Figure 1.
SDS-PAGE analysis of different samples taken during the purification process of esterase LipC.
Lane 1: crude supernatant of pET28a-LipC non-induced culture; Lanes 2 and 6: crude extract of induced culture; Lane 3: purification on Ni-NTA affinity Sepharose column; Lane 4: further purification with DEAE column; Lanes 5 and 6: further purification with Sephadex-G200. Lanes 1 to 5 were stained with Coomassie Brilliant Blue and Lanes 6 was stained with α-naphtyl acetate and Fast Blue for the detection of hydrolase activity.
Table 1.
Purification steps and yields of recombinant esterase from Haloarcula marismortui (ATCC43049).
Figure 2.
MALDI-TOF peptide mass fingerprint (PMF) spectrum.
The PMF analysis was made from fragments of halophilic esterase LipC derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments is shown in red.
Figure 3.
Substrate specificity of the purified esterase LipC.
The enzyme activity was measured towards p-nitrophenyl acylates with different acyl chain lengths (C2–C16) at 45°C and pH 8.5 with 3.4 M NaCl present. Activity for p-NP-C2 was taken as 100%.
Figure 4.
Influences of (A) temperature and (B) pH on the esterase activity of LipC of Haloarcula marismortui (ATCC 43049).
For the pH profile, the activity was measured at constant ionic strength of 3.4 M NaCl and ambient temperature of 22 C. For the temperature profile, the activity was measured in 50 mM Tris-HCl and pH 8.5 with no NaCl added. The values represented the means of results from duplicate experiments and activities at the peak positions were taken as references.
Figure 5.
Effects of salt on p-NP-C2 hydrolysis activity of LipC.
For each salt, the activity assessment was made at 45°C, pH 8.5 in Tris-HCl buffer, All the assays were taken when the enzyme incubated with the salt for more than 1 hour.
Figure 6.
Effects of temperature and [NaCl] on the stability of LipC.
Measurements were made at 1 mg/ml enzyme with (•) and without (▪) 3.4 M NaCl: (a) 22°C, (b) 37°C, (c) 55°C and (d) 60°C. Relative activities (normalised to the value at the start of the measurements) were determined at 50 mM Tris-HCl, pH 8.0 using p-NP-C2 as substrate.
Figure 7.
The CD measurements were made in the presence of different concentrations of NaCl: 0 M (▪), 1.7 M (•), 3.4 M (▴) and 5.1 M (▾). The concentration of LipC was fixed at 0.4 mg/ml (22°C, 20 mM Tris-HCl buffer pH 8).
Figure 8.
Dynamic light scattering from different LipC solutions.
The DLS measurements were made from the esterase in aqueous buffer (20 mM TrisHCl, pH 8) at 22°C (◊), 45°C(Δ), and buffer with 3.4 M NaCl at 22°C (□), 45°C (○). The enzyme concentrations were between 0.5 and 0.7 mg/ml. A, measured immediately; B, measured after 2 days.
Figure 9.
SANS scattering intensity profiles (I) plotted against wave vector (Q).
The two sets of the measured data were from 2 mg/ml LipC in 20 mM Tris-HCl pH 8 (Δ); in 20 mM Tris-HCl pH 8 with 3.4 M NaCl at 45°C (◊). The continuous lines represented the best fitted curves, with the parameters obtained listed in Table 2. Error bars were not added for clarity.
Table 2.
Best fit parameters obtained from analysis of SANS profiles.