Structural and functional characterization of a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina, a psychrophilic bacterium

Background S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. Results A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. Conclusions Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure–function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH. Electronic supplementary material The online version of this article (10.1186/s12934-019-1190-1) contains supplementary material, which is available to authorized users.


Background
Esterases hydrolyze ester bonds and produce cleaved acid and alcohol compounds. Although they have low sequence identity and diverse substrate specificities as well as biological functions, most esterases adopt a common α/β hydrolase fold and contain a highly conserved serine-histidine-aspartic acid catalytic triad at the active site [1][2][3]. S-formylglutathione hydrolase (SFGH, EC 3.1.2.12) is a member of the esterase family and is also known as esterase D. SFGH hydrolyzes S-formylglutathione into glutathione and formic acid and can be broadly considered an esterase, or more specifically, a thioesterase. This enzyme is involved in the glutathionedependent formaldehyde detoxification pathway, which prevents the negative effects of formaldehyde by a series of enzymatic reactions involving formaldehyde dehydrogenase (FALDH, S-hydroxymethylglutathione dehydrogenase) and SFGH, which depend upon the spontaneous binding of glutathione to formaldehyde. Indeed, the expression levels of FALDH (frmA) and SFGH (frmB) in Escherichia coli cells are remarkably increased by 20to 100-fold over basal levels when subjected to formaldehyde treatment [4]. Notably, a double-mutant strain of E. coli with the deletion of both frmB and paralogous yeiG genes, which all encode SFGH, showed a remarkably reduced growth rate in the presence of formaldehyde [5]. Likewise, a mutant strain of Paracoccus denitrificans for the SFGH gene (fghA) was unable to grow on methanol or methylamine, which resulted in the formation of formaldehyde [6]. Considering the importance of this pathway, SFGH genes are widely conserved in diverse organisms, including prokaryotes and eukaryotes. In humans, esterase D, which was subsequently proven to be identical to SFGH, has been identified, characterized, and structurally determined. This enzyme has attracted much attention as its polymorphisms are related to several diseases, including retinoblastoma and Wilson's disease [7][8][9]. In addition, SFGHs were found in other eukaryotes including plants (Arabidopsis thaliana [10,11] and Pisum sativum [12]) and yeast (e.g., Saccharomyces cerevisiae [13] and Candida boidinii [14]). Bacterial SFGHs were also identified in various species including Paracoccus denitrificans [6], Agrobacterium tumefaciens [15], and E. coli [5].
As mentioned above, many studies on SFGHs have been conducted, which have revealed the features of SFGHs. Sequence alignment analyses have shown that SFGH has a conserved sequence motif (GHSMGG) that harbors catalytic serine; thus, this enzyme is also referred to as a serine hydrolase. This serine residue, along with the conserved aspartate and histidine residues, forms a catalytic triad of SFGH. Two conserved residues of leucine and methionine that form an oxyanion hole are also found in close proximity to the catalytic serine. Furthermore, enzymes belonging to the SFGH class generally contain five large blocks of conserved residues [5]. Functional characterization studies demonstrated that SFGH could also exhibit carboxyl esterase activity towards xenobiotic esters (e.g., methylumbelliferyl acetate) in addition to having thioesterase activity [10,11,13]. Many SFGHs are known to be active towards p-nitrophenyl esters or naphthyl ester derivatives as well as formyl glutathione.
In this study, we characterized a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from a psychrophilic bacterium, Shewanella frigidimarina. Shewanella frigidimarina is a facultative anaerobic Gram-negative bacterium with rod-like shape, originally isolated from Antarctic marine environments. To date, only two studies have characterized SFGH from Arctic/ Antarctic organisms: OLEI01171 from Oleispira antarctica [16] and PhEST from Pseudoalteromonas haloplanktis [17,18]. However, the structural and functional properties of S-formylglutathione hydrolase from psychrophiles are still poorly understood. In this context, our study will enrich the pool of SFGH, providing useful information regarding the sequence, biophysical, and enzymatic characteristics of SFGH. Furthermore, the crystal structure of SfSFGH has been determined at 2.32 Å resolution. Several substrate-binding site residue mutants have been generated to alter the substrate specificity and flexibility of SfSFGH. Interestingly, the W182A mutant showed remarkably increased substrate flexibility compared to wild-type SfSFGH. Collectively, these findings provide useful insights to inform the protein engineering of bacterial esterases as industrially useful biocatalysts.

Biochemical characterization of SfSFGH
The sequence of SfSFGH (GenBank I.D.: ABI73260.1) was previously annotated as a carboxylesterase with little additional information. Sequence alignment of SfS-FGH with similar sequences retrieved from PSI-BLAST indicates that these enzymes share highly conserved sequences, including a 'GHSMGG' motif. These enzymes are now classified as a PF00756 esterase family based on sequence homology [15,[19][20][21]. As the 'GHSMGG' motif is a representative feature of the esterase/lipase family V, a phylogenetic tree was generated containing SfSFGH with other enzymes belonging to the esterase/lipase family from I to VIII. As expected, SfSFGH was classified into the esterase/lipase family V and showed a clear separation from other families (Additional file 1: Fig. S1). Furthermore, it is generally known that the 'PAL' motif is one of the representative features of the esterase/lipase family V [22]. Together with SfSFGH, other PF00756 esterases, such as OLEI01171 and PhEst, have the 'PAL' and 'PML' motifs, respectively. Collectively, sequence analysis data suggest that SfSFGH might be a new member of the esterase/lipase family V. In addition, SfSFGH shows significant sequence homology with other bacterial SFGHs (66.43% sequence identity with Pseudoalteromonas haloplanktis TAC125 SFGH, 54.48% with Paracoccus denitrificans SFGH, 50.91% with Agrobacterium tumefaciens SFGH, and 67.93% with E. coli SFGH). We also performed gene cluster analysis of SfSFGH, and found that gene clusters surrounding the SFGH gene were highly conserved in Shewanella species, including S. sediminis, S. woodyi, S. pealeana, and S. piezotolerans (Additional file 1: Fig. S2).
Recombinant SfSFGH protein was expressed in E. coli and purified by His-tag affinity chromatography and size-exclusion chromatography (Additional file 1: Fig.  S3). Analytical ultracentrifugation (AUC) analysis with SfSFGH showed a dimer mass of 62.3 kDa (sedimentation coefficient, 4.209 S; frictional ratio, 1.34). The substrate specificity of recombinant SfSFGH protein was determined using p-nitrophenyl esters with different acyl-chain lengths. As shown in Fig. 1a, SfSFGH showed a preference toward short-chain substrates, including p-NA, p-NB, p-NH, and p-NH, with more than 50% of its maximum activity. However, less than 15% of relative enzymatic activities were detected towards longer-chain substrates, such as p-ND and p-NDD, and almost no enzymatic activity was observed with p-NP. The optimum pH for SfSFGH was determined to be in the range of pH 3.0 to 10.0 (Fig. 1b). SfSFGH showed maximum activity at pH 8.0 and ~ 40% activity at pH 7.0. In contrast, SfS-FGH exhibited almost no enzymatic activity in other pH ranges. The kinetic parameters of SfSFGH were determined using p-NA. SfSFGH exhibited typical Michaelis-Menten kinetics with a hyperbolic plot of reaction velocity as a function of substrate concentration. Based on the Lineweaver-Burk plot with linear regression, the Michaelis-Menten constant (K m ) and maximum velocity (V max ) were 515 µM (± 42.8) and 0.319 µM s −1 (± 0.0254), respectively (Fig. 1c). The calculated k cat of SfSFGH was 0.310 s −1 (± 0.0258), which is almost six times less than that of AtuSFGH and PhEST (1.95 s −1 and 2 s −1 , respectively) [15,17]. Notably, enzymatic activity assay results for several different temperatures showed that SfSFGH has high activity at low temperature (4 °C), but starts to lose activity at ~ 37 °C (Fig. 2).

Overall structure of SfSFGH
The crystal structure of SfSFGH was determined to a resolution of 2.32 Å using a molecular replacement method (Fig. 3). The crystal structure of esterase (OLEI01171) from Oleispira antarctica (sequence identities 83%; PDB cod 3i6y) was used as a template model [16]. SfSFGH was crystallized in space group P2 1 with two monomers in the asymmetric unit. The final model contained 208 water molecules and 554 amino acids. The monomeric structure of SfSFGH includes 12 α-helices and 9 β-strands (Fig. 3a). The β1-β2-β3 strands form an antiparallel β-sheet, and the β4 to β9 strands form a parallel β-sheet. These two β-sheets are linked and assemble into a large central β-sheet. The 12 α-helices surround both sides of the large β-sheet. These features of the overall architecture show considerable similarity with those of the previously reported S-formylglutathione hydrolases (SFGHs). Structural alignments using a Dali search showed that the esterase (OLEI01171) from Oleispira Antarctica (PDB code 3i6y) has the highest Z-score with 50.5 [16,23]. Crystal structures of S-formylglutathione hydrolase from Pseudoalteromonas haloplanktis (PDB code 3ls2), esterase D from Neisseria meningitidis (PDB code 4b6g), and S-formylglutathione hydrolase from Agrobacterium fabrum (PDB code 3e4d) also showed high structural similarities (Table 1) [15,18,24].

Active site of SfSFGH
The putative substrate-binding site of SfSFGH is located at the end of the β6-strand. Including the α2, α8, and α12 helices, the β4-α2 loop, β8-α11 loop, and β9-α12 loop regions together form a groove architecture. The conserved catalytic triad residues of Ser148, Asp224, and His257 are located in this groove (Fig. 4). The catalytic residue Ser148 is located in the short β6-α6 loop region and directly interacts with His257 located in the β9-α12 loop region. Similarly, the residue Asp224 located on the β8-α11 loop region interacts with His257. In addition, the oxyanion hole comprising the backbone amides Leu55 and Met149 can be observed at the neighboring active site. In the apo state of the SfSFGH structure, oxyanion holes are occupied by water molecules. The vicinity of the substrate-binding site is positively charged by the Lys65 and His147. This positively charged area might induce binding of an acidic substrate, such as an acyl group. Moreover, the residues Cys57, Asn61, Trp182, and Phe226 are conserved in the substrate-binding site of SfS-FGH. Especially in SFGH from Saccharomyces cerevisiae, the W197I mutant (Trp182 of SfSFGH) showed highly increased substrate affinities [25]. Likewise, mutation of Trp182 to alanine in SfSFGH affected its activity. Detailed data for the W182A mutant are provided in the next section.

Site-directed mutagenesis to increase the substrate-binding pocket size of SfSFGH
Based on this structural information, we hypothesized that remodeling the substrate-binding pockets of SfS-FGH would allow the enzyme to exhibit broadened substrate specificity. Therefore, several mutants targeting the substrate-binding pocket residues (L55A, L55V, H147A, W182A, and F226A) were designed, constructed, purified, and characterized with respect to their activity (Fig. 5a). Interestingly, the W182A mutant showed substantially increased activity for large and bulky substrates compared to wild-type SfSFGH. Notably, the W182A mutant showed a substantially altered preference for longer substrates (p-NB and p-NH), unlike wild-type SfSFGH (Fig. 5a, b). Likewise, the K m values of W182A for p-NA, p-NB, and p-NO were 1.393 (± 0.136), 0.303 (± 0.049), and 0.459 (± 0.085), respectively. The calculated catalytic efficiency (k cat /K m ) of W182A for p-NB (26.4 s −1 mM −1 ) was 20 times greater than that for p-NA Fig. 2 Effects of temperature on the activity of SfSFGH. a Thermal stability of SfSFGH. SfSFGH was heated at various temperatures, ranging from 25 to 70 °C. Then, 10 μg SfSFGH was collected every 15 min and the residual activity was measured using p-NA as a substrate. Activity is shown relative to the initial activity. b Enzymatic activity of SfSFGH was evaluated at different temperatures. Relative activity of SfSFGH was measured using pNP-C 2 as a substrate after 1 h incubation at each temperature. A two-tailed unpaired Student t test was used to evaluate statistical significance: *P < 0.05 and **P < 0.005. All experiments were performed in triplicate (See figure on next page.) Fig. 3 Crystal structure of SfSFGH. a The overall structure of SfSFGH is shown as a ribbon diagram with α-helices (aquamarine color) and β-strands (salmon color). The catalytic triad residues (Ser148, Asp24, and His257) are shown as stick models. b Multiple sequence alignments of SfSFGH (NCBI reference sequence number WP_011638859.1) with homologous models. The sequences of esterases from the oil-degrading bacterium Oleispira antarctica (PDB code 3i6y; UniProtKB code D0VWZ4), S-formylglutathione hydrolase from Pseudoalteromonas haloplanktis (PDB code 3ls2; UniProtKB code Q3IL66), S-formylglutathione hydrolase from Agrobacterium tumefaciens (PDB code 3e4d; UniProtKB code A9CJ11), esterase D from humans (PDB code 3fcx; UniProtKB code P10768), esterase D from Neisseria meningitides (PDB code 4b6g; UniProtKB code Q9JZ43), and S-formylglutathione hydrolase from Saccharomyces cerevisiae (PDB code 1pv1; UniProtKB code P40363) were used for alignment. The conserved motif found in SFGHs is boxed in red. The catalytic triad residues indicated with a black triangle are conserved in all models. The gatekeeper residue of tryptophan is indicated with a black circle. An alignment was prepared using the program ClustalX and edited with GeneDoc. Corresponding secondary structures are shown above the sequences (1.3 s −1 mM −1 ) ( Table 2). In addition, the W182A mutant also showed acetylation activity towards α-d-glucose penta-acetate whereas the wild-type did not. It is thought that the W182A mutation probably eliminates the steric hindrance of substrate entry and increases the size of the substrate-binding pocket by removing the bulky side chain of Trp182 (Fig. 5c, d). However, other mutations were not effective for changing the substrate specificity and improving the activity of SfSFGH. Therefore, the Trp182 residue is important for substrate selectivity and discrimination in SfSFGH. In conclusion, the W182A mutant of SfSFGH has different substrate specificities and unique biochemical characteristics. Thus, the engineered W182A mutant may be useful for biotechnological fields, such as fine chemical synthesis and industrial pharmaceutics.

Structural stability of SfSFGH
The structural stability of SfSFGH was investigated via thermal and chemical unfolding with circular dichroism and intrinsic fluorescence analyses, respectively. For thermal unfolding, CD signals of SfSFGH were monitored at 222 nm, at temperatures from 20 to 90 °C. In these Far-UV CD measurements, SfSFGH showed a remarkable structural transition at around 45 °C (Fig. 6a), which is consistent with the results of the enzymatic activity assays (Fig. 2a). In the case of OLEI01171, another coldactive esterase, enzymatic activity decreased when the temperature increased, and the aggregation temperature is similar to that of SfSFGH (~ 45 °C) [16]. Structural superposition analysis shows how SfSFGH remains catalytically active at low temperatures. SfSFGH has a more open active site compared with mesophilic homologs (human esterase D, PDB code 3FCX, and Saccharomyces cerevisiae SFGH, PDB code 4FLM) [9,38]. Especially, the β5-α4 loop region (residues 83-112) in SfSFGH has a significantly different conformation with a relatively higher main chain B-factor value (27.9 Å 2 ) than overall B-factor value (24.7 Å 2 ) (Additional file 1: Fig. S4). Our sequence alignment results also show that only cold-active enzymes have a conserved glycine residue in this β5-α4 loop region (Gly96 in SfSFGH and Gly96 in OLEI01171). Collectively, it is thought that the flexibility of the β5-α4 loop region and the open active site are related to the cold-activity of SfSFGH.
Next, chemical unfolding of SfSFGH was monitored based on changes in intrinsic fluorescence with different urea concentrations (from 0 to 5 M). As shown in Fig. 6b, native SfSFGH exhibited a λ max of 341 nm in the absence of urea, and a red shift of λ max to 367 nm was observed in the presence of 5 M urea. Fluorescence intensity was significantly decreased from 1 M urea, and further reduction was nearly proportional to the increase in urea concentration.
In the chemical stability test, SfSFGH showed remarkable residual enzymatic activity in the presence of diverse chemicals (Fig. 6c-e). With organic solvents, SfSFGH could retain ~ 76%, ~ 51%, and ~ 43% of its original activity with 10% EtOH, 30% EtOH, and 30% I-PrOH, respectively. For detergents, SfSFGH exhibited more than 50% of its initial activity with 1% Triton X-100 or SDS up to 1%, although almost no enzymatic activity was observed with 1% Tween-20. Higher SDS concentrations of 2% and 5% decreased the enzymatic activity to 30% and 15% of its original activity, respectively, rather than inducing complete inhibition of enzymatic activity. Notably, SfSFGH incubated with 5 mM PMSF, a serine hydrolase inhibitor, retained ~ 36% of its original activity. To verify the resistance of SfSFGH to hydrogen-bond-disturbing conditions, SfSFGH activity was also examined with urea concentrations ranging from 0 to 5 M. The enzymatic activity of SfSFGH was found to be gradually reduced as the concentration of urea increased. Interestingly, SfSFGH retained more than ~ 50% of its initial activity at all concentrations. Furthermore, SfSFGH maintained its activity (≥ 80%) even after 10 repeated cycles of freezing and thawing (Additional file 1: Fig. S5). Taken together, SfSFGH appears to have notable thermal and chemical stability. Notably, SfSFGH was strongly resistant to organic solvents and detergents such as EtOH, i-PrOH, Triton X-100, and SDS, suggesting that it could be useful in industrial applications.

Conclusion
In this study, a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina was characterized. Regarding enzymatic activity, SfSFGH was found to prefer short-chain substrates and weakly basic pH conditions. A single residue mutation (W182A) changed the substrate preference of SfSFGH to longer-chain substrates. This finding provides valuable information for further protein engineering of SfSFGH to obtain more suitable substrate-binding sites. Remarkably, SfSFGH appears to retain its enzymatic activity in the presence of inhibitory chemicals. These properties of SfSFGH could be very useful in industrial applications that generally require enzymes to show robust activity in harsh conditions.

Protein expression and purification
Shewanella frigidimarina (KCTC 22881, Korean Collection for Type Cultures (WDCM 597), Seoul, Korea) The conserved catalytic triad (Ser148, Asp24, and His257) residues are located on the substrate-binding site. The residues creating a substrate-binding site are presented as a stick model. c Dimerization of SfSFGH is mediated by the α2, α3, and α12 helices. The β1-strand also participates in the dimerization interaction was cultured in marine medium 2216 (BD Difco, USA). Chromosomal DNA was extracted and purified using a DNeasy Tissue and Blood Kit (Qiagen, USA) according to the manufacturer's instructions. The open reading frame of the SfSFGH gene was amplified using polymerase chain reaction with the following primers: forward, 5′-CCT GCT AGC ATG ACA ATT GAA AAT ATG AG-3′; reverse, 5′-CTAA CTC GAG CTT TCC CAG ATG CTC-3′. The PCR product was inserted into a pET21a vector, and pET21a-SfSFGH was cloned into the E. coli BL21(DE3) stain. Transformed cells were grown in LB medium containing 100 μg mL −1 ampicillin at 37 °C until the OD600 reached 0.5. Then, isopropyl-β-d-1-thiogalactoside (IPTG) was added to a final concentration of 1 mM and the cells were incubated for 3 h to overexpress SfSFGH. Effects of mutation on SfSFGH enzymatic activity. a Comparison of relative activities between wild-type SfSFGH and several point mutants (L55A, L55 V, H147A, W182A, and F226A). All mutants are located on the substrate-binding pocket. Assays were carried out using various substrates (p-NA, p-NB, p-NH, p-NO, p-NDe, p-NDo, and p-NPP). The relative activity of wild-type SfSFGH with p-NA was set to 100%. b Increased enzymatic activity for α-NB in the W182A mutant was observed compared with that of wild-type SfSFGH. The relative activity of wild-type SfSFGH with α-NA was set to 100%. c The surface of the substrate-binding site of SfSFGH is colored grey. The catalytic triad residues (Ser148, Asp224, and His257; salmon) and Trp182 (cyan) are presented as a stick model. d Hydrolytic activities towards α-d-glucose penta-acetate, cellulose acetate, and N-acetyl-d-glucosamine. All experiments were performed in triplicate The cells were harvested by centrifugation at 5000 rpm for 20 min at 4 °C. The resulting pellet was then resuspended in binding buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0), followed by sonication. The lysate was centrifuged at 40,000×g for 10 min, and the supernatant was applied to a 1 mL HisTrap FF column under the AKTA Start system (GE Healthcare).
After an extensive washing step with binding buffer, bound SfSFGH was eluted with elution buffer (20 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0). The purified SfSFGH was desalted using a PD-10 column (GE Healthcare) with an assay buffer (20 mM Tris-HCl, 200 mM NaCl, pH 8.0). The molecular mass of SfS-FGH in the native state was estimated using size exclusion chromatography with a Sephacryl S200 HR column (GE healthcare) in an AKTA Start FPLC system (GE healthcare).

Crystallization and data collection of SfSFGH
Initial crystallization screening of SfSFGH was performed using the sitting-drop vapor-diffusion method at 293 K in 96-well crystallization plates (Emerald Bio, Bainbridge processed and scaled using HKL-2000 [26]. Data collection and processing statistics are shown in Table 3.

Structure determination
The SfSFGH crystal belongs to the monoclinic space group of P2 1 , with unit cell parameters of a = 49.4, b = 76.6, c = 64.7 Å, α = γ = 90, and β = 105.3°. The crystal structure in SfSFGH was solved by a molecular replacement method using the MOLREP program from the CCP4i package [27,28]. The crystal structure of esterase (OLEI01171) from Oleispira Antarctica (sequence identity 83%; PDB code 3i6y) was used as a template model [16]. The initial model was built iteratively and then refined using Coot and REFMAC5 [29,30]. The phenix.refine from PHENIX was also used for refinement [31]. The final model had an R work of 17.6% and R free of 23.5%. Molprobity was used for quality validation of the final model [32]. The detailed refinement statistics are provided in Table 3. The atomic coordinates and experimental structure factors of SfS-FGH were deposited in the RCSB Protein Data Bank under accession code 6JZL. All graphical representations of protein structures were prepared using PyMOL [33].

Mass spectrometry and analytical ultracentrifugation
The molecular weight of SfSFGH-His 6 was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF) using Voyager DE STR (Applied Biosystems, NCIRF, Seoul, Korea). To investigate the oligomeric state of SfSFGH in solution, analytical ultracentrifugation (AUC) was performed using a ProteomeLab XL-A (Beckman Coulter, Brea, CA, USA), in buffer conditions of 150 mM NaCl and 20 mM Tris-HCl pH 8.0 at 20 °C. The sample was centrifuged at 40,000 rpm for 10 min and the sedimentation profile was monitored at a wavelength of 280 nm. Data were analyzed using the program SEDFIT [34,35].

Enzymatic assays
In general, the carboxyl esterase activity of SfSFGH was assessed against p-nitrophenyl esters by measuring absorbance at 405 nm using an EPOCH2 microplate reader (Biotek). The standard assay solution consisted of 250 µM substrate solution in 100 mM NaCl and 20 mM Tris-HCl pH 8.0 with 10 µg SfSFGH protein (at a final concentration of ~ 1.03 µM). The substrate specificity of SfSFGH was determined using p-nitrophenyl esters varying in acyl-chain length, e.g., p-nitrophenyl phosphate (p-NP), p-nitrophenyl acetate (p-NA), butyrate (p-NB), hexanoate (p-NH), octanoate (p-NO), decanoate (p-ND), and dodecanoate (p-NDD). The optimum pH of SfSFGH was determined using p-NA with buffers at different pH. Enzymatic activities that differed by pH were measured after incubating the enzyme for 1 h at various pH values ranging from 3.0 to 10.0. The thermal stability of SfSFGH was examined using p-NA. Activity changes by temperature were measured after incubating the enzyme at 25 °C, 37 °C, 50 °C, 60 °C, and 70 °C for 1 h. Aliquots were taken every 15 min to measure the residual activity of SfSFGH at each given temperature. To assess effect of low temperature on the activity of SfSFGH, activity was studied by equilibrating the enzyme at 4 °C. The activity was measured using p-NA as a substrate. The chemical stability of SfSFGH was determined using p-NA with buffers containing diverse chemicals. The residual activities of SfSFGH were measured after incubating the enzyme for 1 h in diverse conditions, such as 10% and 30% ethanol (EtOH), 30% isopropanol (I-PrOH), 0.1% sodium dodecyl sulfate (SDS), urea (from 0 M to 5 M), 1% Triton X-100 (Tx-100), 1% Tween 20 (Tw20), and 5 mM phenylmethylsulfonyl fluoride (PMSF). Measurement of enzymatic activity was performed in the same manner as the esterase activity assay described above.

Thermal and chemical unfolding
The thermal unfolding of SfSFGH was assessed using Far-UV CD measurements on a J-715 spectropolarimeter (JASCO) equipped with a thermostat-containing cell holder. The CD signal was monitored at 222 nm from 20 to 90 °C at a rate of 1.0 °C min −1 . Chemical unfolding of SfSFGH was induced by incubating the protein with increasing concentrations of urea (from 0 to 5 M) at room temperature for 1 h. Fluorescence measurements were performed using a FP-6200 spectrofluorometer (JASCO) in which the samples were excited at 280 nm and emission spectra were recorded in the range of 300 to 400 nm. All fluorescence spectra were recorded at a scan speed of 250 nm min −1 with a 5-nm slit width.

Determination of kinetic parameters
Kinetic assays were performed using p-NA, p-NB, and p-NO as substrates. Enzyme solutions were prepared in 120 mM NaCl and 20 mM Tris-HCl pH 8.0 buffer. Various concentrations of substrate were added to the enzyme solutions, and the reactions were performed at RT. The absorbance changes in the reaction mixtures were monitored using an EPOCH2 microplate reader (Biotek) at 405 nm. Measurements were taken along with a blank, every 10 s for 2 min, and at least three measurements were collected to determine the rate at each substrate concentration. The molar extinction coefficients of p-nitrophenol and hydrolyzed nitrocefin were determined experimentally (p-nitrophenol; 17,154 M −1 cm −1 ). Reaction velocities were determined from the initial linear portion of progress curves, which were generated after subtracting the absorbances of the blank experiments, correcting sample path-length, and converting the absorbance to product concentration [P] with the experimentally obtained extinction coefficient. The kinetic parameters (V max and K m ) of SfSFGH were obtained from the x-and y-intercepts of the Lineweaver-Burk plot. The k cat was calculated using the equation k cat = V max [E] total .

Bioinformatics analysis
For functional classification and sequence analysis of SfS-FGH, proteins related to SfSFGH were identified using a BLAST search on the Protein Data Bank (PDB). All primary sequences were retrieved from the NCBI database in FASTA format. Multiple sequence alignments were performed using Clustal Omega [36] with the enzymes found using BLAST, and the results were rendered using ESPript [37]. For gene cluster analysis, homologous proteins of SfSFGH (sequence identity: ≥ 80%) were collected from a BLAST search on the non-redundant protein sequence database, followed by retrieval of chromosome information for the collected proteins. The gene locus encoding SfSFGH and its surroundings in the chromosome of Shewanella frigidimarina were compared with the corresponding regions in other Shewanella species, including S. sediminis, S. woodyi, S. pealeana, and S. piezotolerans.

Site-directed mutagenesis
To obtain mutants of SfSFGH, mutagenesis was carried out using the Quik-Change site-directed mutagenesis method [19]. Chemically synthesized oligonucleotides were used to substitute each residue (purchased from Cosmogenetech, Korea). In the Quik-Change method, the PCR mixture contained ~ 300 ng DNA template, 4 μL 5× Pfu DNA polymerase master mix, and 1 μM of each primer in a total volume of 20 μL. The mixture was heated at 95 °C for 2 min and then subjected to thermal cycling (18 cycles at 95 °C for 1 min, 66 °C for 1 min, and 72 °C for 7 min). The PCR product was incubated with DpnI at 37 °C for 1 h, subsequently incubated for 10 min at 80 °C, and transformed into E. coli DH5α. Three colonies were randomly selected for sequencing. The verified plasmid was then transformed into E. coli BL21(DE3) for overexpression.