Purification and Characterization of Trehalase From Acyrthosiphon pisum, a Target for Pest Control

Insect trehalases are glycoside hydrolases essential for trehalose metabolism and stress resistance. We here report the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1), its biochemical and structural characterization, as well as the determination of its kinetic properties. The protein has been purified by ammonium sulphate precipitation, first followed by an anion-exchange and then by an affinity chromatography. The SDS-PAGE shows a main band at 70 kDa containing two isoforms of ApTreh-1 (X1 and X2), identified by mass spectrometry and slightly contrasting in the C-terminal region. A phylogenetic tree, a multiple sequence alignment, as well as a modelled 3D-structure were constructed and they all reveal the ApTreh-1 similarity to other insect trehalases, i.e. the two signature motifs (179)PGGRFRELYYWDTY(192) and (479)QWDFPNAWPP(489), a glycine-rich region (549)GGGGEY(554), and the catalytic residues Asp336 and Glu538. The optimum enzyme activity occurs at 45 degrees C and pH 5.0, with K-m and V-max values of similar to 71 mM and similar to 126 mu mol/min/mg, respectively. The present structural and functional characterization of soluble A. pisum trehalase enters the development of new strategies to control the aphids pest without significant risk for non-target organisms and human health.

Automated summary

Insect trehalases, essential for trehalose metabolism and stress resistance, were studied with a focus on the extraction and purification of Acyrthosiphon pisum soluble trehalase (ApTreh-1) and its biochemical and structural characterization. The enzyme showed optimal activity at 45 degrees C and pH 5.0, with specific kinetic properties.