Homology model of EstOF4, produced by MODELLER 9.9 program based on the X-ray structure of esterase Est30 from Geobacillus stearothermophilus (GenBank Number AY186197,Protein Data Bank code 1TQH).

<p>Visualization of the tertiary structure was done by Pymol and represented in the form of ribbons. A: Superimposition of homology modelled structure of EstOF4 (magenta) onto the 3D structure of Est30 (green), the cap-like domain constructed by α2, α5 and α6 was enclosed in the dashed box. B: 3D model of EstOF4, with β-strands and α-helices shown in yellow and red, respectively, and the catalytic triad Ser93, Asp190 and His220 shown in sticks.</p

Effects of different cations and reagents on the activities of EstOF4.

<p>The assay was taken at the optimal condition (50°C and pH 8.5) with p-nitrophenyl caprate as substrate. The activity of EstOF4 in buffers without adding any ions was taken as 100%.</p

The protein isoelectric point of esterase EstOF4 was determined by isoelectric focusing on precast IEF gel.

<p>Lane1: purified esterase EstOF4. Lane M: protein IEF marker contains proteins with pI 3.5 to 10.7.</p

Alignment of EstOF4 sequence with those from previously characterized homologous esterases [AAX86643, AAT57903, AY186197, BAD77330, AAT65181 and ABB90597].

<p>Strictly conserved residues are white letters on grey background, and conservatively substituted residues are boxed. The residues equivalent to the putative acid/base catalysts are indicated by a solid triangle and proteolytic cut site K210 is indicated by black solid circle. The secondary structure elements (helices α, strands β, and helices (η) of 1TQHA (AY186197) are shown above the alignment. The figure was produced using ESPript.</p

Substrate specificity of the esterase EstOF4 against p-nitrophenyl esters and glyceryl esters.

<p>The assay was taken at the optimal condition (50°C and pH 8.5) with various substrates. Each value was averaged from three duplicates. The values shown represent the means ± standard deviations from the triplicate values measured.</p

Effect of pH on the activity of esterase EstOF4.

<p>The reaction was taken at 50°C with pNC6 as substrate in different pH solution: sodium phosphate (pH 7.0 to 8.0), Tris-HCl (pH 8.0 to 9.0), glycine-NaOH (pH 9.0 to 10.5) and Na2HPO4/NaOH (pH 11.0 to 11.5). Each value of the assay was the arithmetic mean of triplicate measurements. Where pH overlapping, the averaged values were used.</p

SDS-PAGE analysis of the purification process of esterase EstOF4 and active staining.

<p>A: Lane 1: crude supernatant of E. coli BL21; Lanes 2: Lysate of induced E. coli BL21 pET-estof4 cells; Lane 3: purification on Ni-NTA affinity column; Lane 4: further purification with Sephadex-G200; Lane M: molecular weight markers. B: Line 1: denaturing gel electrophoresis of protein EstOF4 stained with Coomassie Blue. Line 2: native gel electrophoresis of EstOF4, the gel was stained with a-naphthyl acetate and Fast Blue for detection of hydrolase activity.</p

Effect of temperature on the activity of esterase EstOF4.

<p>The assay was taken at the optimal pH of 8.5 with pNC6 as substrate. Each value was the averaged over three duplicates.</p

Kinetic parameters for hydrolysis of the various p-nitrophenyl esters by purified EstOF4.

<p>All spectrometric assays were performed in the favourite active condition of the enzyme (50°C and pH 8.5).</p

Effects of temperature and [NaCl] on the stability of LipC.

<p>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 <i>p</i>-NP-C2 as substrate.</p