It appears you don't have support to open PDFs in this web browser. To view this file, Open with your PDF reader
Abstract
A key event in the directed evolution of enzymes is the systematic use of mutagenesis and selection, a process that can give rise to mutant libraries containing millions of protein variants. To this day, the functional analysis and identification of active variants among such high numbers of mutational possibilities is not a trivial task. Here, we describe a combinatorial semi-rational approach to partly overcome this challenge and help design smaller and smarter mutant libraries. By adapting a liquid medium transesterification assay in organic solvent conditions with a combination of virtual docking, iterative saturation mutagenesis, and residue interaction network (RIN) analysis, we engineered lipase B from P. antarctica (CalB) to improve enzyme recognition and activity against the bulky aromatic substrates and flavoring agents methyl cinnamate and methyl salicylate. Substrate-imprinted docking was used to target active-site positions involved in enzyme-substrate and enzyme-product complexes, in addition to identifying ‘hot spots’ most likely to yield active variants. This iterative semi-rational design strategy allowed selection of CalB variants exhibiting increased activity in just two rounds of site-saturation mutagenesis. Beneficial replacements were observed by screening only 0.308% of the theoretical library size, illustrating how semi-rational approaches with targeted diversity can quickly facilitate the discovery of improved activity variants relevant to a number of biotechnological applications.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Details


1 Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique (INRS), Université du Québec, 531 Boulevard des Prairies, Laval, QC, Canada
2 Centre Armand-Frappier Santé Biotechnologie, Institut National de la Recherche Scientifique (INRS), Université du Québec, 531 Boulevard des Prairies, Laval, QC, Canada; PROTEO, the Québec Network for Research on Protein Function, Engineering, and Applications, 1045 Avenue de la Médecine, Université Laval, Quebec City, QC, Canada