Cloning and catalytic profile of Hyalomma dromedarii leucine aminopeptidase

Esraa A A Ali; Nahla A Hussein; Amr E El-Hakim; Mahmoud A Amer; Yasser E Shahein

doi:10.1016/j.ijbiomac.2024.131778

Cloning and catalytic profile of Hyalomma dromedarii leucine aminopeptidase

Int J Biol Macromol. 2024 Apr 22;268(Pt 1):131778. doi: 10.1016/j.ijbiomac.2024.131778. Online ahead of print.

Authors

Esraa A A Ali¹, Nahla A Hussein², Amr E El-Hakim¹, Mahmoud A Amer³, Yasser E Shahein⁴

Affiliations

¹ Molecular Biology Department, Biotechnology Research Institute, National Research Centre, Dokki, 12622 Cairo, Egypt.
² Molecular Biology Department, Biotechnology Research Institute, National Research Centre, Dokki, 12622 Cairo, Egypt. Electronic address: nahlahussein@aucegypt.edu.
³ Zoology Department, Faculty of Science, Cairo University, 12613 Giza, Egypt.
⁴ Molecular Biology Department, Biotechnology Research Institute, National Research Centre, Dokki, 12622 Cairo, Egypt. Electronic address: ye.shahein@nrc.sci.eg.

PMID: 38657929
DOI: 10.1016/j.ijbiomac.2024.131778

Abstract

Ticks have harmful impacts on both human and animal health and cause considerable economic losses. Leucine aminopeptidase enzymes (LAP) play important roles during tick infestation to liberate vital amino acids necessary for growth. The aim of the current study is to identify, express and characterize the LAP from the hard tick Hyalomma dromedarii and elucidate its biochemical characteristics. We cloned an open reading frame of 1560 bp encoding a protein of 519 amino acids. The LAP full-length was expressed in Escherichia coli BL21 (DE3) and purified. The recombinant enzyme (H.d rLAP- 6×His) had a predicted molecular mass of approximately 55 kDa. Purification and the enzymatic characteristics of H.d rLAP- 6×His were studied. The purified enzyme showed maximum activity at 37 °C and pH 8.0-8.5 using Leu-p-nitroanilide as a substrate. The activity of H.d rLAP- 6×His was sensitive to β-mercaptoethanol, dl-dithiothreitol, 1,10- phenanthroline, bestatin HCl, and EDTA and completely abolished by 0.05 % SDS. In parallel, the enzymatic activity was enhanced by Ni²⁺, Mn²⁺ and Mg²⁺, partially inhibited by Na⁺, Cu²⁺, Ca²⁺ and completely inhibited by Zn²⁺.

Keywords: Enzyme kinetics; Gene expression; Hyalomma dromedarii; Leucine aminopeptidase; Ticks.