Efficient heterologous expression of Fusarium solani lipase, FSL2, in Pichia pastoris, functional characterization of the recombinant enzyme and molecular modeling.
The gene coding for a lipase of Fusarium solani, designated as FSL2, shows an open reading frame of 906bp encoding a 301-amino acid polypeptide with a molecular mass of 30kDa. Based on sequence similarity with other fungal lipases, FSL2 contains a catalytic triad, consisting of Ser144, Asp198, and His256. FSL2 cDNA was subcloned into the pGAPZαA vector containing the Saccharomyces cerevisiae α-factor signal sequence and this construct was used to transform Pichia pastoris and achieve a high-level extracellular production of a FSL2 lipase. Maximum lipase activity was observed after 48h. The optimum activity of the purified recombinant enzyme was measured at pH 8.0-9.0 and 37°C. FSL2 is remarkably stable at alkaline pH values up to 12 and at temperatures below 40°C. It has high catalytic efficiency towards triglycerides with short to long chain fatty acids but with a marked preference for medium and long chain fatty acids. FSL2 activity is decreased at sodium taurodeoxycholate concentrations above the Critical Micelle Concentration (CMC) of this anionic detergent. However, lipase activity is enhanced by Ca2+and inhibited by EDTA or Cu2+and partially by Mg2+or K+. In silico docking of medium chain triglycerides, monogalctolipids (MGDG), digalactolipids (DGDG) and long chain phospholipids in the active site of FSL2 reveals structural solutions.
University of Sfax, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, BPW 3038 Sfax, Tunisie.
Raida
R
Jallouli
CNRS, Aix Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Goetz
G
Parsiegla
CNRS, Aix Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse UMR7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Frédéric
F
Carrière
University of Sfax, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, BPW 3038 Sfax, Tunisie.
Youssef
Y
Gargouri
University of Sfax, Laboratoire de Biochimie et de Génie Enzymatique des Lipases, ENIS route de Soukra, BPW 3038 Sfax, Tunisie. Electronic address: sofiane_bezzine@yahoo.com.
Sofiane
S
Bezzine
D002118:Calcium / Q000737:chemistry; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D005670:Fusarium / Q000201:enzymology; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry*; D062105:Molecular Docking Simulation; D010843:Pichia; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D040681:Structural Homology, Protein; D013379:Substrate Specificity
D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase; D002118:Calcium
Expression; Fusarium solani lipase 2 (FSL2); In silico docking; Pichia pastoris