Efficient heterologous expression of Fusarium solani lipase, FSL2, in Pichia pastoris, functional characterization of the recombinant enzyme and molecular modeling

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• 作者：Raida Jallouli^a ; Goetz Parsiegla^b ; Fré ; dé ; ric Carriè ; re^b ; Youssef Gargouri^a ; Sofiane Bezzine^a ; ^{sofiane_bezzine@yahoo.com} (Pr.)
• 关键词：NhL1 ; Nectria haematococcae lipase ; FSL ; Fusarium solani lipase ; FSL2 ; Fusarium solani lipase 2 ; NaTDC ; Sodium taurodeoxycholic acid ; TC4 ; Tributyrin ; TC8 ; Trioctanoin ; CMC ; Critical Micelle Concentration
• 刊名：International Journal of Biological Macromolecules
• 出版年：2017
• 出版时间：January 2017
• 年：2017
• 卷：94
• 期：part_PA
• 页码：61-71
• 全文大小：2721 K
• 卷排序：94

文摘

The gene coding for a lipase of Fusarium solani, designated as FSL2, shows an open reading frame of 906 bp encoding a 301-amino acid polypeptide with a molecular mass of 30 kDa. 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 48 h. 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.