Purification and properties of carboxylesterases from the mid-gut of the termite Odentotermes horni. W.

Abstract

Two carboxylesterases (TE-I and TE-II) from the mid-gut of the termite Odentotermes horni. W., have been purified to apparent homogeneity by means of ammonium sulfate fractionation, gel-permeation on Sephadex G-75 and Ultragel AcA-34 and ion-exchange chromatography on DEAE-Sephacel. The homogeneity of the preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and Ouchterlony double immunodiffusion. The apparent molecular weights determined by gel-permeation on Sephadex G-200 was 78,350 and by SDS-PAGE 78,500. In the presence of 2-mercaptoethanol, the proteins were split into two subunits of equal size with subunit molecular weight of about 40,000. The enzymes were found to have a Stokes radius of 3.35 nm. The amino acid analysis of the purified enzymes revealed the presence of a greater number of acidic and neutral amino acids than in other insect carboxylesterases. The isoelectric points of the enzymes, TE-I and TE-II, were 5.4 and 5.6, respectively. The two enzymes were inhibited by organophosphates. The substrate preference and inhibition patterns classify these enzymes as carboxylesterases (EC 3.1.1.1), but the physiological function is unknown. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{k}{}_{\mathrm{<mtext>i</mtext>}}$ and $\text{I}{}_{50}$ values are listed. The product inhibition studies with the enzymes revealed the linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol. In addition, optimum temperature and pH, thermal stability, effect of temperature and pH on $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the two enzymes were determined.