Mutant organophosphorus acid anhydrolase enzymes with stereospecificity on Sarin enantiomers and uses thereof
Inventors
Bae, Sue Y • Guelta, Mark A. • Harvey, Steven P
Assignees
United States Department of the Army • Government of the United States of America
Publication Number
US-10143874-B1
Publication Date
2018-12-04
Expiration Date
2037-09-21
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Abstract
Disclosed herein are non-wild-type organophosphorus acid anhydrolases having three site mutations, methods of production, and methods of use to effectively degrade toxic chemicals such as((RS)-Propan-2-yl methylphosphonofluoridate)(Sarin) and other organophosphorus compounds. Also provided are organophosphorus acid anhydrolase mutants capable of degrading Sarin with distinct selective stereospecificity preferences differing from the wild-type organophosphorus acid anhydrolase
Core Innovation
The invention relates to non-wild-type organophosphorus acid anhydrolases (OPAA) enzymes having three site mutations at positions 212, 215, and 342 of the wild-type sequence SEQ ID NO: 1, specifically substitutions Y212F, I215D, and V342L. These mutants exhibit similar or greater catalytic activity toward the degradation of the toxic chemical Sarin ((RS)-Propan-2-yl methylphosphonofluoridate) compared to the wild-type enzyme, with a distinct selective stereospecificity preference differing from wild-type. The enzyme can catalytically degrade Sarin with higher preference for the P(+) enantiomer.
The problem being addressed is the marginal catalytic activity of the native wild-type OPAA enzyme in degrading Sarin, a highly toxic organophosphorus nerve agent. Wild-type OPAA catalyzes both Sarin enantiomers with similar rates and thus is not effective for enrichment or decontamination purposes targeted specifically at Sarin’s more toxic enantiomers. Current enzymes have limited activity or stereospecificity for effective detoxification and decontamination of Sarin and related organophosphorus compounds.
The core innovation involves engineering the non-wild-type OPAA with targeted amino acid substitutions that modify the substrate binding site, particularly the small pocket formed by residues at positions 212, 215, and 342. This leads to enhanced catalytic efficiency and altered stereoselectivity, with a stronger preference for hydrolyzing the less toxic P(+) enantiomer of Sarin. This enzymatic stereospecificity allows enrichment and isolation of the more toxic P(−) enantiomer and facilitates improved identification of Sarin enantiomers in chromatographic analysis.
Claims Coverage
The patent discloses six claims covering an isolated mutant OPAA enzyme, methods of degrading Sarin using the mutant enzyme, stereospecific activity, and kits incorporating the mutant enzyme. The claims focus on the mutated amino acid sequence SEQ ID NO:2 and its functional properties.
Isolated mutant organophosphorus acid anhydrolase enzyme
An isolated mutant OPAA enzyme comprising the specific amino acid sequence of SEQ ID NO:2, characterized by substitutions Y212F, I215D, and V342L relative to the wild-type sequence.
Method for degrading Sarin using the mutant enzyme
A process involving contacting Sarin ((RS)-Propan-2-yl methylphosphonofluoridate) with the isolated mutant OPAA enzyme comprising SEQ ID NO:2 to catalytically degrade Sarin.
Stereospecific catalytic activity favoring P(+) enantiomer
The mutant OPAA enzyme exhibits catalytic activity toward both Sarin enantiomers but has increased activity towards the P(+) enantiomer compared to the P(−) enantiomer, enabling enrichment of the P(−) enantiomer.
Kit for degrading Sarin incorporating the mutant enzyme
A kit comprising the isolated mutant OPAA enzyme of SEQ ID NO:2, for use in degrading Sarin.
Use of the mutant enzyme in stereospecific hydrolysis enabling P(−) enantiomer enrichment
The isolated mutant OPAA enzyme preferentially hydrolyzes the P(+) enantiomer of Sarin, facilitating enrichment of the P(−) enantiomer.
Method of enriching the P(+) enantiomer of Sarin
A method comprising contacting a Sarin sample with the mutant OPAA enzyme of SEQ ID NO:2 to enrich the P(+) enantiomer.
The independent claims cover the isolated mutant OPAA enzyme characterized by three amino acid substitutions, methods of enzymatic degradation of Sarin using this mutant, stereospecific catalytic activity favoring P(+) enantiomer hydrolysis, and related kits and enrichment methods. These claims define the core inventive features as the specific triple mutation sequence and its stereospecific functional properties.
Stated Advantages
The mutant OPAA enzyme has similar or greater catalytic efficiency for Sarin degradation compared to wild-type, improving practical utility for detoxification.
Distinct stereospecificity favoring hydrolysis of the less toxic P(+) enantiomer allows enrichment and isolation of the more toxic P(−) enantiomer for study or selective degradation.
Enhanced stereospecific enzymatic activity enables improved identification of Sarin enantiomers in analytical chiral chromatographic separations.
Documented Applications
Catalytic degradation of Sarin nerve agent and other organophosphorus compounds such as Soman and Cyclosarin for detoxification and decontamination.
Enzymatic enrichment and isolation of individual Sarin enantiomers, specifically enabling enrichment of the P(−) enantiomer.
Use in kits and compositions for catalytic degradation of Sarin, facilitating environmental and surface decontamination.
Facilitating identification of Sarin enantiomers through enzymatically enriched samples for chiral chromatographic analysis.
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