Smart mutational exploration of the CalB lipase active site using a combination of virtual screening and iterative saturation mutagenesis
One of the key elements for proper directed evolution of protei ns is the cyclic use of mutagenesis and selection processes, giving rise to libraries containing mi llions of mutants. However, analyzing such an important number of mutants is not a trivial task, as the identification of active variant...
| Main Authors: | , , |
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| Format: | Other/Unknown Material |
| Language: | unknown |
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| Online Access: | https://espace.inrs.ca/id/eprint/5794/ http://www.profs.inrs.ca/ndoucet/PEC2016_Final_Program.pdf. |
| Summary: | One of the key elements for proper directed evolution of protei ns is the cyclic use of mutagenesis and selection processes, giving rise to libraries containing mi llions of mutants. However, analyzing such an important number of mutants is not a trivial task, as the identification of active variants among millions of possibilities quickly becomes exhaus tive and inefficient. Here we describe a semi-rational combinatorial approach supported by vi rtual docking to generate smaller and smarter libraries. Because of its ability to perform the sy nthesis of esters in organic media, lipase B from Pseudozyma antarctica (CalB) was used as an indus trially-relevant model system. Since CalB displays very low activity towards bulky substrates, the main goal of this project was aimed at the development of CalB variants with enhanced synthet ic activity towards bulky substrates. Substrate-imprinted docking was used to uncover tar get positions involved with the stabilization of the enzyme-substrate complex, identifying “hot spots” that are most likely to yield active improvements for desired ligands. The Iterative Saturati on Mutagenesis strategy was employed to sequentially incorporate favorable mutations, furth er increasing our chances of selecting improved variants with a concomitant reduction in scr eening effort. We tested a limited number of 164 mutants that explored 6 residue positions in the active-site cavity of CalB. For a single round of mutagenesis and selection against 2 different s ubstrates, a number of variants showed up to 5-fold increase in activity relative to WT CalB. T hese results represent the first stage in the development of additional CalB variants with improved ac tivity towards bulky esters. |
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