Open AccessEngineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase
Author(s) -
Časaitė Vida,
Sadauskas Mikas,
Vaitekūnas Justas,
Gasparavičiūtė Renata,
Meškienė Rita,
Skikaitė Izabelė,
Sakalauskas Mantas,
Jakubovska Jevgenija,
Tauraitė Daiva,
Meškys Rolandas
Publication year - 2019
Publication title -
microbiologyopen
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.881
H-Index - 36
ISSN - 2045-8827
DOI - 10.1002/mbo3.795
Subject(s) - chromogenic , indole test , monooxygenase , substrate (aquarium) , chemistry , enzyme , carboxylic acid , biochemistry , combinatorial chemistry , chromatography , biology , cytochrome p450 , ecology
Here, we present a proof‐of‐principle for a new high‐throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme‐coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro‐chromogenic substrate, which is transformed by the target enzyme to indole‐3‐carboxylic acid. The later compound is converted to indoxyl by a newly identified indole‐3‐carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme‐producing colonies turn blue. Two types of pro‐chromogenic substrates have been tested. Indole‐3‐carboxaldehydes and the amides of indole‐3‐carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine‐tuning of the pro‐chromogenic substrate allows screening enzymes with the desired substrate specificity.