Introduction

Esterase (EC 3.1.1.1.) is a lipolytic enzyme that hydrolyzes ester bonds to carboxylic acid and alcohols. Bacterial lipolytic enzymes were first classified into eight families by Arpigny and Jaeger according to conserved amino acid sequence motifs and biochemical properties [1]. The lipolytic enzymes have been studied, and recently, family XIX was reported [2].

The family IV esterase is also called hormone-sensitive lipase (HSL) because it showed epinephrine-sensitive activity in human adipose tissue [3]. Family IV esterase belongs to an alpha/beta hydrolase and has β-sheet structures covered by α-helices [4]. The family IV esterase has two domains: a cap domain and a catalytic domain. The role of the cap domain is unknown, but there is a report that the cap domain of family IV esterase is deeply related to the recognition of substrates [5], and the catalytic domain has a catalytic triad: serine (S) in the GXSXG motif, aspartic acid (D), and histidine (H) [6].

Esterase has numerous applications. In particular, esterase can be used for chemical reactions, such as transesterification or production of biodiesel [7], and for ester prodrugs, which have been focused on the application for drug delivery systems to avoid metabolism and side effects [8]. For example, the 2nd-generation fluoroquinone antibiotic ciprofloxacin is used for antimicrobial activity against most gram-negative bacteria and many gram-positive bacteria [9]. However, it has low aqueous solubility and intestinal permeability. The introduction of triethylene glycol to ciprofloxacin, which is an ester prodrug, and its use with esterase, increased solubility 400 times, inducing quick hydrolysis and restoration of antimicrobial activity [10]. Additionally, in the case of esterase with enantioselectivity, it is more valuable for the chemical reaction with a specific enantiomer. For example, the statin derivatives—which can be used for various diseases, such as cardiovascular diseases—need esterase with enantioselectivity and regioselectivity to synthesize them [11].

On the other hand, the metagenome is the genome collected from whole microorganisms in an environment [12]. It is also called environmental DNA (eDNA) because it is collected from a specific environment [13]. Many approaches through a metagenomic library were employed to obtain novel genes because it is possible to predict the diversities and properties of the enzyme from the environment. For the reasons described above, many metagenomic studies have been explored, such as Himalayan glacier frozen soil [14], the saline desert of Kutch [15], freshwater lake [16], caves [17], oil-polluted mud flats [18], and compost [19].

Compost has been selected as an object of metagenomic study because it contains various useful enzymes, such as endoglucanase, xylanase, and esterase [20,21,22]. We selected the compost metagenome and reported the properties of some lipolytic enzymes obtained from the compost metagenomic library, such as Est2K, Est7K, Est8L, and Est13L [19, 23, 24]. Recently, a family IV esterase from another compost metagenome was reported, and it showed organic solvent stability [25]. In this study, a novel esterase gene, est15L, was analyzed, and its encoded enzyme, Est15L, was characterized as a novel member of family IV with efficient enantioselectivity.

Materials and methods

Materials

The compounds isopropylthio-β-D-galactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) were purchased from Bioneer (Daejeon, Korea). The substrates p-nitrophenyl (p-NP) esters (C2 ~ C16), glyceryl triesters (glyceryl tributyrate and glyceryl trioleate), acetylthiocholine iodide (ATCI), S-butyrylthiocholine iodide (BTCI), and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HiTrap Q HP (5 mL), t-butyl HIC (1 mL), and HiPrep 16/60 Sephacryl S-200 HR (120 mL) column were purchased from GE Healthcare (Uppsala, Sweden).

A positive esterase clone from the compost metagenomic library

The metagenome was obtained from Yonghyun Nonghyub Compost Factory (Sachon, Korea), and its library was constructed using the fosmid vector [19]. From this library, 19 esterase-positive clones were obtained on LB agar plates containing 1% glyceryl tributyrate for 15 h at 37 ℃. They were mixed, digested with a restriction enzyme, cloned with plasmid pUC19, and 18 positive subclones were obtained [19]. By sequencing, nine different lipolytic enzymes were identified, and some of them were reported [19, 23, 24]. In this study, a positive clone YH-E15 was selected for further study.

Sequence analysis of the insert DNA in the positive clone

DNA sequences of the esterase-positive clone were determined using the Sanger dideoxy method by Solgent (Daejeon, Korea). From this sequence, the ORF similar to esterase was confirmed, and its amino acid sequence was analyzed by BLASTp of NCBI (http://www.ncbi.nlm.nih.gov). Prediction of signal peptide was performed using SignalP 5.0 in CBS (http://www.cbs.dtu.dk/services/SignalP/). Molecular weight and pI were predicted using the ExPASy ProtParam tool (http://web.expasy.org/protparam). Clustal W method of DNA/MAN (Lynnon Biosoft, version 4.11, Quebec, Canada) was used to analyze multiple sequence alignment, and the neighbor-joining method in MEGA version X [26] was used to construct the phylogenetic tree. Similarities between the identified enzyme Est15L and other enzymes were calculated using DNA/MAN.

Preparation of crude enzymes

The clone YH-E15 was cultured in 200 mL of LB broth containing 50 μg/mL of ampicillin and incubated for 15 h at 37 ℃ and 200 rpm. The cell was collected from the cultured medium by centrifuging at 4 ℃ and 6,000 × g for 15 min. The collected pellet was washed two times with 20 mL of 20 mM Tris–HCl (pH 8.0) buffer by centrifugation at 4 ℃ and 6,000 × g for 5 min, resuspended with 5 mL of the same buffer, sonicated (amplitude of 38%, pulse on for 1 s and pulse off for 1 s) three times using a microtip sonicator (VCX500, Sonics & Materials, Newtown, CT, USA), and then centrifuged at 4 ℃ for 15 min at 6,000 × g. The supernatant was collected as a crude extract.

Purification of Est15L

Before the purification step, the crude enzyme was centrifuged for 15 min at 6,000 × g and 4 °C. The supernatant was loaded to a HiTrap Q anion exchange column in a BioLogic LP system (Bio-Rad, Hercules, CA, USA) with 20 mM Tris–HCl (pH 8.0), and the buffer flowed with a linear gradient with a high buffer containing 1 M NaCl at 1 mL/min for 1 h 30 min. The active fractions were pooled, dialyzed with 50 mM sodium phosphate (pH 7.0) containing 1.5 M (NH4)2SO4 for the t-butyl HIC column as a second column, loaded to the column, and eluted with a high buffer containing 1.5 M (NH4)2SO4 with a linear gradient at 1 mL/min for 1 h. To confirm the native molecular mass of the enzyme, Sephacryl S-200 size exclusion chromatography was performed using 50 mM sodium phosphate (pH 7.0) containing 0.15 M NaCl at a flow rate of 0.5 mL/min for 4 h. β-Amylase, bovine serum albumin (BSA), and trypsinogen (200, 66.4, and 24.0 kDa, respectively) were used as standard markers. During purification, active fractions were loaded on 11.5% acrylamide gel, and then SDS-PAGE was performed [27]. The concentration of protein was determined by Bradford assay using BSA as a standard [28].

Enzyme assays

The standard esterase assay was performed using 1 mM p-NP butyrate in 50 mM Tris–HCl (pH 8.0). The amount of p-nitrophenol as the product was observed continuously by kinetic mode in a spectrophotometer (OPTIZEN, K-Lab, Daejeon, Korea) for 2 min at 25 ºC at 400 nm. The molecular extinction coefficient of p-nitrophenol used was 16,400 /M/cm at pH 8.0. The production of 1 µmol p-nitrophenol per minute was defined as one unit of an enzyme.

The acetyl-or butyryl-cholinesterase activity was measured by the Ellman method using ATCI or BTCI, respectively, as the substrate, as previously described [29]. Briefly, the enzyme was added to 100 mM sodium phosphate containing 0.5 mM DTNB and 0.5 mM ATCI or BTCI, respectively, and the absorbance of the reaction mixture was observed continuously at 412 nm for 15 min at 25 ºC using kinetic mode in spectrophotometer (OPTIZEN).

Characterization of the enzyme

The standard enzyme assay was characterized using p-NP butyrate with slight modification. For the optimum temperature experiment, the buffer was preheated to 20, 30, 40, 50, 60, and 70 ℃ prior to assay. For optimum pH, 50 mM Universal buffer (boric acid/ citric acid/ trisodium orthophosphate) for pH 6.0 to 12.0 was used. The molecular extinction coefficients at each pH were used as previously described [18]. For thermostability, the enzyme was heated for 0, 5, 10, 20, 30, and 60 min at 30 and 40  ºC, added to the assay mixture, and its residual activity was measured.

For the p-NP ester specificity, 1 mM of p-NP acetate (C2), p-NP butyrate (C4), p-NP caproate (C6), p-NP octanoate (C8), p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14), and p-NP palmitate (C16) were used as substrates for the esterase assay. The kinetic experiment was performed using 0.05, 0.1, 0.2, 0.5, and 1 mM of C4 and a Lineweaver–Burk plot was constructed for measuring Km and Vmax values.

Ions such as NaCl, KCl, MgCl2, CaCl2, BaCl2, MnCl2, FeCl2, CoCl2, CuCl2, and ZnCl2 were added to the assay solution at 2 or 5 mM to confirm the effects of ions. Methanol, isopropanol, and acetonitrile were added to the assay solution to have a final concentration of 5 or 30% to confirm the effect of the organic solvent. Effects of detergents (such as SDS and Triton X-100) were observed at a concentration of 1%. The effects of inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA), were observed at a concentration of 1 mM.

Lipid hydrolysis activity was measured with a pH shift assay using oils (fish and olive oil) and glyceryl triesters (glyceryl tributyrate and glyceryl trioleate) as substrates [30]. The enzyme was reacted with the substrate in 20 mM Tris–HCl (pH 8.0) containing 0.1% phenol red, and its absorbance at 560 nm was observed continuously using kinetic mode in the spectrophotometer (OPTIZEN) at 25 ºC for 60 min at 5 min intervals. The enantioselectivity was measured using 1% (R)-methyl-3-hydroxy-2-methyl-propionate or (S)-methyl-3-hydroxy-methyl propionate as a substrate for the pH shift assay [18].

In silico 3D modeling

The prediction of 3D models was conducted using SWISS-MODEL of expasy (https://swissmodel.expasy.org/). The predicted structures were transferred to SwissDock (http://www.swissdock.ch/), and docking simulation was constructed with (R)-or (S)-methyl-3-hydroxy-2-methyl-propionate (ZINC accession numbers were 395641 and 395640, respectively). Docking data were analyzed using Chimera version 1.15 (https://www.cgl.ucsf.edu/chimera/).

Results

Sequence analysis and multiple alignments of Est15L

Due to DNA sequencing for the positive clone YH-E15, it was revealed that insert DNA comprised 2,587 bp, and an open reading frame (ORF) was predicted to be an esterase. The ORF was 987 bp in length and named est15L. The encoded Est15L esterase comprised 328 amino acids with molecular weights of 34,770 Da with no signal peptide, and its predicted theoretical pI value was 4.57. Est15L has been deposited under the accession number of OK336712 in GenBank. In BLASTp, Est15L showed the highest homology (85.03%) to alpha/beta hydrolase of Sphingorhabdus sp. (GenBank accession number, MBF6602187) obtained from metagenome-assembled genomes isolated from diarrhea affected cattle B. Conversely, alpha/beta hydrolase of Sphingorhabdus sp. Showed a similar identity (100%) with Est8L (QZA73595), which was obtained from the compost metagenomic library [24]. Est15L showed relatively low identity to other reported enzymes, and enzymatic properties were characterized in further study.

In the phylogenetic tree, it was confirmed that Est15L is a novel member of family IV esterase (i.e., HSL) (Fig. 1).

Fig. 1
figure 1

Phylogenetic tree of Est15L with other esterases/lipase family. The phylogenetic tree was constructed using the neighbor-joining method in MEGA version X with 1,000 bootstrap replications. The accession numbers are accessible using GenBank of NCBI

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Multiple sequence alignment showed several conserved regions—such as HGG (101 ~ 103), DYR (132 ~ 134), GXSAG (174 ~ 178), DPL (268 ~ 270), and GXIH (295 ~ 298)—of which the predicted catalytic residues serine (S), aspartic acid (D), and histidine (H) were underlined (Fig. 2).

Fig. 2
figure 2

Multiple sequence alignment of Est15L with other family IV esterase by Clustal W method of DNA/MAN. The sequences present in this figure are accessible using the accession number through GenBank of NCBI (http://www.ncbi.nlm.nih.gov/Genbank) QZA73595, family IV esterase (Est8L) [Uncultured bacterium]; QZX52242, esterase Est2L [Uncultured bacterium]; PHS53692, esterase [Alteromonas sp.]; ALP75907, lipase/esterase [Parvibaculum sp. enrichment culture]; QGT40748, carboxylesterase E2 [Expression vector PCJH10_pETcc2::E2Aa]; BAC06606, [Pyrobaculum calidifontis]; AKG92633, Est3K [Uncultured bacterium]

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Purification of Est15L

Est15L was bound to HiTrap Q and eluted with the linear gradient (Fig. 3A). Specific activity was increased 5.50 times (24.86 U/mg), compared to the crude extract (4.52 U/mg), with a yield of 24.8%. In size exclusion chromatography using Sephacryl S-200, Est15L was eluted at 61.5 mL, with an increased specific activity of 160.3 U/mg (Fig. 3B; Table 1).

Fig. 3
figure 3

HiTrap Q (A) and Sephacryl S-200 (B) chromatograms of Est15L. The blue, red, and black lines of the HiTrap Q chromatogram indicate the absorbance at 280 nm (A.U.), conductivity (dS/cm), and esterase activity (U/mL), respectively. The size exclusion chromatography by Sephacryl S-200 was performed twice, and pools from the 2nd experiment were used for SDS-PAGE

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Table 1 The specific activity and yield of Est15L at purification steps
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In SDS-PAGE, the predicted band of Est15L, corresponding to about 34.9 kDa, was detected in the fractions from Sephacryl S-200 in an activity-dependent manner (Fig. 4). However, it showed smear bands around the major band, along with some minor bands at 100, 75, 48, and 27 kDa. Est15L was partially purified using the two chromatographies. Est15L did not bind to other resins (such as CHT-II, High S, and t-butyl HIC) and showed low yields of less than 8.7%.

Fig. 4
figure 4

SDS-PAGE of Est15L stained with silver nitrate. An active fraction with the highest activity from HiTrap Q and active fractions from Sephacryl S-200 were used for the experiment. The red arrow in the figure represents the predicted position of the Est15L (~ 34.9 kDa). M, size marker; C, crude extract; Q, the fraction from HiTrap Q (number 54 in Fig. 3A); S, fractions from Sephacryl S-200 (numbers 54, 56, 58, 60, 68, and 72 in Fig. 3B)

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From the elution volume, the molecular mass of Est15L was calculated to be 67.2 ± 8.8 kDa, and it can be predicted that native Est15L existed as a dimeric form (Fig. 3B; Table 2).

Table 2 The molecular mass of native Est15L determined by size exclusion chromatography
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Characterization of Est15L

Est15L was optimally active at 50 ℃ and pH 9.0, indicating Est15L was an alkaline esterase (Fig. 5A, B). In thermostability, Est15L was sensitive to thermal stress with half-lives of 2.7 min at 40 ℃ and 30.3 min at 30 ℃ (Fig. 5C).

Fig. 5
figure 5

The optimum temperature (A) and pH (B), and thermostability (C) of Est15L. For optimum temperature experiment, 50 mM Tris–HCl (pH 8.0) buffer was preheated to designated temperatures. For optimum pH, 50 mM Universal buffer (boric acid/citric acid/trisodium orthophosphate, pH 6.0 to 11.0) was used. For thermostability, Est15L was preincubated for designated times without substrate prior to the standard assay

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Est15L preferred C4 followed by C6, C8, and C2 with relative activities of 69.3, 49.3, and 35.8%, respectively (Fig. 6A). On the other hands, Est15L did not show AChE and BChE activities. In a kinetic study using C4, Km and Vmax values of Est15L were 0.28 ± 0.02 mM and 278.0 ± 10.9 U/mg, respectively (Fig. 6B).

Fig. 6
figure 6

Substrate specificity (A) and Lineweaver–Burk plot (B) of Est15L. For substrate specificity, p-NP esters were used for esterase assay at 1 mM. For the cholinesterase assay, 0.5 mM substrates were used. Kinetic study was performed using 0.05, 0.1, 0.2, 0.5, and 1.0 mM of C4

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In the presence of 30% methanol, isopropanol, and acetonitrile, Est15L activity was significantly inhibited to 12.5, 0.9, and 0.3%, respectively. In detergent, Est15L activity was maintained to 88.2% at 1% Triton X-100. However, it was extensively inactivated to 0.38% at 1% SDS. In the case of inhibitors, Est15L was stable at 1 mM EDTA with a relative activity of 89.9% but was strongly inhibited to 2.5% by 1 mM PMSF (Fig. 7A).

Fig. 7
figure 7

Effects of organic solvents, detergents, inhibitors (A), and ions (B) on Est15L activity. The effectors were added to the standard esterase assay solution

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Est15L was inhibited by metal ions (i.e., 2 mM of Zn2+, Co2+, and Na+) with residual activities of 16.6, 47.3, and 52.5%, respectively. It was also inhibited by 5 mM of Cu2+, Mn2+ and Fe2+ with relative activities of 5.5, 30.1, and 49.8%, respectively. No activations were observed with any metal ions (Fig. 7B).

Est15L efficiently hydrolyzed glyceryl tributyrate with a residual substrate amount of 43.7%. However, no significant hydrolysis activity was observed for olive oil, fish oil, and glyceryl trioleate (Fig. 8A). Interestingly, Est15L showed higher enantioselectivity toward the R-form with a residual substrate amount of 44.6% than toward the S-form with 83.5% after a 60 min reaction (Fig. 8B).

Fig. 8
figure 8

Lipid hydrolysis activity (A) and enantioselectivity (B) of Est15L. Substrates, that is, 1% of oils, glyceryl triesters, and (R)- or (S)-methyl-3-hydroxy-2-metyhl-propionate isomers, were added to the solution for the pH shift assay

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Discussion

In this study, an esterase Est15L, a novel member of family IV (HSL family), was obtained from a compost metagenomic library. Est15L showed the highest similarity (85.03%) to alpha/beta hydrolase of Sphingorhabdus sp. (MBF06602187) and Est8L, and the properties of Est8L were reported in our previous study [24]. Though Est15L and Est8L showed high similarity of amino acid sequence, their properties were different from each other, such as optimum temperature (50 vs. 40 ℃), thermostability (half-lives of 2.7 min at 40 ℃ vs. 3.2 min at 50 ℃), enantioselectivity (R-form vs S-form), and organic solvents effect. It has been reported that some lipolytic enzymes showed different properties in a specific activity, enantioselectivity, ionic effect, and organic solvent effect, despite high similarities of their amino acid sequences [18, 24, 31,32,33,34].

In predictions of 3D structure models, Est15L showed the highest identity (33.89%) to Esterase Crystal structure of Chloramphenicol-Metabolizing Enzyme EstDL 136-Chloramphenicol complex (PDB code: 6iey.1.A) homo-dimer form. Est8L showed the highest identity (33.55%) to Esterase Crystal structure of Chloramphenicol-Metabolizing Enzyme EstDL 136 (PDB code: 6aae.1.A) with homo-dimer form. In the predicted model, the N-terminal of Est15L (M1 ~ A18) was not predicted because it did not identify with other family IV esterase; thus, its structure could not be predicted. The Chain A of Est15L comprised 11 α-helix and 8 β-sheets, and Chain B comprised 9 α-helix and 8 β-sheets (Fig. 9A, C). Moreover, the Chain A of Est8L comprised 12 α-helix and 8 β-sheets, and Chain B of Est8L comprised 11 α-helix and 8 β-sheets (Fig. 9B, D). In the case of β-sheet structures, no difference was found between Est15L and Est8L. However, Est15L has three fewer α-helix structures than Est8L; that is, the motifs F42 ~ T49 of Chain B, A129 ~ E143 of Chain A, and L286 ~ L289 in Est8L were predicted as α-helical structures, whereas Est15L did not show (Fig. 9). Its structural differences might occur in biochemical properties such as optimum temperature, thermostability, and enantioselectivity.

Fig. 9
figure 9

Predicted 3D models of Est15L (A) and Est8L (B) and model template alignments of Est15L (C) and Est8L (D). The predicted 3D models were constructed using SWISS-MODEL (https://swissmodel.expasy.org/). The model template for A and C of Est15L was the Esterase Crystal structure of Chloramphenicol-Metabolizing Enzyme EstDL 136-Chloramphenicol complex (PDB code: 6iey.1.A) and that for B and D of Est8L was the Esterase Crystal structure of Chloramphenicol-Metabolizing Enzyme EstDL 136 (PDB code: 6aae.1.A). The differences between Est15L and Est8L are marked as red star marks

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By using the model templates as above, docking simulations of Est15L and Est8L with (R)- or (S)-methyl-3-hydroxy-2-methyl-propionate were performed. Interestingly, Est15L showed a higher affinity with the (R)-form (ΔG = ‒ 6.31 kcal/mol) than (S)-form (ΔG = ‒ 6.23 kcal/mol) at cap domain for the strongest binding, which is considered to have an important role in recognition of the substrate (Additional file 1: Table S1). However, Est8L, showing (S)-form selectivity, also showed a higher affinity with the (R)-form, (ΔG = ‒ 6.92 kcal/mol) than with the (S)-form, (ΔG = ‒ 6.87 kcal/mol) (Additional file 1: Table S2). In addition, no interaction or binding was predicted between the catalytic triads of the enzymes and their substrates, except that the (S)-form formed a hydrogen bond with Ser176 of Est15L, at a distance of 3.082 Å (ΔG = ‒ 6.22 kcal/mol) (Additional file 1: Figure S1). Collectively, though the docking values could not sufficiently support the selectivity, it is suggested that other bindings may contribute to their selectivity at the same time.

The other family IV esterase from a compost metagenomic library, EstCS1, was recently reported, and it showed similar properties to Est15L, such as optimum temperature (50 ℃), substrate specificity (C4 vs. C3), and optimum pH (9.0 vs. 8.0). However, Est15L showed lower stability (< 12.5% residual activity) toward organic solvents (i.e., at 30% methanol, isopropanol, and acetonitrile) than EstCS1 (> 90.8% residual activity) [25]. Moreover, the enantioselectivity of EstCS1 was not reported [25] (Table 3).

Table 3 Comparison of Est15 and other family IV esterases
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Compared to other esterases characterized to date, Est15L showed low identities from 3.17 to 30.72% [35,36,37,38,39]. The molecular weights of family IV esterases ranged from 30.4 to 41.2 kDa [40,41,42], except for Est2L, which was a fusion-type protein with a molecular weight of 92.5 kDa [43]. This indicates that the molecular weight of Est15L is an average value. Native Est15L was a dimeric form, as most family IV esterases reported [44,45,46], except that AFEST, EST2, and EstE5 were monomeric forms [38, 39, 41, 47], and Cest-2923 and E40 were tetrameric forms [40, 48, 49] (Table 3). On the other hand, REst1 exists in two types: monomeric form and trimeric form [50].

The specific activity of Est15L (160.3 U/mg) was the average of most family IV esterases [35, 36, 51, 52], and was 2.4 times lower than Est8L (388.6 U/mg), which has high similarity to Est15L. Some of family IV esterases had significantly high specific activities: PestE (3,910 U/mg), AFEST (3,000 U/mg), Est22 (2,065 U/mg), and Rv1399c (1,350 U/mg) [38, 39, 53,54,55,56] (Table 3).

The optimum temperature of Est15L was in the range of most family IV esterases from 25 to 60 ℃ [51, 57, 58], although some of them were thermophilic: EstE1 (95 ℃), PestE (90 ℃), and SaHSL (70 ℃) [53, 54, 59, 60]. Est15L was alkaline, likely to most family IV esterases, that is, alkaline, or neutral esterase [51, 61, 62], except for EstE1, EstE7, Sto-Est, Est3, and EstB28 [59, 63,64,65,66,67] (Table 3).

Est15L had no signal peptide, likely most of the family IV esterase, suggesting Est15L is an intracellular esterase, but EstA1 had a signal peptide [42].

In the case of substrate specificity, Est15L preferred short-length substrates, likely most of family IV esterase (C2 to C6) [35, 36, 60, 68]. However, Sto-Est and EstAG1 showed the highest activity toward medium-length C8 [66, 69]. The organic solvents such as methanol, isopropanol, and acetonitrile inhibited most family IV esterases, except some of them were solvent-tolerant, such as EstCS1, EstA1, and PestE [24, 42, 53, 54] (Table 3).

The metal ion effects of family IV esterases are diverse; Est15L showed inhibitions by Cu2+, Zn2+, Mn2+, Co2+, and Na+ and similar patterns with Est8L, except for Mn2+ and Na+. Similar effects of metal ions were reported for SAestA, EstKT7, DMWf18-543, and DMWf18-558 [57, 68, 70] (Table 3).

Est15L showed great enantioselectivity toward the R-form (38.90% higher than the S-form), whereas Est8L showed an S-form preference. The R-form enantioselectivity of Est15L was similar to PestE, a family IV esterase; however, Est15L was different from PestE in specific activity (160.3 vs. 3,910 U/mg), optimum pH (9.0 vs. 7.0), substrate preference (C4 vs. C6), and optimum temperature (50 vs. 90 ℃) [53, 54] (Table 3). Enantioselective esterases can be used for purifying and enriching the specific enantiomer [71]. For example, the lipolytic enzyme from pseudomonas cepacia is a popular catalyst for hydrolysis, transesterification, and esterification of racemic mixtures of secondary alcohols to synthesize important enantiomers [72]. Est15L can be used for purifying the racemic mixtures to enrich the (S)-form enantiomer by selective hydrolysis of the (R)-form enantiomer.

Collectively, Est15L was sensitive to organic solvents, ions, and thermal stress, but it showed good specific activity and enantioselectivity. In this study, Est15L was obtained from a compost metagenomic library, and its enzymatic properties were characterized. Est15L was a novel protein and showed the highest similarity (85.03%) with an alpha/beta hydrolase of Sphingorhabdus sp. and Est8L. Though Est15L and Est8L showed high similarity, their properties were different from each other: optimum temperature (50 vs. 40 ℃), thermostability (half-lives, 2.7 min at 40℃ vs. 3.2 min at 50 ℃), specific activity (160.3 vs. 388.6 U/mg), and enantioselectivity (R- vs. S-form). Est15L was sensitive to organic solvents such as methanol, isopropanol, and acetonitrile, whereas Est8L was relatively tolerant to them. On the other hands, Est15L had great enantioselectivity with a residual substrate amount of 43.68% for the R-form. We tracked why these differences are occurred by predicting 3D structures and docking simulations, and we found structural differences between Est15L and Est8L, i.e., three less α-helices and longer cap domain of Est15L, which showed higher affinity with R-form in docking simulation. Although Est15L is less stable to ionic and thermal stress than Est8L, the enantioselectivity of Est15L will be more valuable for chemical applications. These results suggest that Est15L is a novel member of family IV esterase and a potential candidate of the chemical reaction or ester prodrugs with enantioselectivity.