2.1 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 Pacific 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).
2.2 Homology modeling and putative structure analysis
The three-dimensional (3D) structures of Est2 and Est4 were modeled using the I-TASSER server (https://zhanglab.ccmb.med.umich.edu)(Roy et al. 2010). Structural figures were generated using PyMOL software (http://pymol.sourceforge.net).
2.3 Cloning, expression and purification
The est2 and est4 genes were amplified 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 purified 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 OD600 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 purified by Ni-NTA affinity chromatography columns.
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 verified mutant recombinant plasmids were transferred into competent E. coli Rosetta (DE3) cells for expression.
2.5 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 purified 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 defined as the amount of esterase required to release 1 µmol of p-NP per minute from the p-NP ester.
2.6 Enzyme characterization assay
The substrate specificity of the enzymes was determined using p-NP esters (Sigma, unless otherwise stated) as the substrates, including p-NP acetate (C2), p-NP butyrate (C4), p-NP hexanoate (C6) (TCI, Japan), p-NP octanoate (C8), p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14) and p-nitrophenyl palmitate (C16). The kinetic parameters were obtained by measuring the initial reaction rates of hydrolysis of p-NP butyrate (C4) and p-NP hexanoate (C6) at various concentrations ranging from 0.1 to 2.0 mM. GraphPad Prism 7.0 software (GraphPad Inc., USA) was used to calculate the kinetic parameters of the enzymes by analyzing the slopes of the Michaelis–Menten equations.
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 p-nitrophenolate. 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 (Ba2+, Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Ni2+, Sr2+, and Zn2+) and the chelating agent ethylenediaminetetraacetic acid (EDTA) were examined at a final 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 final 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 defined 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 significant.
2.7 Nucleotide sequence accession numbers
The locus tags of the est2 and est4 genes are JF766282 and JF766284, respectively.