Multilayer enzyme-coupled magnetic nanoparticles as efficient, reusable biocatalysts and biosensors

Abstract

Herein we report the development of a highly active, magnetically retrievable and reusable biocatalyst using multilayer enzyme coupled-magnetic nanoparticles (MNPs) prepared by layer-by-layer assembly using two well-studied enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOX), as a model enzyme system. We show that by combining the use of a biocompatible linker as well as biospecific immobilisation, the first layer enzyme in our HRP(1)-MNP system retains the native activity of the enzyme in solution, and the overall catalytic activity of the multilayer enzyme system, HRP(x)-MNP, increases linearly with the increasing number of enzyme layers. Furthermore, the HRP(x)-MNP system can be conveniently retrieved by using an external magnetic field and reused for 10 consecutive cycles without apparent reduction of catalytic activity. We also report the development of a novel coupled bienzyme, GOX/HRP(x)-MNP, system that can perform bi-enzymatic reactions to couple the colourless GOX-catalyzed reaction to the chromophoric HRP-catalyzed reaction via H(2)O(2) production. This model bienzyme-MNP system can be used for simple, rapid colorimetric quantification of micromolar glucose.

Magnetic metal–organic frameworks as scaffolds for spatial co-location and positional assembly of multi-enzyme systems enabling enhanced cascade biocatalysis

“…Several attractive materials and successful strategies have been proposed leading to the design of an arsenal of multienzyme systems for mimicking the natural metabolic pathways in vitro. Examples are the preparations of hydrogels, 15,22 hourglass shaped nanochannel reactor, 23 mesoporous silica nanoparticles, 18,24 formation of inorganic nanocrystal-protein complexes 25 and self-assembled crystals, 26 microbeads, 27,28 DNA nanostructures, 4,5,11,[29][30][31] polymersomes, 14,32 nanoparticles, 33 nanobers, 13,16,34 graphene, 9,35 and metal-organic frameworks (MOFs). 17,36,37 In contrast to other scaffolds, MOFs, which are formed by the self-assembly of metal ions and organic linkers, feature ultrahigh surface area and porosity, uniform pores with tunable sizes, surfaces with variable chemistries, and structural diversity.…”

“…32 Garcia adopted a layer-by-layer assembly strategy using biotin-avidin interactions to achieve the desired spatial co-localization of GOx and HRP on magnetic Fe 3 O 4 nanoparticles. 33 Liu's group constructed the same bienzyme system based on the formation of the Cu 2+ -protein nanocrystal complexes. 25 Polyelectrolyte doped hollow bers were prepared by Zhang's group to accomplish the accurate control of enzyme positions in two multienzyme systems.…”

Facile preparation of magnetically functionalized graphite nanosheets for porcine pancreatic lipase immobilization

“…Enzyme as an important group of biocatalyst is superior over chemical catalyst because of its high effectiveness, high specificity, and green reaction conditions (Garcia et al 2011). Among the enzymes from abundant sources, lipases (glycerol ester hydrolases E.C.3.1.1.3) have been widely used including enantioselective hydrolysis, chiral resolution, esterification, synthesis of enantioenriched monomers, and macromolecules for polymerization, and other enzymatic reactions (Jaeger and Reetz 1998;Omprakash and Toyoko, 2005).…”

Nanobiocatalyst advancements and bioprocessing applications

“…In a cascade reaction, two enzymes immobilized in a short distance can accelerate the biocatalysis reactions since the diffusion paths of intermediates are reduced (figure 5b). Glucose oxidease-horseradish peroxidase immobilization onto magnetic NPs was used to demonstrate the concept [132].…”