Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

Alaa A A Aljabali,George P Lomonossoff,Nicole F Steinmetz,J Elaine Barclay,David J Evans

doi:10.1039/c2nr31485a

Alaa A A Aljabali, George P Lomonossoff + Show 3 more

Open Access

https://doi.org/10.1039/c2nr31485a

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Abstract

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.

Highlights

The enzymes horseradish peroxidase (HRP) and glucose oxidase (GOX) have been sequentially co-immobilised in polyelectrolyte multilayers on silica nanoparticles to facilitate the overall enzymatic reaction: the GOX layer generates hydrogen peroxide in the presence of b-D-glucose that diffuses toward the HRP layer to be reduced to water in the presence of o-dianisidine (HRP substrate).[6]
The approach was used to oxidize hydroxyl-containing carbohydrate within the HRP and GOX to create reactive aldehyde groups, which were conjugated to acid dihydrazide (ADH) and subsequently coupled to activated carboxylate ester groups on the cowpea mosaic virus (CPMV) external surface (Fig. 1)
The enzymes HRP and GOX have been covalently coupled to the CPMV capsid external surface via the carbohydrate residues on the enzyme surface

Summary

Introduction

Enzyme–nanoparticle conjugates that exploit the catalytic activity of the bound enzyme have been reported for various applications,[1,2] especially for biosensing.[3,4,5] For example, the enzymes horseradish peroxidase (HRP) and glucose oxidase (GOX) have been sequentially co-immobilised in polyelectrolyte multilayers on silica nanoparticles to facilitate the overall enzymatic reaction: the GOX layer generates hydrogen peroxide in the presence of b-D-glucose that diffuses toward the HRP layer to be reduced to water in the presence of o-dianisidine (HRP substrate).[6]. The ability to immobilise enzymes on a support is of importance as it enables easy separation of reaction products from the enzyme, facilitates the construction of porous multilayers and 5640 | Nanoscale, 2012, 4, 5640–5645 the development of micro-fluidic and lab-on-a-chip type devices.[11,12] The advantages of immobilisation of enzymes onto virus nanoparticles include multiple attachment sites, defined geometry and the potential for the construction of permeable, multilayer catalytic arrays

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