Two Novel Esterases from Deep-Sea Sediment Reveal Key Residues for Thermostability

Yi Ding Shanghai Jiao Tong University Xiao-chen Yang Second Institute of Oceanography Ministry of Natural Resources Ying-yi Huo Zhejiang University Shu-ling Jian Second Institute of Oceanography Ministry of Natural Resources Xue-we Xu Second Institute of Oceanography Ministry of Natural Resources Heng-lin Cui (  cuihenglin@ujs.edu.cn ) Jiangsu University Yue-hong Wu Second Institute of Oceanography Ministry of Natural Resources https://orcid.org/0000-0002-15938662


Introduction
The marine environment is one of the largest pools of novel enzymes that could provide potential pro ts in many industrial elds. Metagenomics screening has become an e cient method for exploring novel His-Asp/Glu catalytic triad located on the catalytic domain is responsible for ester bond hydrolysis (Li et al. 2015). The conserved HGG motif of family IV esterases is involved in the formation of oxyanion holes and plays a key role in stabilizing the tetrahedral intermediate of the reaction (Mandrich et al. 2008). The cap domain located on the entrance of the catalytic pocket impacts the maintenance of enzyme stability, activity and speci city (Mandrich et al. 2005).
In this study, we described the expression, puri cation and biochemical characterization of two novel esterases, Est2 and Est4, that were screened from a deep-sea sediment metagenomic library (Jiang et al. 2012). The two enzymes had high amino acid sequence identity. Mutagenesis analysis identi ed two residues in Est2, Asp18 and Lys289, which had signi cant impacts on the catalytic features of the enzyme. The Asp18Asn mutation of Est2 signi cantly affected the thermostability and catalytic activity of the enzyme. The residue Lys289 was identi ed as the key residue to determine the discrepancy of surface potential and thermostability between Est2 and Est4.

Sequence and polygenetic analysis
Two putative esterase genes with high amino acid sequence identity, designated est2 and est4, were previously obtained from a deep-sea sediment metagenomics library using the subcloning method (Jiang et al. 2012). The deep-sea sediments were collected from the skirt of a seamount located in the Paci c Ocean at a depth of 5 886 m. Their deduced amino acid sequences were analyzed by the BLASTp program (https://blast.ncbi.nlm.nih.gov). Sequence alignment of the amino acid sequences of multiple proteins was performed using Clustal X version 2.0 (Larkin et al. 2007) and ESPript 3.0(Robert and Gouet 2014). The corresponding phylogenetic tree was constructed using the neighbor-joining method with MEGA version 7.0 software (Kumar et al. 2016).

Cloning, expression and puri cation
The est2 and est4 genes were ampli ed by polymerase chain reaction using the primers listed in Table 1. The PCR products were digested by NdeI and HindIII (New England BioLabs, USA). The puri ed digested fragments were ligated into the pET28b(+) (Novagen, Germany) expression vector that had been digested with the same enzymes. The recombinant plasmids were transformed into Escherichia coli (E. coli) Rosetta (DE3) cells.
Recombinant E. coli Rosetta (DE3) strains were cultivated in LB medium containing 50 µg/mL kanamycin and 34 µg/mL chloramphenicol at 37°C until the OD 600 reached 0.8. Protein expression was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 20 h at 16°C. The cells were harvested by centrifugation at 4°C and 10 000 ×g, and were resuspended in 10 mM imidazole buffer containing 500 mM NaCl and 20 mM Tris/HCl (pH 8.0) for sonication. Protein samples were collected from the supernatant of cell lysates by centrifugation at 4°C and 18 000 ×g and were puri ed by Ni-NTA a nity chromatography columns.

Mutagenesis
The point mutants Est2-Asp18Asn (Est2-D18N) and Est2-Lys289Glu (Est2-K289E) were constructed by site-directed mutagenesis using wild-type recombinant plasmid as the template with the Fast Mutagenesis System (Transgene Biotech, China) via whole-plasmid PCR in 20 reaction cycles of 94°C for 20 s, 55°C for 20 s and 72°C for 3 min. The primers used for PCR are listed in Table 1. The veri ed mutant recombinant plasmids were transferred into competent E. coli Rosetta (DE3) cells for expression.

Enzyme activity assay
Enzyme activity was evaluated by measuring the UV absorption at 405 nm of p-nitrophenol at 40°C for 2 min with a DU800 ultraviolet-visible spectrophotometer (Beckman, USA). The 1 mL standard reaction mixture contained 10 µL of 100 mM p-nitrophenyl (p-NP) hexanoate, 980 µL of phosphate buffer (100 mM, pH 7.0), and 10 µL of the puri ed enzyme. All values were determined in triplicate, and reactions with the added thermally inactivated enzyme were used as controls. One unit of enzyme activity was de ned as the amount of esterase required to release 1 µmol of p-NP per minute from the p-NP ester.
The optimum pH for enzyme activity was measured over a pH range from 3.0 to 10.0 with four different buffers, including 100 mM citrate buffer (pH 3.0-6.0), 100 mM phosphate buffer (pH 6.0-7.5), 100 mM tricine buffer (pH 7.5-9.0), and 50 mM CHES buffer (pH 9.0-10.0). The reactions at different pH values were measured at 348 nm, the pH-independent isosbestic wavelength of p-nitrophenol and pnitrophenolate. The optimum temperature for enzyme activity was measured over a range of 15-60°C with an interval of 5°C. The thermostability of Est2 and Est4 was analyzed by measuring the residual activity after incubating the enzymes at different temperatures (ranging from 30°C to 60°C) in 100 mM phosphate buffer (pH 7.0) for 1 h.
The effects of various metal ions (Ba 2+ , Ca 2+ , Co 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , Sr 2+ , and Zn 2+ ) and the chelating agent ethylenediaminetetraacetic acid (EDTA) were examined at a nal concentration of 10 mM. The effects of various detergents were determined using 1% (v/v) Triton X-100, Tween-20, Tween-80, and 1% (w/v) SDS. The effects of various organic solvents were examined using acetone, acetonitrile, ethanol, N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), glycerol, isopropanol, and methanol at a nal concentration of 15% (v/v). All tests were performed in 100 mM Tris/HCl buffer (pH 7.5), and the values obtained without additives in the reaction mixture were de ned as 100%. Data were presented as the mean ± SD. Statistical analyses were performed with two-tailed unpaired Student's t-tests. P values less than 0.05 were considered statistically signi cant.

Nucleotide sequence accession numbers
The locus tags of the est2 and est4 genes are JF766282 and JF766284, respectively.

Sequence analysis of Est2 and Est4
The sequences of est2 and est4 were both 921 bp and encoded 306 amino acids with 96% sequence identity. The identi ed esterases in the GenBank nr database that shared the highest amino acid sequence identity (77-78%) with Est2 and Est4 were three metagenome-derived esterases, namely, FLS10,  (Table 2).
Polygenetic analysis validated that Est2 and Est4 are new members of microbial esterases in family IV (Fig. 2).

Expression and characterization of wild-type Est2 and Est4
Est2 and Est4, with molecular weights of 32.5 kDa, were successfully expressed in E. coli Rosetta (DE3) and puri ed using Ni-NTA a nity chromatography columns. Est2 and Est4 had higher activities toward relatively short-chain p-NP esters (< C10) with the highest activity toward p-NP hexanoate than those with long-chain p-NP esters (≥ C10) (Fig. 3a). The catalytic activity of the two enzymes can be retained under a wide range of temperatures from 15°C to 45°C (Fig. 3b) and pH values from 6.0 to 8.5 (Fig. 3c). Both Est2 and Est4 exhibited the highest activity at pH 7.0-8.0 and 40°C.
Thermostability analysis showed that Est2 and Est4 maintained approximately 80% and 70% of their initial activities, respectively, after incubation at 40°C for one hour (Fig. 3d). Est4 completely lost catalytic activity, while Est2 retained 30% of its initial activity after incubation at 50°C for one hour.

Structural model analysis
The 3D structures of Est2 and Est4 were modeled using the I-TASSER server. The protein structures of both enzymes contain 8 b-sheets and 9 a-helices. The structures could be divided into two domains: a catalytic domain (residues 45-305) with a canonical α/β-hydrolase fold consisting of eight parallel β strands surrounded by ve α helices and a cap domain (residues 1-44) (Fig. 4). The global structures of Est2 and Est4 did not exhibit signi cant differences, while Est4 contained more negative surface potentials than Est2 (Fig. 5).

Mutagenesis analysis of Est2
Two mutants of Est2 (Est2-D18N and Est2-K289E) were designed to explore the mechanism of catalytic and structural discrepancies between Est2 and Est4. The kinetic parameters, substrate speci city, optimum pH and temperature as well as the thermostability of mutants were measured. The mutants Est2-D18N and Est2-K289E showed similar substrate speci city as well as optimum pH and temperature to the wild-type enzyme (Fig. 3a, 3b & 3c). Compared with the wild-type enzyme, mutant Est2-D18N had a reduction in both Kcat and substrate a nity of p-NP hexanoate by more than 2.0-fold (Table 3). In addition, the thermostability of the mutant Est2-D18N was signi cantly reduced compared with that of wild-type Est2. The mutant Est2-D18N completely lost activity after incubation at 40°C for one hour (Fig.  3d). Mutant Est2-K289E exhibited little deviation from the Kcat and the Km of wild-type Est2. The thermostability and surface potential of Est2-K289E were more similar to those of Est4 than Est2.

Discussion
Two novel esterases, Est2 and Est4, which shared 96% amino acid sequence identity, were expressed and characterized in this study. The two enzymes exhibited similar catalytic features, including substrate speci city, optimum pH and temperature, as well as global structure (Fig. 3 and Fig. 4). A notable discrepancy in their catalytic feature is their thermostability. Est2 can sustain 50°C treatment for one hour, while Est4 completely loses its catalytic capability under the same conditions (Fig. 3d). To investigate the structural basis of the different thermostabilities of the enzymes, we attempted to obtain their crystal structures for many times. Unfortunately, crystals did not appear and grow (data not shown). Thus, we constructed and compared structural models of Est2 and Est4. The model comparison results suggested that Est2 and Est4 had different surface potentials of the molecule (Fig. 5a and 5b). This might result from the different amino acids of the two enzymes that were located on the surface of the molecules. Among the twelve pairs of different amino acids between Est2 and Est4, 6 pairs contained different electrical potentials and polarities. The 6 amino acids in Est2 were the neutral polar amino acids Gln4, Gly63, Thr180 and Gln187, the negatively charged polar amino acid Asp18, and the positively charged polar amino acid Lys289. In Est4, they are the negatively charged polar amino acids Glu4 and Glu289, the polar amino acid Asn18, the nonpolar amino acids Ala63 and Ile180, and the positively charged polar amino acid Arg187. The amino acid residues at positions 18 and 289 had signi cant differences in charge between the two enzymes.
To analyze the mechanism of different catalytic features between Est2 and Est4, mutants Est2-D18N and Est2-K289E were induced and expressed. The residue Asp18 was located at the cap domain of Est2. The Lys289 was located at the a9-helix. The mutation of residues Asp18 and Lys289 both changed the surface potential ( Fig. 5c and 5d) and reduced the thermostability of wild-type Est2 (Fig. 3d). In previous studies, the surface potential was considered to be crucial for the salinity tolerance of esterase that was isolated from halophilic archaea (Muller-Santos et al. 2009). In this study, the thermostability of mutants was reduced in accordance with the changes in the surface potential of Est2, indicating that the surface potential might also determine the thermostability of the enzyme. The thermostability and surface potential of mutant Est2-K289E were similar to those of Est4 (Fig. 3d, and Fig. 5b & 5d), indicating that residue Lys289 may play a key role in determining the discrepancy of surface potential and thermostability between Est2 and Est4.
The cap domain has been reported to have an effect on the substrate speci city and catalytic activity of enzymes (Holmquist 2000). In this study, the mutation of residue Asp18 located at the cap domain dramatically reduced the thermostability (Fig. 3d) and catalytic activity of Est2 (Table 3). An increase in the Km value and a decrease in the kcat value toward p-NP hexanoate were observed (from (55.0±3.0) μM and (352.4±2.9) s -1 to (147.2±14.4) μM and (159.1±4.4) s -1 , respectively), suggesting that both the capability of substrate binding and the turnover rate of the enzyme-substrate complex to product were decreased (Table 3). Decreases in both kcat and Km values toward the p-NP butyrate substrate indicated that the turnover rate of the enzyme-substrate complex decreased while the substrate a nity of p-NP butyrate was increased by the mutation. Compared with mutant Est2-K289E, mutant Est2-D18N had dramatic impacts on the thermostability and catalytic activity of the enzyme. This might result from changes in both the surface potential and mobility of the cap domain attributed to the mutation of Asp18 to Asn18.
In summary, two novel family IV esterases, Est2 and Est4, were expressed and characterized from a deepsea sediment metagenomic library. The two esterases showed high amino acid sequence identity (96%) and shared most catalytic features except thermostability. The structural modeling and mutagenesis of Est2 and Est4 provided insight into the determinants of thermostability. Moreover, residue Asp18 in the cap domain of Est2 showed a marked impact on the thermostability and catalytic activity of Est2.
Residue Lys289 determined the differences between Est2 and Est4 in the surface potential and thermostability. The characterization and mechanistic analysis of these enzymes should provide a basis for further exploration of their potential biotechnological applications.

Con ict of interest
The authors declare no con ict of interest.

Ethical approval
This study does not include any experimental procedure performed on humans or animals.    Table 4 Effects of various metal ions and chelating agents on the activity of Est2 and Est4 Amino acid sequence alignment of Est2 and Est4 with related lipolytic enzymes. Sequence alignment was performed using the ClustalX and ESPript programs. Identical and similar residues among groups are indicated in white text on a red background and in red text on a white background, respectively. Solid circles indicate the locations of the residues involved in the oxyanion hole (glycine (G)). The catalytic active site residues (serine (S), glutamic acid (E), and histidine (H)) are indicated by triangles. The conserved HGGG and GDSAG motifs, in which the oxyanion hole and catalytic triad are located, respectively, are outlined with boxes. The purple arrows indicate the locations of residues Asp18 and Lys289 of Est2.

Figure 2
Neighbor-joining phylogenetic tree based on amino acid sequences of Est2 and Est4. Sequence alignment was performed using ClustalX. The phylogenetic tree was constructed by MEGA software. Bootstrap values were based on 1 000 replicates, and values higher than 50% are shown in the tree. The scale bar measured the number of amino acid substitutions per site.

Figure 3
Characterization of Est2, Est4, and two Est2 mutants. a. Substrate speci city was determined using the p-NP esters, including p-NP acetate (C2), p-NP butyrate (C4), p-NP hexanoate (C6), p-NP caprylate (C8), p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14), and p-NP palmitate (C16). All of the tests were performed at 40°C and pH 7.0. b. The effects of pH on the activity were determined by using p-NP hexanoate as the substrate at 40°C in different buffers: 100 mM citrate buffer (pH 3.0-6.0), 100 mM phosphate buffer (pH 6.0-7.5), 100 mM tricine buffer (pH 7.5-9.0), and 50 mM CHES buffer (pH 9.0-10.0). c. The effects of temperature on the activity were determined at various temperatures at pH 7.0 using p-NP hexanoate as the substrate. The highest activity was taken as 100% d. Thermostability of Est2, Est4 and two mutants of Est2 at different temperatures. The relative activities of Est2 and Est4 are based on their initial activities, which are 100%. Data are presented as the mean ± SD (n = 3).

Figure 4
The 3D structural models of Est2 (a) and Est4 (b). Models were constructed by I-TASSER server. The catalytic domains of Est2 are shown in red and yellow, while the catalytic domains of Est4 are shown in sky blue and purple. The cap domains of both are shown in dark blue. The catalytic triad residues are indicated as stick models.