7-days of FREE Audio papers, translation & more with Prime
7-days of FREE Prime access
7-days of FREE Audio papers, translation & more with Prime
7-days of FREE Prime access
https://doi.org/10.1016/j.bej.2019.01.023
Copy DOIJournal: Biochemical Engineering Journal | Publication Date: Feb 2, 2019 |
Citations: 2 |
We recently described LipJ, a mesophilic esterase clustering in a specific clade of bacterial lipase family I.5, bearing the same structure but several distinct sequence motifs. Two mutagenesis approaches were used here to modify LipJ by addition of common features of family 1.5 lipases, searching for changes on the activity profile. Iterative saturation mutagenesis of a residue possibly involved in Zn2+ coordination produced a LipJ variant –H110N– displaying a remarkable shift in substrate specificity, possibly boosted by changes in number and distance of relevant polar interactions for Zn2+ coordination. Also, variants E27 A, G136 A and M139Q were obtained by site directed mutagenesis as single, double or triple mutants, showing a general shift in specificity towards higher temperature and longer chain-length substrates, all of them displaying higher activity than LipJ on C7 substrates. The results obtained suggest that the N-terminal region of LipJ may indeed contribute to substrate accommodation, whereas mutations at the pentapeptide displayed maximum activity at 50 °C, with the triple mutant showing activity even at 80 °C, greatly enhanced by Mn2+. 3D modelling and molecular docking analysis of selected mutants provided clues for a better accommodation of medium chain-length substrates, and for a modified Zn2+ environment, in agreement with the substrate and temperature profile shifts observed.
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.