Structure and Function of a Recombinant Thermoalkalophilic Lipase from Archaeoglobus fulgidus

• Main Authors: Hsiao-Jung Liu, 劉曉蓉
• Other Authors: Guan-Chiun Lee
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/5v86p2

碩士 === 國立臺灣師範大學 === 生命科學研究所 === 97 === A novel lipase gene (AFL) from Archaeoglobus fulgidus has been previously cloned and functionally expressed in E. coli. AFL is a thermoalkalophilic lipase. Its three-dimensional structure has been already resolved by X-ray crystography. AFL is composed of 474 amino acid residues with a N-terminal signal peptide (18 amini acid residues) and mature lipase gene (456 amino acid). The N-terminal of AFL contains the catalytic triad and the substrate binding site (tunnel) is located at the C-erminal domain. According to the informations of three-dimensional structure, protein engineering and biochemical characters assay were performed to investigate the structure-function relationship of AFL. In the N- and C-terminal domain interface, K184, D370 and E372 form a electrostatic interaction network to stabilize these two domains. The activities of the mutants K184A, D370N and E372Q dromatically decreased. The optimal temperatur of mutant K184A became narrowly at 90℃ as compared with the broad range one (70-90℃) of wild-type AFL. A32, S332 and E339 located in the substrate binding tunnel were predicted to be involved in the substrate specificity of AFL. Through site-directed mutagenesis and activity assay the substrate specificity of the mutant A32W changed to favor the hydrolysis of middle chain-length esters as compared with the long-chain specificity of wild-type AFL, while the activities of S332W and E339W were lower than wild-type AFL. In the C-terminal domain, there is a putative divalent cation binding site composed of D405, D409 and D431. From kinetics assay, thermostabilty assay and thermo-dynamic assay, the binding of calcium benefited the substrate affinity of AFL at high temperature, and also enhanced the thermostability at the 90℃. On the other hand, magnesium did not affect the substrate affinity and stability of AFL. Through interfacial activation assay using triglycerides as substrate, AFL is proved to be a true lipase rather than a carboxylesterase. A drastic increase in lipase activity occurred when the solubility limit of tricaprylin was exceeded. This means that the apparent rate of hydrolysis correlate with the degree of micellar formation. Consistent with observations on other lipases, the lid conformation may change from closed to open form in the presence of lipid interface. Site-directed mutagenesis assays were performed to identify D405, D409 and D431 which form the cation binding site. These results revealed the structural basis of the thermoalkalophilic characteristics and the catalytic mechanism of AFL, and provides important clues for the engineering of AFL in biotechnological applications.