Fig 1.
Characterization of the esterase LgEstI.
(A) The effect of temperature on enzyme activity. Enzyme activity was measured at a temperature range of 20–60°C every 15 min for 1 h. The activity value obtained at 20°C was defined as 100% (refer to S2 Table). (B) The effect of pH on enzyme activity. Enzyme activity was determined in citrate buffer (pH 3.0–5.0), phosphate buffer (pH 6.0–7.0), Tris-HCl buffer (pH 8.0), and glycine buffer (pH 9.0–10.0) at 25°C. The activity at pH 8.0 using pNA was defined as 100%. The activity at pH 8.0 was used as a reference and percentage levels at each pH relative to the reference are shown. (C and D) Effect of organic solvents on LgEstI activity in the presence of pNA. Residual activity of LgEstI after incubating for 1 h with the indicated chemicals. Activity without the addition of chemical compounds was defined as 100% and relative activity was expressed. All data are presented as mean ± standard deviation (s.d.) (n = 3). The activity without denaturant was used as the reference and percentage levels relative to the reference are shown. Statistical significance (p-values) was calculated using a t-test. p values were indicated by ns, not significant (p > 0.05), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001). Statistical analyses were performed in GraphPad Prism software v.8.3.0.
Fig 2.
Overall structure of LgEstI and dimer state.
(A) Cartoon representation of the LgEstI monomer with a front view (left) and 90° rotated view (right). The CAP domain made up of three triangular helices; α1, α2, and α8 are shown with blue, while the α/β hydrolase domain is indicated with red for helices and yellow for strains. The residues of the catalytic triad (Ser159, Asp256, and His286) are represented by a stick. (B) Size exclusion chromatography (SEC) profile of LgEstI. Measured molecular mass of LgEstI was 70.91 kDa, which is close to the sequence-based molecular weight of the dimer (72.05 kDa). (C) Overall dimer presentation of LgEstI and the dimer interface is indicated with a dotted square. (D) Stereo view of the dimer interface of LgEstI. Residues involved in the dimeric interface of LgEstI are represented with sticks. The chains are differentiated with color: red for chain A and cyan for chain B, and marked after the residue number with parentheses.
Fig 3.
Characterization of the active site of LgEstI.
(A) Representation of the LgEstI substrate access channel. Illustrated and surface views of the enzyme. (B) Superimposed structure of LgEstI wild-type (green) and mutant F207A (magenta). Residues around the substrate access channel are depicted with sticks. (C) Comparative activity of the wild-type LgEstI and mutant enzymes with various substrates. The activity value obtained with wild-type LgEstI was defined as 100%. All data are presented as mean ± standard deviation (s.d.) (n = 3). The activity of LgEstI wild-type against pNA was used as the reference and percentage levels to the reference are shown. Statistical significance (p values) was calculated using a t-test. P values were indicated by *** (p < 0.001), and **** (p < 0.0001). Statistical analyses were performed in GraphPad Prism software v.8.3.0.
Fig 4.
Comparison of the substrate access channel.
(A) The surface area and volume of esterase and lipase based on the molecular surface are represented with black surfaces. The Computed Atlas of Surface Topography of proteins (CASTp) was employed to determine protein volume (probe radius of 1.4 Å). (B) Electrostatic potential surface and diagrammatic representation of the substrate access channel of the enzymes. The surface charge distribution is displayed as blue for positive, red for negative, and white for neutral. The channel of each enzyme is highlighted with yellow dots. Bulky residues blocking the channel of LgEstI, rPPE, and E40 are indicated with sticks.
Table 1.
Structural homolog search result for LgEstI from a DALI search (DALI-Lite server).
Fig 5.
Active site and substrate specificity of LgEstI.
(A) A close-up view of the active site illustrating the coordination between the active site residues and the acetate moiety connected to nucleophilic S159. Hydrolysis of 4-methylumbelliferyl (4-MU) acetate and phosphate by LgEstI was measured (B) and observed (C). Control indicates LgEstI only. All data are presented as mean ± standard deviation (s.d.) (n = 3). Statistical significance (p values) was calculated using a t-test. P values were indicated by ns, not significant (P > 0.05), * (p < 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001). Statistical analyses were performed in GraphPad Prism software v.8.3.0.
Fig 6.
Immobilization of LgEstI and activity.
(A) Scanning electron microscope (SEM) image of LgEstI-CLEAs. Representative images at 30 kX (upper) and 100 kX (lower) are shown. (B) The reusability of LgEstI-CLEAs was assessed by measuring residual activity after repeated washing. pNA was used as a substrate and the activity of soluble LgEstI was defined as 100%. Statistical significance (p values) was calculated using a t-test. P values were indicated by ns, not significant (P > 0.05), and * (p < 0.05).