Purification of high molecular weight thermotolerant esterase from Serratia sp. and its characterization

Abstract

In the present study, an extracellular esterase from Serratia sp. was purified 24.46 fold using an initial ammonium sulphate precipitation step (optimized concentration of 30-40%), followed by Diethylaminoethyl cellulose (DEAE-cellulose) chromatography and size exclusion Sephadex G-200 column chromatography steps. The molecular weight of the esterase using native polyacrylamide gel electrophoresis (PAGE) was determined to be 236 kDa and by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was found to be 60 kDa suggesting that the enzyme was a tetramer of 4 subunits. The purified esterase was able to catalyze the hydrolysis of p-nitrophenyl esters, especially p-nitrophenyl acetate. Maximum esterase activity was achieved in 0.15 M Tris-HCl buffer of pH 8.5 at 50 °C after 10 min. The enzyme was stable for at least 8 h at 4 and 35 °C but the half-life was determined to be 4.5 h at 50 °C and 3 h at 60 °C. The esterase activity was inhibited by detergents (1 mM) (Triton X-100, Tween 60, Tween 80, ethylenediamine tetraacetic acid and SDS) except Tween 20. The esterase activity was inhibited by organic solvents (1 mM) such as ethanol, methanol, acetone, acetonitrile and was stable in the presence of glycerol, isopropanol but the organic solvent dimethyl sulfoxide (DMSO) significantly (p < 0.05) enhanced esterase activity. The matrix-assisted laser desorption ionization-time of flight mass spectrometry showed that the enzyme exhibited similarity with the pimeloyl-[acyl carrier protein] methyl ester esterase of Serratia marcescens.

Keywords: Diethylaminoethyl cellulose; Esterase; Matrix-assisted laser desorption ionization-time of flight mass spectrometry; SDS-PAGE; Tris–HCl buffer; p-NPA.

Fig. 1

Elution profile of purified esterase from Serratia sp. after DEAE-Cellulose column chromatography. 22–29 fractions showed maximum absorbance at 280 nm and specific activity

Fig. 2

Elution profile of purified esterase from Serratia sp. after Sephadex G-200 column chromatography. 13–21 fractions showed maximum absorbance at 280 nm and specific activity

Fig. 3

a Native-PAGE of a purified esterase from Serratia sp. L 1: dialyzed esterase enzyme, L 2: Purified esterase enzyme, L 3: SERVA Native protein marker (high range molecular weight). b SDS-PAGE of a purified esterase from Serratia sp. L 1: BR BIOCHEM BLUeye prestained SDS protein marker (medium range molecular weight), L 2,3: purified esterase enzyme, L 4: dialyzed esterase enzyme

Fig. 4 a

Effect of pH of Tris–HCl buffer on the activity of purified esterase from Serratia sp. 0.15 M concentration of Tris–HCl buffer was used. p-NPA was used as substrate to monitor ester hydrolysis. Values are mean ± S.D. of triplicate experiments. b Effect of incubation temperature on the activity of purified esterase from Serratia sp. p-NPA was used as a substrate to monitor ester hydrolysis. Values are mean ± S.D. of triplicate experiments

Fig. 5

Stability of purified esterase from Serratia sp. The esterase activity was determined at different temperatures i.e., 4, 35, 50, and 60 °C over a period of 8 h at intervals of 1 h. The assay was performed in 0.15 M Tris–HCl buffer of pH 8.5 using p-NPA as substrate. Values are mean ± S.D. of triplicate experiments

Fig. 6

Effect of detergents on the activity of purified esterase from Serratia sp. p-NPA was used as a substrate to monitor ester hydrolysis. Relative activities were expressed as percentages of control activity (without detergent) plotted for each detergent. Values are mean ± S.D. of triplicate experiments. ***p < 0.001; **p < 0.01 and *p < 0.05 as compared to control

Fig. 7

Effect of different solvents on the activity of esterase from Serratia sp. p-NPA was used as a substrate to monitor ester hydrolysis. Relative activities were expressed as percentages of control activity (without metal ion) plotted for each organic solvent. Values are mean ± S.D. of triplicate experiments. ***p < 0.001; **p < 0.01 and *p < 0.05 as compared to control

Fig. 8

a MALDI-TOF MS spectrum of tryptic digested peptides of purified esterase from Serratia sp. b 3-D structure of esterase from Serratia sp. predicted using SWISS MODEL and active site residues i.e.,serine (green), aspartate (blue), histidine (red)