Enzymes embedded in barrier coatings for active and intelligent packaging

Enzymes are versatile biocatalysts which are increasingly being used for industrial purposes. However, the technical application of an enzyme is often limited by insufficient long-term stability under real life processing conditions and due to recycling problems. These weak spots can be avoided by immobilizing the enzymes. Moreover, the immobilization also provides the opportunity for improving the catalytic features and for avoiding protein contamination in the product.

Objective of the development

Figure 1: Scanning electron microscope image of p(styrene-co-AUPDS-1%)-nanoparticles, 100,000 times enlarged.

The focus of the project presented here is the development of active and intelligent packaging materials for monitoring the quality and shelf life of foodstuffs.This comprises the development of an active barrier layer to determine putrefaction gases in foodstuffs on paper and plastic materials in packaging for foodstuffs. We intend to achieve these targets by means of enzymatically modified nanocarrier systems.

With our NANOCYTES® technology we can couple biomolecules such as peptides, antibodies or enzymes to particulate systems in the nanometer range (Fig. 1). Here the basic properties and advantages of the conjugates are founded on their small size and the resulting volume/surface effect. For customer-specific applications we adapted bioconjugation strategies: through customized particle surfaces and the selection of suitable coupling strategies enzymes can be immobilized on the particle surface whilst retaining their full activity.

Linker-mediated coupling of silica nanoparticle surfaces

Figure 2: Fluorescence assays to prove enzyme activities. A) By means of color reaction (blue) it is possible to prove successful coupling of glucose oxidase with surfmer-nanoparticles; B) bicinchoninic acid-assay: assay for the quantitative, photometric determinations of proteins.

Amino and carboxylized silica nanoparticles were coupled to various different oxidoreductases such as laccase, glucose oxidase and catalase. Covalent links between the particle surface and the enzyme are generated by means of linker-mediated synthesis techniques. Through selection of suitable linker molecules it is possible to create molecular spacers to ensure the activity of the enzyme and reduce unspecific coupling. Determination of the concentration and activity of the coupled enzymes is carried out through suitable fluorescence assays (Fig. 2).

One-step enzyme coupling with "surfmers"

Figure 3: Ultrasound treatment for the re-suspension of the surfmer nanoparticles after centrifugation.

A new method for the manufacture of polymer particles with surface-active functional anchor sites is emulsion polymerization through utilization of polymerizable tensides, also called surfmer (derived from the words surfactant and momomer). The customized anchor sites of these polymer active ester surfmer particles are particularly suited for the coupling of biomolecules, due to the fact that the nitrogen-nucleophilic structural units of the enzymes can be linked in a single process step. The advantage of this process is that during the copolymerization process with a co-monomer the utilized polymerizable tensides are built into the polymer structure. As a result, the tenside does not separate from the particle surface and causes agglomeration during further utilization of the polymer particles (Fig. 3). In addition, the active ester unit which functions as the anchor point provides ideal reactivity with sensitive biomolecules whilst ensuring maximum stability during the manufacture, storage and transport of such polymer particles.

Immobilized enzymes

Figure 4: Enzyme activity of glucose oxidase with specific or non-specific coupling to p(mma-co-muPDs-3%)-nanoparticles.

On both particle types the enzymes laccase, glucose oxidase and catalase were immobilized and their enzyme activities were compared with each other. The immobilized enzymes on both the silica nanoparticles and the surfmer particles showed enzymatic activities upon being coupled. As an example the chart shows the activity of glucose oxidase immobilized on surfmer particles and on hydrolized surfmer particles. On the surfmer particles the enzyme couples specifically onto the designated bonding site. On hydrolized surfmer particles the enzyme bonds unspecifically as specific bonding is no longer possible due to the hydrolysis of the reactive groups.

Benefits for consumers

The motivation for the utilization of intelligent packaging materials is to increase consumer safety. These packaging materials will enable consumers to examine the shelf life and quality of the foodstuffs.


The project "Enzymes embedded in barrier coatings for active and intelligent packaging – ENZYCOAT II" was funded within the framework of the micro- and nanotechnology program MNT-ERA.NET of the German Federal Ministry of Education and Research (BMBF), Promotional reference 16SV3689.