Nanoparticle-attached enzyme cascades for accelerated multistep biocatalysis
Inventors
Medintz, Igor L. • Vranish, James N. • Ancona, Mario • Susumu, Kimihiro • Diaz, Sebastian A.
Assignees
Publication Number
US-11512305-B2
Publication Date
2022-11-29
Expiration Date
2037-12-13
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Abstract
A nanoparticle (for example, quantum dot) 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.
Core Innovation
The invention concerns a method of using nanoparticles, such as quantum dots, as substrates for immobilizing enzymes involved in consecutive reactions within an enzymatic cascade. This immobilization on nanoparticles results in a significant increase in the rate of catalysis and the final product yield compared to enzymes that are not immobilized. Typically, enzymes that carry out consecutive reactions are immobilized on surfaces to improve stability and catalytic efficiency, but immobilization on large surfaces often diminishes enzymatic activity. In contrast, immobilization on nanoparticles can enhance enzymatic activity due to their surface properties and ability to tightly bind enzymes via metal affinity coordination, for example, through hexahistidine tags.
The problem addressed is how to harness the enhancement effects of nanoparticle-bound enzymes within complex multistep enzymatic cascades. Industrial processes often require multiple sequential enzymatic steps to convert reactants into desired products. While immobilization of single enzymes on nanoparticles was known to enhance activity, demonstrating such enhancement for multi-enzyme cascades was lacking. This invention thus provides methods and embodiments where multiple enzymes forming a cascade are immobilized on nanoparticles, either individually or as clusters, enabling enhanced catalytic rates and substrate channeling effects to improve overall cascade efficiency.
Further, embodiments include enzymatic cascade clusters where nanoparticles are closely associated, each bound to multiple enzymes forming the cascade, with enzymes immobilized using polyhistidine sequences. The proximity of nanoparticles and enzyme co-localization facilitates greater catalytic enhancement, stabilizes enzymes, and facilitates substrate channeling without the need for excessive mixing. The invention includes methods of conducting cascade reactions by using nanoparticle clusters loaded with enzymes, contacting them with substrates, and allowing the enzymatic conversion to proceed under conditions minimizing stirring or mixing to preserve substrate channeling.
Claims Coverage
The patent includes one independent method claim that covers the main inventive aspects of conducting enzymatic cascades using nanoparticles and enzyme clusters.
Enzymatic cascade cluster with nanoparticles closely associated
A cascade cluster comprising multiple nanoparticles associated as a cluster, each bound to multiple enzymes configured as an enzymatic cascade with at least two different enzymes, and the nanoparticles are closely associated such that each nanoparticle is separated from its nearest neighbor by no more than about one nanoparticle diameter.
Performing cascade reaction with minimized mixing
Conducting a multistep enzyme cascade by contacting the cascade cluster with substrate and allowing the reaction to proceed so that each enzyme acts in succession to produce an end product, while minimizing stirring or mixing to preserve reaction enhancement.
Cross-linking of nanoparticles using enzymes with multiple polyhistidine tags
At least one enzyme in the cascade contains multiple polyhistidine tags that mediate cross-linking of nanoparticles into the cluster, enhancing the spatial arrangement of the enzymes and nanoparticles.
The independent claim covers the use of closely associated nanoparticle-enzyme clusters forming enzymatic cascades with specific structural organization and methods of conducting reactions that exploit minimized mixing and cross-linking via polyhistidine tags to improve catalytic efficiency and substrate channeling.
Stated Advantages
Metal nanoparticles can be functionalized with various surface ligands allowing tuning of surface properties.
Enzymes can be tightly and site-specifically bound to nanoparticles via genetically incorporated hexahistidine tags, allowing uniform orientations.
Attachment to nanoparticles can enhance individual enzyme activity and stabilize oligomeric enzyme structures at low concentrations.
The enhanced activity of bound enzymes can be harnessed in cascades, with enzymes either bound or unbound depending on effectiveness.
Co-localization of enzymes on nanoparticles enables substrate channeling that further enhances reaction kinetics.
Enzymes can be assembled on nanoparticles in controlled ratios and orientations, permitting pathway optimization.
Nanoparticles have large surface areas accommodating numerous enzymes leading to improved total turnover numbers.
Nanoparticle binding stabilizes enzymes and substrates/intermediates accumulate near the surface, facilitating substrate channeling.
If magnetic nanoparticles are used, they can facilitate catalyst removal or sequential addition to control chemistry.
Documented Applications
Use in industrial and pharmaceutical biocatalysis to enhance multi-step enzymatic cascade reactions, improving catalyst durability and total turnover.
Conducting cascades of enzyme-catalyzed reactions in cell-free environments with easy separation of products from nanoparticle-bound enzymes.
Enzymatic detection of metabolites and small molecules in clinical and other samples with increased enzyme longevity and enhanced signal production rates.
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