Self-assembled nanoplatelet-enzyme bioconjugates providing for increased biocatalytic efficiency
Inventors
Medintz, Igor L. • Breger, Joyce • Walper, Scott • Stewart, Michael H.
Assignees
Publication Number
US-11795483-B2
Publication Date
2023-10-24
Expiration Date
2037-12-13
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
A nanoplatelet serves as a substrate for immobilizing enzymes involved in consecutive reactions as a cascade. This results in a significant increase in the rate of catalysis as well as final product yield compared to non-immobilized enzymes or enzymes immobilized to quantum dots.
Core Innovation
The invention relates to the use of nanoplatelets (NPLs) as substrates for immobilizing enzymes involved in consecutive reactions configured as an enzymatic cascade, where the product of one enzyme serves as the substrate for the next. These nanoplatelets cluster together, with enzymes bound to their surface through metal coordination chemistry, notably via oligohistidine tags binding to zinc-rich NPL surfaces, resulting in enhanced catalytic efficiency and product yield compared to enzymes free in solution or immobilized on spherical nanoparticles like quantum dots.
The background problem addressed is that while enzyme immobilization enhances stability and allows enzyme reuse, attaching enzymes to large surfaces typically reduces their activity. Although immobilization on nanoparticles can increase enzyme activity, improvements remain needed. This invention resolves this by using quasi-two-dimensional nanoplatelets, which, due to their geometry and surface properties, allow greater enzyme loading, stabilization, and enhanced substrate channeling, culminating in improved enzymatic activity and resistance to parasitic enzyme interference.
This method of creating enzymatic cascade clusters involves close association of nanoplatelets, separated on average by no more than one nanoplatelet diameter, promoting substrate channeling and increased overall cascade reaction efficiency without the need for stirring or mixing. The assembly order and batch versus sequential methods further influence activity, demonstrating that ordered and simultaneous enzyme assembly onto NPL clusters optimizes catalytic performance.
Claims Coverage
The patent contains two independent claims describing methods for conducting enzymatic cascade reactions using nanoplatelet clusters and preparing such enzymatic cascade clusters with defined assembly characteristics.
Method of conducting an enzymatic cascade reaction using nanoplatelet clusters
Provides a cascade cluster comprising closely associated nanoplatelets each bound to multiple different enzymes arranged as an enzymatic cascade. The method contacts the cluster with substrate and allows the cascade reaction to proceed sequentially without stirring or mixing, wherein nanoplatelets are separated by no more than about one nanoplatelet diameter on average.
Method of preparing enzymatic cascade clusters with crosslinked nanoplatelets
Forms enzymatic cascade clusters by contacting nanoplatelets with multiple different enzymes, where at least one enzyme has multiple hexahistidine tags effective to cross-link nanoplatelets into a cluster closely associated such that nanoplatelets are separated on average by no more than one nanoplatelet diameter. The contacting can be performed either batch-wise (simultaneous mixing) or sequentially.
The claims cover methods of assembling and utilizing clusters of nanoplatelets densely functionalized with enzymes arranged as cascades, where physical proximity and enzyme binding strategies enhance catalytic efficiency and product yield in enzymatic cascade reactions without the need for active mixing.
Stated Advantages
Metal-NPLs can be functionalized with various surface ligands providing different charges, polarities, and steric bulk.
Enzymes bind easily and tightly via genetically incorporated hexahistidine tags, allowing uniform enzyme orientation.
NP attachment can enhance the activity of individual bound enzymes and stabilize oligomeric enzyme structures.
Co-localization of enzymes on NPLs fosters substrate channeling, increasing reaction kinetics.
Enzymes can be assembled on NPLs in controlled ratios and orientations to tune pathway catalytic rates.
NPLs support greater enzyme surface loading and cluster formation compared to spherical nanoparticles, resulting in improved enzymatic efficiency.
Enzyme-NPL clusters exhibit resistance to fouling and off-site reactions from parasitic enzymes.
The method provides a more durable catalyst with improved total turnover number, contrasting conventional immobilization that sacrifices enzyme efficiency for stability.
Documented Applications
Industrial chemical reactions benefiting from enzymatic biocatalysis, where the bioconjugates improve enzyme stability and catalytic efficiency.
Conducting enzyme cascade reactions in a completely cell-free environment with easy separation of products from nanoparticle-bound enzymes.
Enzymatic detection of metabolites and small molecules in clinical and other samples, with enhanced enzyme longevity and signal production.
Commercial enzyme use in industry and pharmaceutical synthesis to catalyze chemical transformations using NP-enzyme constructs.
Interested in licensing this patent?