For esterase activity of EaEST, p-nitrophenyl esters of different acyl chain lengths including p-nitrophenyl acetate (C2, p-NA), butyrate (C4, p-NB), hexanoate (C6, p-NH), octanoate (C8, p-NO), decanoate (C10, p-NDec), dodecanoate (C12, p-NDo), and phosphate (p-NP) were used as substrates. The release of p-nitrophenol was measured at 405 nm using an EPOCH2 microplate reader (Biotek, USA). Regioselectivity of EaEST was also studied by using 1-naphthyl phosphate (1-NP), 1-naphthyl acetate (1-NA), 1-naphthyl butyrate (1-NB), and 2-naphthyl acetate (2-NA). The absorbance was measured at 310 nm.
The kinetic constants of EaEST toward acetic acid perhydrolysis were measured using a monochlorodimedone (MCD) assay at 25°C. All reactions contained 0.047 mM of MCD, 149 mM of potassium bromide, and appropriated amounts of the enzyme. The concentrations of acetic acid were varied to 1.4 M. The reaction was initiated with the addition of 9.9 mM of hydrogen peroxide. Enzyme activity was determined by the halogenation of MCD (ε = 19.9 mM−1 cm−1 at 290 nm) as described previously [35 ]. The data were fit to the Michaelis-Menten equation using nonlinear regression (GraphPad Prism 5 Software, San Diego, CA, USA).
The optimal temperature and pH were investigated in the assay mixture containing 20 mM Tris-HCl, 100 mM NaCl (pH 8.0), 0.5 mM p-NA, and 10 μg of EaEST. The optimal pH was studied by measuring enzyme activity of EaEST from pH 3.0 to pH 10.0 at 25°C. Following buffers were used including 50 mM citrate-NaOH (pH 3.0–6.0), 100 mM phosphate-NaOH (pH 7.0), 50 mM Tris-HCl (pH 8.0), and 20 mM glycine-NaOH (pH 9.0–10.0). The optimal temperature was examined at 20, 40, 45, 50, 55, 60, and 80°C. Thermostability of EaEST was measured by incubating the enzyme at 0, 20, 40, 50, and 60°C for 1 h. Each aliquot was taken every 15 min for measuring the residual activity.
The effects of NaCl and glycerol additions on EaEST were determined by incubating the enzyme with various concentrations of NaCl (0–5 M) or glycerol (0–5 M) at 25°C for 1 h. For chemical stability of EaEST, the effects of ethanol, isopropanol (i-PrOH), SDS, Tween 20, Triton X-100, and phenylmethylsulfonyl fluoride (PMSF) were determined. For enantioselectivity analysis, a pH shift-colorimetric assay was carried out with (R)- and (S)-methyl-3-hydroxy-2-methylpropionate in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl in 100 μl reaction mixture. The absorbance spectra were recorded from 350 nm to 600 nm. This pH shift-colorimetric assay was also used for the hydrolysis of phenyl acetate, 2-phenylethyl acetate, and 2-methylbutyl acetate. In addition, the hydrolysis of glyceryl tributyrate, glyceryl trioleate, olive oil, and fish oil were measured with this assay. Fluorescence analysis was executed using a Jasco FP-8200 spectrofluorometer (MD, USA). EaEST samples were incubated with different concentration urea (0–5 M) for 1 h. After excitation at 280 nm, emission spectra were recorded from 300 nm to 400 nm using a 5 nm slit width and a scan speed of 250 nm/min.
To prepare immobilized forms of EaEST, a purified EaEST (2 mg) was precipitated with 80% ammonium sulfate and crosslinked with 50 mM glutaraldehyde with gentle inverting for 12 h. Then, suspension was centrifuged at 13,000 rpm at 4°C for 30 min and the resulting immobilized EaESTs were washed 3 times with 20 mM Tris-HCl (pH 8.0), 100 mM NaCl. Activity of immobilized EaEST was monitored by measuring the hydrolysis of p-nitrophenyl acetate (C2, p-NA). Thermal stability of immobilized EaEST was investigated at 80°C and the activity of soluble EaEST was set to 100%. To examine reusability, immobilized EaEST was retrieved by simple centrifugation after each reaction. After repeated washing steps (usually 3 times), new substrate was added for another cycle and the activity of immobilized EaEST was measured. For surface morphology of immobilized EaEST, a scanning electron microscope (SUPRA 55VP, Carl Zeiss, Jena, Germany) was used. Samples were prepared by fixation process with 0.05 M cacodylate buffer (pH 7.2) containing 1% osmium tetraoxide (OsO4) at 4°C and consecutive cycles of dehydration by ethanol solutions. After drying with hexamethyldisilazane solution, samples were mounted on metal stubs and sputtered with gold.
The kinetic constants of EaEST toward acetic acid perhydrolysis were measured using a monochlorodimedone (MCD) assay at 25°C. All reactions contained 0.047 mM of MCD, 149 mM of potassium bromide, and appropriated amounts of the enzyme. The concentrations of acetic acid were varied to 1.4 M. The reaction was initiated with the addition of 9.9 mM of hydrogen peroxide. Enzyme activity was determined by the halogenation of MCD (ε = 19.9 mM−1 cm−1 at 290 nm) as described previously [35 ]. The data were fit to the Michaelis-Menten equation using nonlinear regression (GraphPad Prism 5 Software, San Diego, CA, USA).
The optimal temperature and pH were investigated in the assay mixture containing 20 mM Tris-HCl, 100 mM NaCl (pH 8.0), 0.5 mM p-NA, and 10 μg of EaEST. The optimal pH was studied by measuring enzyme activity of EaEST from pH 3.0 to pH 10.0 at 25°C. Following buffers were used including 50 mM citrate-NaOH (pH 3.0–6.0), 100 mM phosphate-NaOH (pH 7.0), 50 mM Tris-HCl (pH 8.0), and 20 mM glycine-NaOH (pH 9.0–10.0). The optimal temperature was examined at 20, 40, 45, 50, 55, 60, and 80°C. Thermostability of EaEST was measured by incubating the enzyme at 0, 20, 40, 50, and 60°C for 1 h. Each aliquot was taken every 15 min for measuring the residual activity.
The effects of NaCl and glycerol additions on EaEST were determined by incubating the enzyme with various concentrations of NaCl (0–5 M) or glycerol (0–5 M) at 25°C for 1 h. For chemical stability of EaEST, the effects of ethanol, isopropanol (i-PrOH), SDS, Tween 20, Triton X-100, and phenylmethylsulfonyl fluoride (PMSF) were determined. For enantioselectivity analysis, a pH shift-colorimetric assay was carried out with (R)- and (S)-methyl-3-hydroxy-2-methylpropionate in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl in 100 μl reaction mixture. The absorbance spectra were recorded from 350 nm to 600 nm. This pH shift-colorimetric assay was also used for the hydrolysis of phenyl acetate, 2-phenylethyl acetate, and 2-methylbutyl acetate. In addition, the hydrolysis of glyceryl tributyrate, glyceryl trioleate, olive oil, and fish oil were measured with this assay. Fluorescence analysis was executed using a Jasco FP-8200 spectrofluorometer (MD, USA). EaEST samples were incubated with different concentration urea (0–5 M) for 1 h. After excitation at 280 nm, emission spectra were recorded from 300 nm to 400 nm using a 5 nm slit width and a scan speed of 250 nm/min.
To prepare immobilized forms of EaEST, a purified EaEST (2 mg) was precipitated with 80% ammonium sulfate and crosslinked with 50 mM glutaraldehyde with gentle inverting for 12 h. Then, suspension was centrifuged at 13,000 rpm at 4°C for 30 min and the resulting immobilized EaESTs were washed 3 times with 20 mM Tris-HCl (pH 8.0), 100 mM NaCl. Activity of immobilized EaEST was monitored by measuring the hydrolysis of p-nitrophenyl acetate (C2, p-NA). Thermal stability of immobilized EaEST was investigated at 80°C and the activity of soluble EaEST was set to 100%. To examine reusability, immobilized EaEST was retrieved by simple centrifugation after each reaction. After repeated washing steps (usually 3 times), new substrate was added for another cycle and the activity of immobilized EaEST was measured. For surface morphology of immobilized EaEST, a scanning electron microscope (SUPRA 55VP, Carl Zeiss, Jena, Germany) was used. Samples were prepared by fixation process with 0.05 M cacodylate buffer (pH 7.2) containing 1% osmium tetraoxide (OsO4) at 4°C and consecutive cycles of dehydration by ethanol solutions. After drying with hexamethyldisilazane solution, samples were mounted on metal stubs and sputtered with gold.
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