Catalytic biomaterials: Engineering organophosphate hydrolase to form self-assembling enzymatic hydrogels

Abstract

Organophosphate (OP) neurotoxins have contaminated the environment, contributed to millions of poisoning annually, and have been used as chemical weapons. Biomaterials incorporating the native activity of the OP hydrolase (OPH) enzyme are of interest for applications including OP sensing, environmental bioremediation and prophylactic decontamination. We have engineered and characterized four novel hydrogel-forming OPH variants by genetically fusing the OPH enzyme with α-helical leucine zipper domains (H), unstructured soluble linker domains (S) and polyhistidine purification tags. The appended H domains form physical cross-links between the enzymes and enable self-assembly of the enzymes into hydrogels. The addition of the H and S fusions significantly increased the expression levels of soluble protein. OPH constructs with biterminal H domains form hydrogels at lower protein weight percents and exhibit higher enzymatic activity than those variants modified with a single H domain fusion. Polyhistidine tags were not useful for purification but they were not benign, as the addition of the 6His tags increased the hydrogel-forming abilities of the proteins with a concomitant reduction in both the kcat and KM values. Active enzymatic hydrogels could be made from concentrated unpurified crude protein lysates, significantly simplifying the processing and utilization of the biomaterials. And, a simple proteinaceous bioactive surface coating exhibiting OPH activity is demonstrated. The hydrogels were stable over long-term storage, as activity was retained after cold storage in buffer after 5 months. These new protein constructs further show the use of rational protein design to create novel, bifunctional, self-assembling units for the formation of catalytic biomaterials. © 2010 The Author. Published by Oxford University Press. All rights reserved.