Secretory lipase from the human pathogen Leishmania major: Heterologous expression in the yeast Pichia pastoris and biochemical characterization

• Published in: Biochimie 2018-03, Vol.146, p.119-126
• Main Authors: Ben Ayed, Saoussen, Ali, Madiha Bou, Bali, Aymen, Gargouri, Youssef, Laouini, Dhafer, Ben Ali, Yassine
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biocatalysis; Biochemistry, Molecular Biology; Cloning, Molecular; Enzyme Inhibitors/pharmacology; Enzyme Stability; Gene Expression; Heterologous expression; Humans; Hydrogen-Ion Concentration; Leishmania; Leishmania major/enzymology; Leishmania major/genetics; Life Sciences; Lipase; Lipase/antagonists & inhibitors; Lipase/chemistry; Lipase/genetics; Lipase/metabolism; Microbiology and Parasitology; Models, Molecular; Parasite; Parasitology; Pichia/genetics; Protein Conformation; Sequence Alignment; Temperature
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/j.biochi.2017.12.002

Leishmaniasis is a parasitic reticuloendotheliosis whose pathogen is a zooflagellate belonging to the genus Leishmania transmitted by the bite of an infected phlebotome. Recently, a unique secretory lipase from the human pathogen Leishmania donovani Ldlip3 has been identified and characterized. This lipase has a high identity with a putative triacylglycerol lipase of Leishmania major (Lmlip2). In the present study, Lmlip2 was expressed in the eukaryotic heterologous expression system Pichia pastoris as tagged enzyme of 308 amino acids. Maximal protein production was reached after 2 days of fermentation. Optimal Lmlip2 lipase activity was measured using the pH stat technique at pH 8 at 26 °C using vinyl esters and triacylglycerols (true lipids) as substrates. Moreover, biochemical characterization of Lmlip2 contained in culture supernatant, illustrates that L. major secreted lipase is active and stable at low temperatures especially 26°and prefer neutral pH; concerning substrate specificityLmlip2 presents a preference for short chains lipid substrates vinyl esters such as VC2, VC3 and VC4 likewise, it is capable to hydrolyze long chain triacylglycerols like olive oil. Metal ions and surfactants tested in this study decrease Lmlip2 activity. Further studies are needed to clarify the relation between the lipase activity and the virulence. Thus, it could lead to the identification of novel targets to block cutaneous Leishmaniasis in human hosts. •Leishmania major lipase Lmlip2 is expressed in the eukaryotic expression system Pichia pastoris•Lmlip2 is a confirmed secretory lipase.•Lmlip2 is active and stable at low temperatures and prefers neutral pH.