeny.fr platform (custom mode with 16 as the maximum number of iterations). A lipase from Candida cylindracea (gi: 1325988) was used as an outgroup.

| Cloning of BaEstB
The coding sequence of the BaEstB was amplified using primers ForwBaEstB (5′gaattcatggaatctatccgtctgtc3′) and RevBaEstB (5′tcta gaccctattccgtcgctggta3′) with cutting sites underlined for EcoRI and XbaI, respectively, which allowed the subcloning into the expression vector (pPICZαA); the two cytosines in bold were added to put the sequence in frame with the myc-and poly-His tags. The PCR conditions were as follows: 95°C 5 min (one cycle); 95°C 45 s, 55°C 60 s and 72°C 2 min (30 cycles) and a final extension step of 72°C 7 min (one cycle), on a thermocycler (Axigen-Maxigene) and the amplification was carried out with Pfu DNA polymerase (Jena Biosciences). The amplified product was purified with the Column DNA Gel Extraction Spin Kit (Thermo Scientific), ligated into vector pJET (pJET-BaEstB) and transformed into E. coli DH5α electrocompetent cells for subsequent sequencing.
The genomic sequence of BaEstB was obtained using the Genome Walker Kit (Clontech) following the manufacturer's instructions using oligonucleotides pDNRlib Fwd: 5′ ACCATGGAATCTATCCGTCT 3′ and pDNRlib Rev: 5′ AGAAAATGTCATTACGGTGG 3′. The PCR conditions were as follows: 95°C-5 min (one cycle), 95°C 1 min; 57°C 30 s; 73°C 2 min (30 cycles); and a final extension step of 73°C for 5 min, using the same apparatus and enzyme as described above. The amplified fragments were purified as described above for sequencing.

| Expression of BaEstB in Pichia pastoris X-33 and enzyme purification
pJET-BaEstB was digested with EcoRI and XbaI, and the released BaEstB cDNA fragment was inserted into pPICZαA (Invitrogen, USA) digested with the same enzymes. This construct was designated as pPICZαA/BaEstB and was transformed into E. coli DH5α. Restriction digestion and DNA sequencing verified the identity of the insert. This construction was linearized with SacI, and P. pastoris X-33 cells were transformed by electroporation for 5 ms at 2,000 volts employing an Eporator apparatus (Eppendorf). Positive transformants were selected for their ability to grow on Yeast-Peptone-Dextrose (YPD) plates containing zeocin at a final concentration of 100 μg/ml. P. pastoris X-33 transformed with the empty vector pPICZαA was used as a negative control. Integration of the BaEstB gene into the P. pastoris X-33 genome was confirmed by colony PCR analysis using 5′ and 3′ AOX1 primers, following instructions of Easy Select Pichia Expression Kit BaEstB was purified from a 96-hr culture supernatant using Nickel affinity chromatography. Briefly, the supernatant was recovered by centrifugation at 1,500g for 10 min and concentrated in a 30 kDa cutoff amicon (GE healthcare). This concentrated preparation was loaded into the Ni-column and eluted with a Tris-HCl 10 mmol/L pH 7.5 and 0.2 mol/L imidazol solution. The purified enzyme was dialyzed and the protein was stored at 4°C until further use.

| SDS-PAGE and zymogram
Proteins from the crude extracts and from various purification steps were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (1970) using 4.5% stacking gel and 12.0% separating gel. The protein bands were stained with Coomassie brilliant blue R-250. Page Ruler plus was used as molecular weight marker (ThermoScientific).
Esterase activity was assayed and visualized on zymograms, using 12.5% polyacrylamide gels (PAGE). Esterase activity staining was performed as described by Karpushova, Brümmer, Barth, Lange, & Schmid (2005). The gels were finally incubated for 5 min at room temperature in developing solution consisting of 3 mmol/L 2-naphthyl acetate, 1 mmol/L Fast Garnet TR (Sigma) and 100 mmol/L sodium phosphate buffer, pH 7.5. The positive esterase activity was detected by the appearance of orange-colored bands in the gels.
The rhodamine B assay, specific for lipases, was conducted according to previous reports (Kouker & Jaeger, 1987). A stock solution with 2.5% (w/v) of olive oil was prepared and autoclaved.
Rhodamine B (1 mg/ml) was dissolved in distilled water and filter sterilized (MILLEX ® GV 0.2 μm). A solution containing 0.8% agar in buffer 10 mmol/L Tris-HCl pH 7 was autoclaved and after cooling down to 60°C, 31.25 ml of the olive oil solution and 10 ml of the rhodamine B solution were added per L. This was poured in Petri dishes and when solidified, small wells were carved, filled with the different samples and incubated at 30°C for 16 hr, when the appearance of a fluorescent halo was detected under UV light (350 nm) in the positive control sample (a Candida cylindracea lipase, Sigma L1754).
The biochemical characterization of BaEstB was performed using 2-naphthyl acetate as substrate. All measurements were performed in triplicate.

| Optimum temperature and stability of BaEstB
Optimum temperature for the enzyme was investigated by measuring the esterase activity at different temperatures (10-60°C) with increments of 10°C. The thermal stability of the esterase was determined by measuring the residual activity of enzyme after a 60-min preincubation employing the aforementioned temperature range with same increments. Specific activities were determined under the same condition mentioned above and using 2-naphthyl acetate as substrate. All measurements were performed in triplicate.

| pH optimum and pH stability of BaEstB
Optimum pH of the enzyme was determined by varying the pH of the assay reaction mixture using the following buffers: 10 mmol/L Tris-HCl (pH 3.0-7.0) or 10 mmol/L carbonate-bicarbonate (pH 8.0-10.0).
The stability of the esterase was determined after preincubating the enzyme in different buffer solutions (pH 3.0-10.0) for 60 min. The residual enzyme activity was then determined under the standard assay conditions. All measurements were performed in triplicate.

| Kinetic parameters via isothermal titration calorimetry (ITC)
ITC experiments were performed on a Malvern ITC200 instrument by titrating 2-naphthyl acetate into the enzyme suspension within the sample cell. Each single-injection consisted in 38 μl of 2-naphthyl acetate (170 μmol/L) and 37 pM of purified BaEstB. All measurements were performed under the following conditions: temperature 30°C, stirring speed of 750 rpm, reference power of 8 μcal/s, and 90 min allowing the heat signal return to the baseline. The data were analyzed using Origin 7.0v software for kcat, Km, and ΔH determinations.

| Effect of metal ions, NaCl, solvents, detergents, and EDTA on BaEstB activity
In all the experiments described below, the specific activities were determined using 10 μl of diluted (1/1,000) pure enzyme (10.08 mg of protein/ml), 2-naphthyl acetate as substrate and 10 mmol/L Tris-HCl buffer (pH 7.0). All measurements were performed in triplicate.

| Effect of metal ion on BaEstB activity
The effect of several metal ions (Ba 2+ , Mg 2+ , Fe 2+ , Co 2+ , Ni 2+ , Mn 2+ , Zn 2+ , Cu 2+ , Ca 2+ Ag 2+ , K + , Li + , Al + ) on the BaEstB activity was evaluated in the presence of 10 mmol/L of each metal ion and pH 7. Additionally, the BaEstB stability was determined incubating the enzyme for 1 h at 30°C in Tris-HCl buffer pH 7 in presence of the metal ions. The specific activity was measured and the residual activity was calculated.

| Effect of NaCl on BaEstB activity
BaEstB activity was determined in different salinity conditions. Specific activity was measured by incubating the BaEstB in Tris-HCl buffer at 30°C with the addition of NaCl to final concentrations of 0.1, 0.25, 0.5, 1.0, and 2.0 mol/L in the reaction mixtures. The halostability (residual activity expressed in percentage) was also evaluated by incubating the BaEstB 1 hr at 30°C in Tris-HCl buffer in presence of 0.1, 0.25, 0.5, 1.0, and 2.0 mol/L NaCl.

was measured by incubating
BaEstB in Tris-HCl buffer at 30°C with the addition of the solvents in the reaction mixture.

| Effect of detergents and EDTA on BaEstB activity
The effect of several detergents (Tween 20, Tween 80, SDS, Triton X-100, and CTAB) and EDTA on the BaEstB activity was evaluated.
Residual activity, expressed in percentage, was calculated by incubating the BaEstB in Tris-HCl buffer for 1 hr at 30°C in presence of 5% (w/v) of each compound (Yan et al., 2014).

| Dynamic light scattering (DLS)
DLS experiments were conducted at 30°C using a Malvern Zetasizer Nano ZSP system. Translational diffusion coefficients (TDC) were obtained via measurements of the decay rates of scattered light and the autocorrelation curves. The hydrodynamic radius (H R ) of the BaEstB population was calculated from TDC on basis of the Stokes-Einstein equation assuming a spherical geometry of the molecules. Samples were centrifuged at 12,000g for 5 min before the measurements. Two set of experiments, (1) BaEstB without substrate and (2) BaEstB in presence of substrate (2-naphthyl acetate) were addressed; performing 12 scans for 10 s in both cases. DLS analysis of BaEstB with 2-naphthyl acetate was performed under the same conditions mentioned above considering its optimum pH and temperature. The data set replicates were analyzed using the DTS 5.10 software. Additionally, unfolding assays by temperature were performed in a range between 10 and 70°C. All measurements were performed in triplicate.

| Nucleotide sequence of BaEstB
A sequence with homology to the α/β hydrolase superfamily was identified from a cDNA library from B. adusta as described in materials and methods. The cDNA sequence consisted of a 969 nucleotide (nt) ORF (GenBank accession number KX580963) and its genomic counterpart comprised 1,133 nt which contained three introns of 57, 50, and 57 nt, respectively (FS1 supplementary material). The first twenty-one hits of the PSI-Blast showed approximately 40%-49% identity and 87%-93% coverage that were annotated as fungal hypothetical proteins or α/β hydrolases (FS2 supplementary material). The 253th hit was the first with an assigned function showing 26% identity and 60% coverage with an esterase from Acinetobacter sp. (GeneBank BAB68337.1). Therefore, PSI-BLAST analysis suggests that this sequence belongs to the α/β hydrolase superfamily, however the majority of the proteins recovered from the PSI-BLAST are hypothetical or uncharacterized proteins and further studies are needed to assign its function. As a first approach to unravel BaEstB function, we decided to study its structure through bioinformatics analyses.  (Table 1), this analysis strongly suggests that BaEstB is a member of this HSL family due to its structural similarity (Table 1, see TM-score, Coverage and RMSD values).

| BaEstB modeling
In order to obtain a more accurate three-dimensional model, we submitted a new modeling round (I-TASSER IDs: S248560 and S277038) using PDB 4ZRS and PDB 4WY8 as templates. The best model was obtained using PDB 4WY8 as template ( Figure 1a Again, HSL were the closest structural neighbors. The results above suggest that BaEstB belongs to HSL family. Enzymes with lipolytic activity are classified into blocks C, L, H or X depending on their amino acid similarities and the presence of conserved motifs involved in the enzymatic catalysis (see ESTHER database) (Lenfant et al., 2013;Marchot & Chatonnet, 2012 Lenfant et al., 2013). According with this, it is not rare that PDB 2O7R was identified by I-TASSER as a template during the first BaEstB modeling round, probably being BaEstB a member of block H.
The HSL family is composed of esterases and lipases, and its members are widely represented in bacteria, plants, and animals (Tao et al., 2013). The HSL sequences identified in bacteria share high amino acid sequence similarity with the LIPE genes, which encode mammalian HSLs (Holm, 2003;Holm, Osterlund, Laurell, & Contreras, 2000).

HSL
Ranking of proteins is based on TM-score of the structural alignment between the query structure and known structures in the PDB library. TM: TM-score. RMSD is the RMSD between residues that are structurally aligned by TM-align. Cov.: Represents the coverage of the alignment by TM-align and is equal to the number of structurally aligned residues divided by length of the query protein.
T A B L E 1 Top 10 identified structural analogs in PDB alignments with the previous fungal HSL allowed identifying the cap (purple) and the catalytic domains (green) in BaEstB (Figure 1a), as well as the catalytic triad, which was located in the respective canonical position. BaEstB cap domain comprises residues 1-39 and 209-238, while the catalytic domain includes residues 40-193 and 244-316 and shows the distinctive molecular topology described for other HSL: an α/β-hydrolase fold with a central β-sheet of eight mostly parallel strands surrounded by α-helices (Rozeboom, Godinho, Nardini, Quax, & Dijkstra, 2014;Yang, Qin, et al., 2015). The critical residues involved in the catalysis of BaEstB were identified in the following positions: S163, D252, and H285 (Figure 17b), being the canonical catalytic residues for HSL family. The three-dimensional superposition between PDB 4WY8 and BaEstB showed that the average RMSDs for the catalytic triad (S163, D252, and H285 in BaEstB vs. S164, D261, and H291 in PDB 4WY8) are 1.75, 5.28, and 4.68 Å,

respectively.
Regarding the main structural characteristics of both, PDB 4WY8 and 4WY5, the critical residues located in the center of the substratebinding pocket were identified in BaEstB also by structural alignments.
BaEstB showed a higher similarity to RmEstB (4WY8) than to RmEstA   (Yang, Qin, et al., 2015) that cap domains are the worst alignment regions, however they share their tertiary structure as Figure 2b shows. Particularly, BaEstB does not exhibit sequence similarity in the cap domain with other HSL, not even with the fungal HSL (RmEstA and RmEstB).

| Structural alignment analysis
From the alignment of the analyzed sequences, the HSL canonical catalytic triad (Ser-His-Asp) was located (Figure 3). In BaEstB, Ser163 is the nucleophile, His285 is the proton acceptor/donor, and Asp252 is the amino acid stabilizing His285. Additionally, the three signature motifs for HSL proteins were identified in BaEstB, and they were named as Block 1, 2, and 3 ( Figure 3). The conserved tetrapeptide motif His-Gly-Gly-Gly (Block 1 in Figure 3) involved in the oxyanion cavity formation was identified in the aligned sequences and it was located upstream of the active site. BaEstB sequence shows a conservative substitution in this motif ( 77 His-Gly-Gly-Ala 80 ), being this structural characteristic never reported for these proteins. It has been demonstrated that an Ala residue downstream of this signature motif is necessary to create the oxyanion cavity (Ngo et al., 2013;Yang, Qin, et al., 2015). We could find it in position 164 of BaEstB, being this amino acid extensively conserved in these sequences including the F I G U R E 3 Structural alignment of BaEstB with HSL deposited in PDB: 4WY8, 4WY5, 4OU4, 1JKM, 4J7A, 3ZWQ, 1LZL, 1JJi and 1QZ3. The catalytic triad (Ser-His-Asp) is indicated with black triangles, while the amino acids located in the center of the substrate-biding pocket are marked with black asterisks. The alignment shows the typical domains in HSL esterases. Identical residues are shaded in black, and conserved residues are shaded in gray. The conserved catalytic motif is underlined. The three signature motifs for HSL proteins named Block 1, 2, and 3 are indicated as black dotted boxes. The putative catalytic nucleophile and acid/base are identified by a black filled arrow Rhizomucor miehei′s HLS (Ala163 and Ala165 in RmEst A and RmEstB, respectively); being Gly78, Gly79, and Ala164 the three amino acids involved in the oxyanion cavity formation in BaEstB.
On the other hand, the nucleophilic Ser163 is located in the characteristic 160 Gly-X-Ser-X-Gly 164 pentapeptide sequence (Block 2 in Figure 3). Some authors extend this block three more residues (Ngo et al., 2013), however these positions are less conserved in the block although it can define a consensus extended motif Gly-X-Ser-X-Gly-Gly-Asn-Leu. Gly and Leu being the most conserved and that are present in BaEstB: 160 Gly-X-Ser-X-Gly-Gly-Ala-Ile 167 . A conservative change (Leu for Ile) and a nonconservative change (Asn for Ala) are present in BaEstB, while similar substitutions are found in the other sequences. A third conserved block Asp-Pro-X-X-Asp (Block 3 in Figure 3) involved in catalysis is common in HSL. BaEstB Block 3 is composed by 252 Asp-Val-Ala-Ala-X-X-Ala 258 . The insertion of two Ala residues, is a distinctive structural characteristic for BaEstB since in the other sequences it is not present. Moreover, in BaEstB and in both R. miehei′s HSL, a Pro residue is changed by Val. This substitution may be typical of fungi, since it is rarely found in bacteria (i.e., PDB 4OU4 of

P. putida). Additionally, a nonconservative change is found in BaEstB,
where an Asp is replaced by Ala. However, the positions X-X in the Asp-Pro-X-X-Asp motif is conserved (L and R) among all the sequences studied except in BaEstB (where we found M and R). Given this analysis, we cannot consider that the third block is conserved in BaEstB.

| Sequence phylogenetic analysis
The phylogenetic analysis based on the amino acid sequence showed that BaEstB is not grouped directly with any HSL member considered in the phylogeny reconstruction (Figure 4). This result is not surprising because BaEstB is a fungal protein with some distinctive structural characteristics as mentioned above. This relation could be supported by the low similarity coefficients observed in the multiple structural alignments. BaEstB is not directly related with RmEstA and RmEstB, which are also fungal proteins but from another distant Phylum (Glomeromycota, subphylum Mucoromycotina), which form a clade with bacterial HSL (Figure 4). The phylogeny suggests that these proteins presumably have a common ancestor since prokaryotic and

| Expression and purification of BaEstB in P. pastoris
Oligonucleotides were designed to amplify the coding sequence of the putative esterase to be inserted in pJET, an intermediate vector.
The cDNA was then subcloned in the expression vector pPICZαA to be transformed in P. pastoris as has been described in material and methods section. Several independent colonies were induced with 0.5% methanol in BMMY medium and a qualitative assay using 2-naphthyl acetate was performed to select a colony that produced the best activity. P. pastoris wild type strain X-33 and a strain X-33 transformed with the empty vector showed no esterase activity. Three out of six colonies were selected to perform a quantitative assay from culture supernatants. Strain BaEstB5 was selected for further experiments.
From this clone, BaEstB was purified through nickel affinity chromatography with 2.9-fold purification with recovery of 17% and specific activity of 31.58 U/mg using 2-naphthyl acetate ( Table 2). The purified enzyme showed a single protein band both on SDS-PAGE and in a zymogram with an estimated molecular mass of 38.3 kDa (Figure 5a,b).
The purified enzyme was used for its biochemical characterization.

| Substrate specificity
Since the α/β hydrolase family comprises both esterases and lipases, we performed a specific assay for lipases using rhodamine B together with commercial olive oil and monitoring the appearance of a fluorescent halo in ultra thin agar plates at λ 350 nm (Kouker & Jaeger, 1987 which is a polymer of xylopyranosyl residues. The difference on this activity is proposed to be due to the architecture of the neighboring sugars that allow the enzyme to better position itself in order to hydrolyze the acetate group (see Figure 12 in Biely, 2012) (Biely, 2012). As mentioned above, in mammals, HSL have bulky substrates such as cholesteryl esters. Taken together, these ideas suggest that BaEstB could be involved in unesterifying ergosterol esters, which have been detected by Yuan, Kuang, Wang, and Liu (2008) in different fungal tissues.

| Effect of temperature and pH on BaEstB activity
Optimal temperature and pH were determined for BaEstB over a temperature range from 10°C to 70°C and pH 4-11. This enzyme showed an optimal activity at 45°C (31.58 ± 0.1 U/mg), whereas at 30°C and 50°C still conserves 69% and 99% of activity, respectively. At lower temperatures, 20°C, it only shows 52% of the optimal activity and at 70°C it is almost inactivated. (Figure 6a). Thermostability of the enzyme was also determined. After incubation at different temperatures, we could determine that BaEstB conserves almost 81% of its activity after incubation at 40°C for 1 hr. Incubation at 10°C to 30°C showed around 97% of its activity, whereas incubation at 50°C abolished the activity to 5% (Figure 6b). HSL counterparts in Rhizomucor show similar optimal temperatures between 45 and 50°C (Liu et al., 2013;Yan et al., 2014), while other esterases from metagenomic libraries have their optimal temperatures around 30°C (Jeon et al., 2012;Li et al., 2014Li et al., , 2015. Considering the temperature, BaEstB is a mesophilic HSL esterase, compared with a thermostable esterase from Thermoanaerobacter tengcongensis with optimal temperature at 70°C (Rao et al., 2011).
The purified BaEstB showed relatively high activity under alkaline conditions, and exhibited optimum activity at pH 7.0 in 50 mmol/L Tris-HCl (100%). The activity diminished only to 91% at pH 10; whereas at pH 11, the activity decreased to 53%. At the acidic pH 4, the enzyme has only 45% of activity ( Figure 6c). We also determined the stability of the enzyme at different pH by incubating it in different buffers for 1 hr and then determining the activity. The most stable condition was found to be pH 7.0 showing 100% of activity, whereas at pH 6 the enzyme retained 69% of the activity. At more basic pH (from 8 to 10), the enzyme showed to be more stable since retained more than 90% of activity. However, at acidic pH 4, only around 20% residual activity was found ( Figure 6d). As it was reported by Liu et al. (2013) for the R. miehei HSL enzymes, BaEstB also shows better activity in pHs around neutrality although it retains 50% of its activity even at pH 11. Compared with other HSL esterases, BaEstB retains its activity in a wider alkaline pH range. BaEstB optimal pH of 7.0 is lower than other esterases like that of an oil-degrading bacterium (pH 8.5) (Mizuguchi et al., 1999), Pseudoalteromonas sp (pH 8) (Cieśliński et al., 2007) or Thermoanaerobacter tengcongensi (pH 9.5) (Rao et al., 2011), but higher than an esterase from the basidiomycete Pleurotus sapidus (pH 6) (Linke et al., 2013).

| Effects of metals, solvents, and detergents on BaEstB activity
The effects of various chemicals on the enzymatic activity were evaluated by measuring initial and residual activity after incubation ( Values represent the means of three replicates ± standard error. 2 mol/L NaCl indicating a slight tolerance to salinity. In contrast, an esterase isolated from a metagenomic library has a high halotolerance showing more activity in presence of salt (Selvin et al., 2012).
The enzyme activity of BaEstB was significantly inactivated in the presence of methanol, isopropanol, ethanol, acetone, DMSO, and hexane. In the presence of 1-butanol and chloroform, the reduction was not as severe, showing 50% and 82%, respectively (Table 5). However, in all the cases, the residual activity was low. Furthermore, the enzyme's activity was significantly abolished in the presence of different detergents such as Tween 20, Tween 80, Triton X-100, SDS and CTAB, as well as a chelant agent, EDTA (Table 6).  Figure 8b suggests that the dimeric conformation of BaEstB is maintained during its interaction with the substrate (the same H R value was obtained). Figure 8c shows the relationship between hydrodynamic radius and temperature. Hydrodynamic radius values increase while temperature is higher, indicating the unfolding of BaEstB. The hydrodynamic radius at 70°C suggests that BaEstB is totally unfolding and it is in correspondence with its specific activity at this temperature (0 IU/mg protein). Other HSL enzymes, as PDB 4J7A isolated from a metagenome (Ngo et al., 2013) and PDB 4WY5 from R. miehei (Yang, Qin, et al., 2015), have been also reported as dimers, the latter forming a tetrameric structure through hydrogen bonding; supporting our results that show that BaEstB is constituted as a dimer.

CONCLUSIONS
In this work, we report for the first time a HSL from a Basidiomycete fungus. The genomic sequence contains three small introns which is not unusual for Basidiomycetes. Through three-dimensional modeling studies and phylogenetic analysis, we conclude that BaEstB is an ortholog of the previously described RmEstB HSL of R. miehei. Traditional phylogenetic analysis together with Blastx results, T A B L E 6 Specific activity and residual activity in (%) of BaEstB in the presence of detergents suggests that a number of fungal hypothetical proteins could belong to the HSL family.

ACKNOWLEDGMENTS
We are grateful to Dr. Agustín López Munguía for providing bench space at his laboratory and to Rocío Rodríguez-Hernández for contributing with experimental material. We feel thankful to Jorge Yáñez, Santiago Becerra, Paul Gaytán, Eugenio López for synthesis of DNA and DNA sequencing. Part of the research was performed at the LabDP-UAEM.