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Title: A High‐Throughput in Vitro Assay System for the Detection of the Enzymatic Dihydroxylation of Aliphatic Olefins
Abstract Rieske dioxygenase enzymes can perform thecis‐dihydroxylation of aliphatic olefins, representing a potential green alternative to established methods of performing this important transformation. However, the activity of the natural enzymes in this context is low relative to their more well‐known activity in thecis‐dihydroxylation of aromatics. To enable the engineering of dioxygenase enzymes for improved activity in the dihydroxylation of aliphatic olefins, we have developed an assay system to detect the relevant diol metabolites produced from whole‐cell fermentation cultures. Optimization studies were carried out to maximize the sensitivity of the assay system, and its utility in thein vitroscreening of enzyme variant libraries was demonstrated. The assay system was utilized in screening studies that identified Rieske dioxygenase variants with significantly improved activity in the dihydroxylation of aliphatic olefins relative to the wild‐type enzyme.  more » « less
Award ID(s):
2147098
PAR ID:
10575515
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
Advanced Synthesis and Catalysis
Date Published:
Journal Name:
Advanced Synthesis & Catalysis
Volume:
366
Issue:
21
ISSN:
1615-4150
Page Range / eLocation ID:
4392 to 4398
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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