Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB

Enzyme immobilization for intelligent packaging

Enzymes are versatile biocatalysts that are increasingly used in industrial applications. However, the technical application of an enzyme is often limited by poor long-term stability under real process conditions and by difficulties in recycling. These weaknesses can be circumvented by immobilising the enzymes. In addition, immobilisation offers the possibility of both improving the catalytic properties and avoiding protein contamination in the product.

 

Benefits for the consumer

The motivation for using intelligent packaging materials is to increase safety for the consumer. With the help of packaging, consumers will in future be able to check the shelf life and quality of the food on offer directly while shopping.

Active packaging materials for monitoring the quality and shelf life of food

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

The project focused on the development of active and intelligent packaging materials for monitoring the quality and shelf life of food. This included the development of an active barrier layer for the detection of putrefactive gases from food on paper and plastics in food packaging. We achieved these goals with the help of enzymatically modified nano-carrier systems.

With our NANOCYTES® technology, we can couple biomolecules such as peptides, antibodies or enzymes to particulate systems on the nanometer scale (Fig. 1). Here, the basic properties and advantages of the conjugates are based on their small size and the resulting volume / surface effect. For customer-specific applications we develop adapted bioconjugation strategies: Customized particle surfaces and the selection of suitable coupling strategies allow the immobilization of enzymes on the particle surface while maintaining their full activity.

Fluorescence assays to prove enzyme activities.
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.

Link-mediated coupling on silica nanoparticle surfaces

Amino- and carboxy-functionalized silica nanoparticles were coupled to a number of different oxidoreductases such as laccase, glucose oxidase and catalase. Link-mediated synthesis techniques were used to generate covalent bonds between particle surface and enzyme. Molecular spacers can be created by selecting the appropriate linker molecules to ensure the activity of the enzyme and to reduce unspecific binding. The determination of the concentration of the coupled enzymes and their activity was carried out by appropriately matched fluorescence assays.

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

One-step enzyme coupling with "surfmers"

A new method of producing polymer particles with surface-active functional anchor points is emulsion polymerization using polymerizable surfactants, so-called surfmers (surfactant monomer). The tailor-made anchor sites of these polymeric active ester surfmer particles are particularly suitable for binding biomolecules, since nitrogen-nucleophilic structural units of the enzymes can be bound in only one process step. This process has the advantage that the polymerizable surfactants used are incorporated into the polymer backbone during copolymerization with a co-monomer. Further use of the polymer particles produced in this way therefore does not result in the splitting off of the surfactant and the associated agglomeration. The active ester unit as anchor group also offers optimum reactivity with sensitive biomolecules and at the same time ensures maximum stability during production, storage and transport.

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

Immobilized enzymes

The enzymes laccase, glucose oxidase and catalase were immobilized on both particle types and their respective enzyme activities were compared. Enzymes immobilized on both silica nanoparticles and surfmer particles showed enzymatic activity after coupling. As an example, the activity of glucose oxidase immobilized on surfmer particles and on hydrolyzed surfmer particles is shown in the adjacent figure. On the surfmer particles the enzyme couples specifically to the intended binding site. On the hydrolysed surfmer particles the enzyme binds non-specifically, since the hydrolysis of the reactive groups means that specific binding is no longer possible.

Promotion

The results were developed in the project "Enzymes embedded in barrier coatings for active and intelligent packaging - ENZYCOAT II", which was funded by the German Federal Ministry of Education and Research (BMBF) within the framework of the micro- and nanotechnology program MNT-ERA.NET under the funding code 16SV3689.