Screening method for the discovery and directed evolution of oxygenase enzymes
AssigneeCalifornia Institute of Technology
InventorsFrances H. Arnold, John Joern, Takeshi Sakamoto, Ulrich Schwaneberg
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Abstract
The present invention relates to screening methods for oxidation enzymes, particularly mono- and dioxygenases. According to the methods of the invention, a product of an oxidation reaction is converted into a phenol or a catechol, which is easily detected by a Gibbs assay. This conversion allows for a sensitive and efficient assay. Both high-throughput liquid-phase and solid-phase methods using these principles are provided. Also described are method for detecting phenolic ether-products and sulfhydryl products from oxidation reactions, also using a Gibbs assay.
Claims62
A method for detecting an oxidation enzyme comprising the steps of:
- (a) contacting a test enzyme with a substrate and an oxygen donor to promote the formation of a cis-dihydrodiol from the substrate and the oxygen donor;
- (b) subjecting the cis-hydrodiol to acidic conditions to promote the formation of a phenol from the cis-dihydrodiol;
- (c) contacting the phenol with 2,6-dichloroquinone-4-chloroimide (Gibbs reagent) under neutral conditions to promote the formation of a detectable composition;
- and
- (d) testing for the detectable composition, wherein the presence of the detectable composition indicates that the test enzyme is an oxidation enzyme.
A method for detecting an oxidation enzyme comprising the steps of:
- (a) contacting an test enzyme with a substrate to promote the formation of a cis-dihydrodiol from the substrate;
- (b) contacting the cis-hydrodiol with cis-dihydrodiol dehydrogenase to promote the formation of a catechole from the cis-hydrodiol;
- (c) contacting the catechol with 2,6-dichloroquinone-4-chloroimide (Gibbs reagent) to promote the formation of a detectable composition;
- and
- (d) testing for the detectable composition, wherein the presence of the detectable composition indicates that the test enzyme is an oxidation enzyme.
A method for detecting an oxidation enzyme comprising the steps of:
- (a) contacting an test enzyme with a substrate to promote the formation of a product from the substrate;
- (b) contacting the product with an agent to promote the formation of a modified product, wherein the modified product is selected from the group consisting of a phenol and a catechol;
- (c) contacting the modified product with 2,6-dichloroquinone-4-chloroimide (Gibbs reagent) to promote the formation of a detectable composition;
- and
- (d) testing for the detectable composition, wherein the presence of the detectable composition indicates that the test enzyme is an oxidation enzyme.
A method for detecting an oxidation enzyme comprising the steps of:
- (a) contacting test enzyme with a substrate to promote the formation of a phenol ether, wherein the hydroxyl-group is attached to the aromatic part of the phenol ether;
- (b) contacting the phenol ether with 2,6-dichloroquinone-4-chloroimide (Gibbs reagent) to promote the formation of a detectable composition;
- and
- (c) testing for the detectable composition, wherein the presence of the detectable composition indicates that the test enzyme is an oxidation enzyme.
A method for detecting an oxidation enzyme comprising the steps of:
- (a) contacting a test enzyme with a substrate to promote the formation of a phenol ether, wherein the hydroxyl-group is attached to the ether part of the phenol ether;
- (b) allowing the phenol ether to dissociate into an aldehyde and a phenol;
- (c) contacting the phenol with 2,6-dichloroquinone-4-chloroimide (Gibbs reagent) to promote the formation of a detectable composition;
- and
- (d) testing for the detectable composition, wherein the presence of the detectable composition indicates that the test enzyme is an oxidation enzyme.
Dependencies are inferred from the claim text.