Multi-Enzyme Assembly on T4 Phage Scaffold

Abstract

Over the past two decades, various scaffolds have been designed and synthesized to organize enzyme cascades spatially for enhanced enzyme activity based on the concepts of substrate channeling and enhanced stability. The most bio-compatible synthetic scaffolds known for enzyme immobilization are protein and DNA nanostructures. Herein, we examined the utility of the T4 phage capsid to serve as a naturally occurring protein scaffold for the immobilization of a three-enzyme cascade: Amylase, Maltase, and Glucokinase. Covalent constructs between each of the enzymes and the outer capsid protein Hoc were prepared through SpyTag-SpyCatcher pairing and assembled onto phage capsids in vitro with an estimated average of 90 copies per capsid. The capsid-immobilized Maltase has a fourfold higher initial rate relative to Maltase free in solution. Kinetic analysis also revealed that the immobilized three-enzyme cascade has an 18-fold higher converted number of NAD + to NADH relative to the mixtures in solution. Our results demonstrate that the T4 phage capsid can act as a naturally occurring scaffold with substantial potential to enhance enzyme activity by spatially organizing enzymes on the capsid Hoc.

Catalytic Kinetics Considerations and Molecular Tools for the Design of Multienzymatic Cascade Nanoreactors

“…Compared to free enzymes in solution, the immobilized enzymes exhibited up to 18-fold enhanced catalytic activity demonstrating the potential of icosahedral T4 phage as potential platform for enzyme assembly. [74] Without doubt VLPs are attractive in the medical area especially for vaccination and therapeutic cargo delivery. However, there are associated drawbacks like reusability, time consuming production process including purification and precise analysis as well as lower storage stability at normal temperatures.…”

Emerging Solutions for in Vivo Biocatalyst Immobilization: Tailor-Made Catalysts for Industrial Biocatalysis

“…These three enzymes are part of a four-enzyme pathway, converting maltoheptaose and 6-phosphogluconolactone . The enzymes were linked to the SpyTag, and the outer capsid protein Hoc was fused to SpyCatcher . By detecting the formation of the byproduct NADH, it could be demonstrated that the virus capsid-based immobilization of the enzyme cascade resulted in an 18-times higher activity as compared to the respective enzymes in solution.…”

“…204 The enzymes were linked to the SpyTag, and the outer capsid protein Hoc was fused to SpyCatcher. 205 By detecting the formation of the byproduct NADH, it could be demonstrated that the virus capsid-based immobilization of the enzyme cascade resulted in an 18-times higher activity as compared to the respective enzymes in solution. Furthermore, Bustos-Jaimes and coworkers described the generation of a POI nanocarrier based on the parvovirus B19 VLP, whose shell is composed of the structural protein V2.…”

Enzymatic activity of individual bioelectrocatalytic viral nanoparticles: dependence of catalysis on the viral scaffold and its length

“…[21][22][23][24][25][26] Multi-enzyme systems, where differing enzymes work in cascade, have also been assembled on DNA origamis 27,28 and viral particles. [29][30][31][32][33][34][35] In many instances, these artificial enzymatic cascades showed better activity than the cascaded enzymes freely cooperating in solution. [36][37][38][39] This improvement was initially assigned to a more efficient channeling of the metabolites between the immobilized cascade enzymes.…”