Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.


Introduction
Carboxylesterases (EC 3.1.1.1) are ubiquitous enzymes found in a wide range of organisms, including some viruses [1]. The commonly known mechanism of carboxylesterases is the formation of an alcohol and a carboxylate resulting from the reaction between a carboxylic ester with water molecules. To date, bacterial carboxylesterases have been identified and characterized from various sources and metagenomic samples [2,3]. Thus, the classification and grouping of bacterial carboxylesterases have been continuously changed and updated. Recently, Hitch and Clavel [4] suggested a new classification method for bacterial lipolytic enzymes using 35 families and 11 true lipase subfamilies based on their sequences and conserved motifs. This updated classification system and representative sequences for each group were used for sequence analysis and grouping of new target esterases.
Exiguobacterium antarcticum is a psychrotrophic bacterium isolated from microbial mats in Antarctica. The complete genome sequence of E. antarcticum has been deposited in the National Center for Biotechnology Information database (GenBank: AY186197.1) [5].

Bioinformatic Analysis of EaEst2
Carboxylesterases are a ubiquitous protein family found in all organisms, including some viruses. EaEst2 originates from the psychrotrophic bacterium E. antarcticum and is assumed to have unique structural features relative to carboxylesterases from mesophiles and thermophiles. Comparative sequence analysis was performed on the EaEst2 sequence and other representative sequences for each family of bacterial lipolytic enzymes using a phylogenetic tree ( Figure S1). Based on the phylogenetic analysis, the EaEst2 protein can be classified into carboxylesterase Family XIII because it shows a high sequence similarity of 65.04% with Est30 (GenBank ID: AAN81911.1), which is in the same clade. Est30 is a thermostable carboxylesterase derived from G. stearothermophilus, and its crystal structure (PDB code 1TQH) was determined in 2004 [6,7]. Additionally, our sequence analysis revealed that EaEst2 possesses a conserved pentapeptide, GLSLG, in its amino acid sequence from 91-95, including a catalytic Ser residue, which is a typical consensus motif in carboxylesterase Family XIII enzymes [16] (Figure 1).

Figure 1.
Multiple sequence alignment of EaEst2. Multiple sequence alignment of EaEst2 and selected structural homologs including carboxylesterase from Geobacillus stearothermophilus (Uni-ProtKB code Q06174; PDB code 1TQH), esterase from Bacillus licheniformis (UniProtKB code Q65EQ1; PDB code 6NKG), carboxylesterase from Bacillus subtilis, strain168 (UniProtKB code O32232), and thermostable monoacylglycerol lipase from Bacillus sp. (UniProtKB code P82597; PDB codes 3RM3 and 4KEA). The catalytic triad residues (Ser93, Asp190, and His220) are indicated by red triangles, and the consensus motif is colored in blue. Secondary structures obtained from the crystal structure of EaEst2 are presented above the aligned sequences.

Biochemical Characterization of EaEst2
The substrate specificity of recombinant EaEst2 was examined using various ester compounds and other possible substrates. First, the relative preference of EaEst2 for different acyl chain lengths showed that EaEst2 had the strongest activity toward p-nitrophenyl acetate (C2), which had the shortest chain length among the p-nitrophenyl esters tested ( Figure 2A). Activity gradually decreased as the length of the substrate increased. Additionally, EaEst2 was thought to have no activity against the substrate, which was longer than p-nitrophenyl dodecanoate (C12). Among the naphthyl derivatives, EaEst2 showed strong regioselectivity for 1-naphthyl ester substrates. EaEst2 activity was much higher toward 1-naphthyl acetate (1-NA) and 1-naphthyl butyrate (1-NB) than that toward 2-naphthyl acetate (2-NA) ( Figure 2B). The optimal pH of EaEst2 was investigated over a pH range from 3.0 to 10.0. EaEst2 showed its maximal activity at pH 7.0, whereas only ~40% of its maximal activity was retained at pH 8.0 ( Figure 2C). Under optimal conditions, the enzymatic activity of EaEst2 showed a hyperbolic curve for Michaelis-Menten kinetics, with kinetic parameters (Vmax, Km, and kcat/Km) determined using p-NA. Vmax and Km values of 5.59 µM s −1 and 0.59 mM were obtained, respectively ( Figure 2D). Multiple sequence alignment of EaEst2 and selected structural homologs including carboxylesterase from Geobacillus stearothermophilus (UniProtKB code Q06174; PDB code 1TQH), esterase from Bacillus licheniformis (UniProtKB code Q65EQ1; PDB code 6NKG), carboxylesterase from Bacillus subtilis, strain168 (UniProtKB code O32232), and thermostable monoacylglycerol lipase from Bacillus sp. (UniProtKB code P82597; PDB codes 3RM3 and 4KEA). The catalytic triad residues (Ser93, Asp190, and His220) are indicated by red triangles, and the consensus motif is colored in blue. Secondary structures obtained from the crystal structure of EaEst2 are presented above the aligned sequences.

Biochemical Characterization of EaEst2
The substrate specificity of recombinant EaEst2 was examined using various ester compounds and other possible substrates. First, the relative preference of EaEst2 for different acyl chain lengths showed that EaEst2 had the strongest activity toward p-nitrophenyl acetate (C2), which had the shortest chain length among the p-nitrophenyl esters tested ( Figure 2A). Activity gradually decreased as the length of the substrate increased. Additionally, EaEst2 was thought to have no activity against the substrate, which was longer than p-nitrophenyl dodecanoate (C12). Among the naphthyl derivatives, EaEst2 showed strong regioselectivity for 1-naphthyl ester substrates. EaEst2 activity was much higher toward 1-naphthyl acetate (1-NA) and 1-naphthyl butyrate (1-NB) than that toward 2-naphthyl acetate (2-NA) ( Figure 2B). The optimal pH of EaEst2 was investigated over a pH range from 3.0 to 10.0. EaEst2 showed its maximal activity at pH 7.0, whereas only~40% of its maximal activity was retained at pH 8.0 ( Figure 2C). Under optimal conditions, the enzymatic activity of EaEst2 showed a hyperbolic curve for Michaelis-Menten kinetics, with kinetic parameters (V max , K m , and kcat/K m ) determined using p-NA. V max and K m values of 5.59 µM s −1 and 0.59 mM were obtained, respectively ( Figure 2D).
For the chemical stability of EaEst2, the results showed that EaEst2 was highly activated in the presence of 10 and 30% ethanol, with approximately 180 and 160% of the initial activity, respectively. However, the enzyme activity of EaEst2 was almost completely lost in the presence of other chemicals such as sodium dodecyl sulfate (SDS), Tween 20, Triton X-100, isopropyl alcohol (Iso-PrOH), and urea ( Figure 3A). Compared with previously studied esterases from the same species (E. antarcticum), EaEst2 showed a greater ethanol tolerance of up to 30% and potential for industrial application [17]. In addition, the activity of EaEst2 was highly stable up to 37 • C after 1 h incubation ( Figure 3B). However, EaEst2 activity was completely lost within 15 min at 50 and 60 • C. The circular dichroism (CD) results supported the thermal stability of EaEst2, as shown by its T m value, implying that 50% of the secondary structure of EaEst2 was denatured at 52 • C ( Figure 3C,D). For the chemical stability of EaEst2, the results showed that EaEst2 was highly activated in the presence of 10 and 30% ethanol, with approximately 180 and 160% of the initial activity, respectively. However, the enzyme activity of EaEst2 was almost completely lost in the presence of other chemicals such as sodium dodecyl sulfate (SDS), Tween 20, Triton X-100, isopropyl alcohol (Iso-PrOH), and urea ( Figure 3A). Compared with previously studied esterases from the same species (E. antarcticum), EaEst2 showed a greater ethanol tolerance of up to 30% and potential for industrial application [17]. In addition, the activity of EaEst2 was highly stable up to 37 °C after 1 h incubation ( Figure  3B). However, EaEst2 activity was completely lost within 15 min at 50 and 60 °C. The circular dichroism (CD) results supported the thermal stability of EaEst2, as shown by its Tm value, implying that 50% of the secondary structure of EaEst2 was denatured at 52 °C ( Figure 3C,D).  We subsequently used a pH-dependent colorimetric assay to examine the hydrolytic properties of EaEst2 with respect to carbohydrates and lipids. Significant EaEst2 hydrolytic activity was detected only against mannose pentaacetate (Man-Ac5) and glyceryl tributyrate (GTB), and EaEst2 also showed weak activity against glucose pentaacetate (Glu-Ac5) and galactose pentaacetate (Gal-Ac5) ( Figure 4A,B). However, no activity was We subsequently used a pH-dependent colorimetric assay to examine the hydrolytic properties of EaEst2 with respect to carbohydrates and lipids. Significant EaEst2 hydrolytic activity was detected only against mannose pentaacetate (Man-Ac5) and glyceryl tributyrate (GTB), and EaEst2 also showed weak activity against glucose pentaacetate (Glu-Ac5) and galactose pentaacetate (Gal-Ac5) ( Figure 4A,B). However, no activity was observed against other compounds. We also conducted an EaEst2 enantioselectivity analysis using a pH shift assay with (R)-and (S)-Roche esters (methyl-3-hydroxy-2-methylpropionate). As shown in Figure 4C,D, a color change to yellow was observed only in the enzyme mixture containing the (S)-Roche ester, indicating that EaEst2 prefers the (S)-enantiomer of the chiral ester to the (R)-enantiomer. These results were confirmed by measuring absorbance spectra. Collectively, our biochemical studies indicated that EaEst2 has promising hydrolytic properties and stability and can be used for diverse industrial applications.

Overall Structure of EaEst2
The crystal structure of EaEst2 has been determined at a 1.74 Å resolution in the ligand-free form ( Figure 5A). The structure was refined to an Rwork of 19.6% and an Rfree of 21.3%. The asymmetric unit of the crystal (space group P212121) contained one EaEst2 molecule and 169 water molecules. The overall structure of EaEst2 comprised seven β-strands and 10 α-helices and adopted a classical α/β hydrolase fold in the core domain, which was a twisted β sheet surrounded by α-helices and a cap domain ( Figure 5B). Three putative catalytic residues, S93, D190, and H220, were located on the β4-α4, β6-α8, and β7-α9 loops, respectively. Notably, the substrate binding pocket of EaEst2 was covered by a cap domain comprising α2-, α5-, and α6-helices, forming a long and narrow shape ( Figure 5C).

Overall Structure of EaEst2
The crystal structure of EaEst2 has been determined at a 1.74 Å resolution in the ligandfree form ( Figure 5A). The structure was refined to an R work of 19.6% and an R free of 21.3%. The asymmetric unit of the crystal (space group P2 1 2 1 2 1 ) contained one EaEst2 molecule and 169 water molecules. The overall structure of EaEst2 comprised seven β-strands and 10 α-helices and adopted a classical α/β hydrolase fold in the core domain, which was a twisted β sheet surrounded by α-helices and a cap domain ( Figure 5B). Three putative catalytic residues, S93, D190, and H220, were located on the β4-α4, β6-α8, and β7-α9 loops, respectively. Notably, the substrate binding pocket of EaEst2 was covered by a cap domain comprising α2-, α5-, and α6-helices, forming a long and narrow shape ( Figure 5C). For molecular characterization, EaEst2 was determined to be approximately 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry ( Figure 6A,B). Unlike most carboxylesterases, which are thought to exist as dimers in solution, the functional unit of EaEst2 was determined to be a monomer by sizeexclusion chromatography [18] (Figure 6C,D). The molecular weight calculated from the correlation equation was approximately 28.46 kDa, and the sequence-based computed molecular weight was 27.69 kDa. For molecular characterization, EaEst2 was determined to be approximately 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry ( Figure 6A,B). Unlike most carboxylesterases, which are thought to exist as dimers in solution, the functional unit of EaEst2 was determined to be a monomer by size-exclusion chromatography [18] (Figure 6C,D). The molecular weight calculated from the correlation equation was approximately 28.46 kDa, and the sequence-based computed molecular weight was 27.69 kDa.
Structural homolog search using the DALI server showed that EaEst2 had the highest structural similarity (Z-core of 41.8) to thermophilic carboxylesterase Est30 from G. stearothermophilus (PDB code 1TQH [7]), which has hydrolysis activity even at 70 • C. In addition, the carboxylesterase from Bacillus stearothermophilus (PDB code 1R1D), lipase from the goat rumen metagenome (PDB code 4DIU), and monoglyceride lipase from Bacillus sp. H257 (PDB codes 4KE6 [19] and 3RLI [20]) showed significant structural similarities to the EaEst2 structure (Table 1). Structural information can also be used for further protein engineering of EaEst2. In addition, a structural comparison of EaEst2 and Est30 may provide useful insights into their different temperature-dependent activities and stabilities.  Structural homolog search using the DALI server showed that EaEst2 had the highest structural similarity (Z-core of 41.8) to thermophilic carboxylesterase Est30 from G.stearothermophilus (PDB code 1TQH [7]), which has hydrolysis activity even at 70 °C. In addition, the carboxylesterase from Bacillus stearothermophilus (PDB code 1R1D), lipase from the goat rumen metagenome (PDB code 4DIU), and monoglyceride lipase from Bacillus sp. H257 (PDB codes 4KE6 [19] and 3RLI [20]) showed significant structural similarities to the EaEst2 structure (Table 1). Structural information can also be used for further protein engineering of EaEst2. In addition, a structural comparison of EaEst2 and Est30 may provide useful insights into their different temperature-dependent activities and stabilities.

BHET Hydrolysis Activity of EaEst2
Notably, we found that EaEst2 had strong activity in BHET hydrolysis. HPLC analysis showed a decrease in peak height for BHET after enzymatic reaction with EaEst2. The newly generated peak at 3.17 min of retention time was considered MHET, regarded as a degradation intermediate since the polarity of the eluted compound was higher than that for BHET and lower than that for the TPA molecule (Figure 7). In addition, subsequent MHET degradation by EaEst2 was not observed. Collectively, the EaEst2 enzyme can degrade BHET into MHET by cleavage of the ester bond and cannot utilize MHET as a substrate. Previous studies showed that MHET released during PET degradation is a possible PET degrading enzyme inhibitor [14,[22][23][24]. This implied that MHET-degrading enzymes are needed to complete the decomposition of PET into TPA and ethylene glycol (EG). Thus, it is possible to utilize EaEst2 enzymes in PET degradation research or industries that need to degrade BHET specifically. sis showed a decrease in peak height for BHET after enzymatic reaction with EaEst2. The newly generated peak at 3.17 min of retention time was considered MHET, regarded as a degradation intermediate since the polarity of the eluted compound was higher than that for BHET and lower than that for the TPA molecule (Figure 7). In addition, subsequent MHET degradation by EaEst2 was not observed. Collectively, the EaEst2 enzyme can degrade BHET into MHET by cleavage of the ester bond and cannot utilize MHET as a substrate. Previous studies showed that MHET released during PET degradation is a possible PET degrading enzyme inhibitor [14,[22][23][24]. This implied that MHET-degrading enzymes are needed to complete the decomposition of PET into TPA and ethylene glycol (EG). Thus, it is possible to utilize EaEst2 enzymes in PET degradation research or industries that need to degrade BHET specifically.

Immobilization of EaEst2
Enzyme immobilization is an effective strategy to improve the stability and recyclability of free enzymes. The immobilization of EaEst2 has been characterized for biotechnological and industrial applications. EaEst2 was immobilized as a crosslinked enzyme aggregate (CLEA) by precipitation with ammonium sulfate and crosslinking with glutaraldehyde. Scanning electron microscopy (SEM) images of the CLEAs show the morphological structure of the amorphous aggregate of EaEst2 ( Figure 8A). Figure 8B shows the reusability of the immobilized EaEst2 after p-NA hydrolysis. Immobilized EaEst2 retained more than 100% of its initial activity after nine reutilization cycles. The thermostability of soluble and immobilized EaEst2 was determined by incubation at 50 °C for various intervals. Notably, immobilized EaEst2 exhibited significantly enhanced activity and stability. Approximately −70% of its initial activity was retained after exposure to 50 °C for 60 min. However, all of its soluble enzymatic activity was lost after only 15 min ( Figure 8C). In

Immobilization of EaEst2
Enzyme immobilization is an effective strategy to improve the stability and recyclability of free enzymes. The immobilization of EaEst2 has been characterized for biotechnological and industrial applications. EaEst2 was immobilized as a crosslinked enzyme aggregate (CLEA) by precipitation with ammonium sulfate and crosslinking with glutaraldehyde. Scanning electron microscopy (SEM) images of the CLEAs show the morphological structure of the amorphous aggregate of EaEst2 ( Figure 8A). Figure 8B shows the reusability of the immobilized EaEst2 after p-NA hydrolysis. Immobilized EaEst2 retained more than 100% of its initial activity after nine reutilization cycles. The thermostability of soluble and immobilized EaEst2 was determined by incubation at 50 • C for various intervals. Notably, immobilized EaEst2 exhibited significantly enhanced activity and stability. Approximately −70% of its initial activity was retained after exposure to 50 • C for 60 min. However, all of its soluble enzymatic activity was lost after only 15 min ( Figure 8C). In addition, the chemical stability of the immobilized EaEst2 was higher than that of the soluble enzyme, especially in 30% EtOH conditions ( Figure 8D). These results suggest that immobilized EaEst2 can be effectively reutilized for potential industrial applications. addition, the chemical stability of the immobilized EaEst2 was higher than that of the soluble enzyme, especially in 30% EtOH conditions ( Figure 8D). These results suggest that immobilized EaEst2 can be effectively reutilized for potential industrial applications.

Expression and Purification of Recombinant EaEst2
The gene encoding carboxylesterase in the E. antarcticum genome was cloned into the pET-28a vector using NheI and XhoI restriction enzymes and then heterologously expressed in Escherichia coli BL21 (DE3) cells with the 6×-histidine tag at the N-terminal of the EaEst2 amino acid sequence. The recombinant E. coli cells containing EaEst2 were cultured in Luria-Bertani (LB) medium with kanamycin (50 µg/mL) to an OD600 of 0.6 at 37 °C. Subsequent culture was performed by adding 1 mM of isopropyl-β-D-1-thiogalactoside (IPTG) for 4 h. Cell pellets were then collected via centrifugation at 2000× g rpm for 15 min, after which the cells were disrupted by sonication in a cell lysis buffer (5 mM imidazole, 20 mM Tris-HCl, 200 mM NaCl, pH 7.5). Thereafter, the supernatant was separated by centrifugation at 16,000× g rpm for 40 min and subsequently loaded onto a HisTrap column (GE Healthcare, Chalfont Saint Giles, UK) for purification. The recombinant EaEst2 protein was subsequently eluted using a high-concentration imidazole buffer (300 mM imidazole, 20 mM Tris-HCl, 200 mM NaCl, pH 7.5), and the pooled sections were transferred into the final buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.5).

Expression and Purification of Recombinant EaEst2
The gene encoding carboxylesterase in the E. antarcticum genome was cloned into the pET-28a vector using NheI and XhoI restriction enzymes and then heterologously expressed in Escherichia coli BL21 (DE3) cells with the 6×-histidine tag at the N-terminal of the EaEst2 amino acid sequence. The recombinant E. coli cells containing EaEst2 were cultured in Luria-Bertani (LB) medium with kanamycin (50 µg/mL) to an OD 600 of 0.6 at 37 • C. Subsequent culture was performed by adding 1 mM of isopropyl-β-D-1-thiogalactoside (IPTG) for 4 h. Cell pellets were then collected via centrifugation at 2000× g rpm for 15 min, after which the cells were disrupted by sonication in a cell lysis buffer (5 mM imidazole, 20 mM Tris-HCl, 200 mM NaCl, pH 7.5). Thereafter, the supernatant was separated by centrifugation at 16,000× g rpm for 40 min and subsequently loaded onto a HisTrap column (GE Healthcare, Chalfont Saint Giles, UK) for purification. The recombinant EaEst2 protein was subsequently eluted using a high-concentration imidazole buffer (300 mM imidazole, 20 mM Tris-HCl, 200 mM NaCl, pH 7.5), and the pooled sections were transferred into the final buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.5).
The chemical stability of EaEst2 was investigated using p-NA substrate in buffers containing diverse chemicals. The residual activity of EaEst2 was measured by spectrophotometric assay after incubating the enzyme for 1 h under diverse conditions, including 10 and 30% ethanol (EtOH), 0.2% sodium dodecyl sulfate (SDS), 1% Tween 20 (Tw20), 1% Triton X-100 (Tx-100), 30% isopropanol (I-PrOH), and 5 M urea. The enzymatic activity against p-NA (C2) in buffer alone was defined as 100% of relative activity. All experiments were performed in triplicate, and the standard deviations were calculated and presented in the figures.

Size-Exclusion Chromatography (SEC)
To investigate the functional unit in the aqueous state, size-exclusion chromatography (SEC) was performed using a Superdex 200 10/100 GL column (Cytiva, Marlborough, MA, USA). The protein standard mix used to generate standard and correlation graphs included bovine thyroglobulin (approximately 670 kDa), g-globulins from bovine blood (approximately 150 kDa), chicken egg grade VI albumin (approximately 44.3 kDa), and ribonuclease A type I-A from the bovine pancreas (approximately 15 kDa). The protein standard mix and EaEst2 were eluted with 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl buffer at a 0.5 flow rate.

Determination of Thermal Stability
To assess the effect of temperature changes on EaEst2 activity, activity was measured by incubating the enzyme at 0, 15, 25, 37, 50, and 60 • C for 1 h. Each aliquot was obtained every 15 min to measure the residual activity against p-nitrophenyl acetate (C2), as described above. The structural denaturation profile was recorded using CD. The secondary structure and thermal unfolding of EaEst2 were evaluated by scanning at 190-280 nm from 5 to 95 • C. Melting temperature (T m ) was calculated using the molar ellipticity value at 222 nm from the thermal denaturation profile.

Crystallization of EaEst2
Preliminary crystallization screening of EaEst2 was performed in a 96-well plate using the sitting-drop vapor-diffusion method. Commercial crystallization suites MCSG I-IV (Anatrace, Maumee, OH, USA), PGA Screen (Molecular Dimensions, Catcliffe, UK), and a customized suite SGC were used for the initial screening. In the reservoir of each well, 80 µL of crystallization solution was aliquoted, and 400 nL of the purified protein (8 mg/mL) and an equal volume of reservoir solution were mixed in each subwell of the plates using an automatic liquid handling robot (Mosquito; SPT Labtech, Melbourn, UK). The crystal of native EaEst2 was grown using a 0.1 M HEPES: NaOH (pH 7.5) and 20% (w/v) PEG 8000 mixed solution, and a single crystal appeared after 1-month incubation at 296 K. The obtained crystals were used directly for X-ray diffraction experiments without further optimization.

X-ray Diffraction Data Collection and Structure Determination
A single crystal of EaEst2 was transferred into a cryoprotectant, a mixture of reservoir solution and glycerol, such that the final concentration of glycerol was 25%. The crystal was then placed on a goniometer with a 100 K liquid nitrogen stream at the synchrotron beamline 5C (BL-5C) of the Pohang Accelerator Laboratory (PAL, Pohang, Republic of Korea). Native diffraction data were successfully collected using 360 images rotating at oscillations of 1 • per frame on an Eiger 9M detector (Dectris, Baden, Switzerland). HKL-2000 software (HKL Research Inc., Charlottesville, VA, USA) [25] was used for data processing, indexing, integration, and scaling. The crystal structure of EaEst2 was determined to be P2 1 2 1 2 1 with unit cell parameters of a = 50.362 Å, b = 67.789 Å, c = 89.617 Å, and α = β = γ = 90 • . In addition, the crystal structure was considered an EaEst2 monomer structure in an asymmetric unit because the Matthews coefficient value was calculated as 2.76 Å 3 Da −1 with 55.5% solvent content [26]. The phasing and generation of electron-density maps were carried out in CCP4 [27] using the program MOLREP [28]. The structure of the carboxylesterase Est30 (PDB code: 1TQH) was used as a template for molecular replacement. Iterative refinement was performed with additional manual model building in Coot [29] and Phenix refinement [30] using the Phenix package [31]. The final model was validated using Mol-Probity [32] and deposited in the Protein Data Bank (PDB) under the accession code 8HEA. The X-ray diffraction and refinement statistics are presented in Table 2 and all structural figures were generated by PyMOL [33]. Table 2. X-ray diffraction data collection and refinement statistics.

BHET Hydrolysis Activity
To verify whether EaEst2 has activity against bis(2-hydroxyethyl) terephthalate (BHET), a 500 mM BHET stock solution was prepared by dissolving it in dimethyl sulfoxide (DMSO). Terephthalic acid (TPA), which is known as one of the final products of BHET, was equally prepared to generate standard HPLC data. The enzyme activity assay was performed in a reaction buffer (100 mM phosphate-NaOH and 100 mM NaCl) at pH 7.5 with 2.5 mM (final concentration) of BHET. The enzyme reaction was initiated by the addition of 100 µg enzyme and incubated at 37 • C for 1 h. The reaction was terminated by adding the same volume of acetonitrile, which was then injected into an Eclipse plus C18 reverse-phase column (150 mm × 4.6 mm, 3.5 µm; Agilent Technologies, Santa Clara, CA, USA) for HPLC analysis.

Immobilization of EaEst2
To prepare the CLEAs of EaEst2, a purified EaEst2 (0.5 mg) was incubated in phosphate buffer (pH 7.5) with 80% ammonium sulfate and 50 mM glutaraldehyde with gentle inverting for 12 h. Thereafter, the suspension was centrifuged at 13,000× g rpm at 4 • C for 30 min, and the resulting immobilized EaEst2 was washed thrice with 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. Immobilized EaEst2 activity was monitored by measuring the hydrolysis of p-nitrophenyl acetate (C2, p-NA). The thermal stability of immobilized EaEst2 was investigated at 80 • C, and the activity of soluble EaEst2 was set to 100%. To examine its reusability, immobilized EaEst2 was retrieved by simple centrifugation after each reaction. After repeated washing steps (usually thrice), a new substrate was added for another cycle, and the activity of immobilized EaEst2 was measured. For SEM analysis of CLEAs-EaEst2, CLEAs were fixed by 1% osmium tetraoxide in a 50 mM sodium cacodylate buffer (pH 7.2) for 2 h at 4 • C. Fixed CLEAs were then dehydrated using 30, 50, 70, 80, 90, and three times of a 100% ethanol series for 10 min each at 25 • C. Dehydrated CLEAs were then incubated in 100% hexamethyldisilazane for 10 min and further dried in an oven for a minimum of 16 h. The samples were mounted onto metal stubs and sputtered with platinum. SEM analysis was performed using a Carl Zeiss SUPRA 55VP instrument (Carl Zeiss, Oberkochen, Germany).

Conclusions
In conclusion, these biochemical activity assays revealed the broad substrate specificity of on various molecules containing ester groups such as p-nitrophenyl acetate (C2), mannose pentaacetate (Man-Ac5) glyceryl tributyrate (GTB), and (S)-Roche esters. Additionally, for the chemical stability of EaEst2, the increased activity to 180 and 160% of the initial activity was examined with 10 and 30% ethanol, respectively. Notably, EaEst2 showed degradation activity on BHET into MHET as one of the PET decomposition steps. In HPLC analysis, EaEst2 is specifically active on BHET and cannot utilize MHET as a substrate. Furthermore, immobilization and reusability for EaEst2 showed the potential of EaEst2 for industrial application. More specifically, the EaEst2 enzyme can be used to disassemble fine chemicals with ester bonds and decompose environmental pollutants. Collectively, these findings may be useful for the development and modification of new BHET-degrading enzymes using previously known esterase enzymes.