Enhancing promiscuous chemistries of a Schiff-base forming enzyme by divergent evolution
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Date
2020Type
- Book Chapter
Abstract
Directed evolution has emerged as a powerful technique for the rapid tailoring of enzymes toward particular synthetic demands, spawning a number of enzymes capable of complex chemical transformations. During random mutagenesis of a protein, changes in fitness must be assayed in order to quantify and understand the relative effect a given mutation has, and the assay employed must be carefully chosen to report directly on the transformation of interest. Here, we describe a series of medium-throughput screening techniques that have been utilized for the evolution and engineering of an artificial carboligase, RA95.5-8, resulting in improvement of catalytic efficiency of a number of promiscuous chemistries. The methods make use of common analytical chemistry equipment and low-cost materials, and may help inspire development of novel screening workflows for related transformations. Show more
Publication status
publishedEditor
Book title
Enzyme Engineering and Evolution: Specific Enzyme ApplicationsJournal / series
Methods in EnzymologyVolume
Pages / Article No.
Publisher
Academic PressSubject
Directed evolution; Protein engineering; Screening; Assay; Artificial enzymes; Carboligase; HPLC; UV/VisOrganisational unit
03492 - Hilvert, Donald (emeritus) / Hilvert, Donald (emeritus)
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