Photochemical Surface Functionalization of Synthetic Fibresby Excimer-UV-Lamp Irradiation
Photochemical treatments allow modifying the surface properties of synthetic fibres such as polyesters, polyamides or polyolefines significantly, if the fibres are irradiated in the presence of different reactive media1.
These processes are based on a homolytic bond cleavage initiated by the absorption of energetic photons and leading to the generation of radicals on the irradiated polymer surface. By grafting reactions suitable molecules can be added to these radicals. The use of common broadband UV sources has the disadvantage of non-specific excitation of polymeric substrate and reactive medium, and may also result in a thermal loading of the substrate and material damage. In contrast, monochromatic UV lamps, e.g. the so-called excimer lamps, allow a selective irradiation taking advantage of substrate and reagent specific absorption bands. In the case of poly (ethylene terephthalate) (PET) the use of a monochromatic KrCl-excimer lamp with a wave length of 222 nm is appropriate, because PET strongly adsorbs radiation between 220 nm and 250 nm. In the presence of oxygen (air) such irradiations initiate a hoto-oxidation of the substrate resulting in high surface energies. Using reactive media other than oxygen, the surface properties of the substrate can be engineered to demand. Allylic compounds are characterized by their marked affinity for addition reactions to radicals.
Enzymatic processes are of growing interest in modern catalysis technique with widespread potential applications. They are used on large scale e.g. in the food or textile industry, as additives in washing agents, and also in medical (diagnostics and therapeutics) or analytical chemistry. The use of enzymes has many advantages compared to conventional, non-enzymatic processes. Enzymes can be used in catalytic concentrations at low temperatures and pH-values near to neutral. Their high substrate selectivity allows a very careful treatment of the goods. The world-wide sale of industrial enzymes was estimated at 1.7 billion EUR in 2002 and is expected to reach 3.0 billion US $ in 2008.
An important step in minimizing the costs of enzyme application is the immobilization of these biocatalysts on suitable carrier materials. In the literature, numerous methods for enzyme immobilization on different carriers have been described. The binding can be of adsorptive, ionic or covalent nature. In general these techniques are conjugated with a high preparative and economic expense.
The oxidoreductase catalase catalyses the degradation of hydrogen peroxide into oxygen and water in human and animal metabolism and has also an essential function in most aerobic microorganisms. This enzyme is also of technical interest. Free or immobilized catalase preparations are e.g. used after textile bleaching processes before the following dyeing step or after milk sterilization to remove exceeding H2O2.
The use of textile carrier materials has several advantages: Textile fabrics are very cheap compared to other commercially available inorganic and organic carrier materials such as aluminum silicates, porous glass or different hydrophilic polymers. Their special structure guarantees an optimal substrate turn-over. The flexibility of fabrics allows reactor constructions of arbitrary geometry and a quick removal of the catalyst without any residues after the enzymatic reaction.
Based on former results, investigations were carried out concerning the use of monochromatic UV-light for the photochemical induced immobilization of the prot ein catalase. Besides their cross-linking properties, bifunctional reactive compounds were used as anchor molecules between synthetic polymers and the enzyme. The aim of this work was the development of a new, rapid and easy immobilization technique in contrast to conventional, wet chemical methods using textile fabrics as carrier materials which are advantageous due to their low costs and their special properties in terms of catalysis mentioned above. Latest investigations aim at the photochemical fixation of e.g. proteins or biopolymers on the polymer surface resulting in highly functionalized synthetic fibre materials with widespread applications in catalysis, medicine, textile industry and food technology.
- Photochemical Immobilization of catalase on textile carrier materials:
UV-light is able to cleave bonds in absorbing materials like PET, enzymes or cross-linking agents yielding radicals. These radicals can react with neighboured radical species forming new covalent bonds. In the presence of all reactants, a cross linked structure surrounding the polymer material is achieved. The procedure of the photochemical immobilization is divided up in three working steps (wetting, irradiation and washing):
• The polymers PET and PA 6.6 are wetted with a surfactant based aqueous emulsion of the reactive cross-linking medium and catalase avoiding the use of organic solvents.
• During the irradiation the monochromatic UV-light 222 nm generates radicals not only on the polymer surface, but also on the enzyme molecule surface. Using argon as inert atmosphere the photo oxidation of the polymer and also an oxidative damage of the catalase could be avoided. In a second step the addition of the radicals to double bonds of the cross-linking agents takes place and finally the bifunctional reactive chemicals form a cross-linked film surrounding the polymer surface.
• After the irradiation the samples must be washed to remove non-covalently fixed protein and cross-linking agent.
An easy qualitative test for the successful fixation of proteins like catalase is the colour reaction with ninhydrin. In the case of treated PET the reaction is positive. The test does not work for PA 6.6 due to the fact that the amino groups of the polymer react with ninhydrin themselves. Therefore UV-Vis-spectroscopy was used as a qualitative test for the immobilization of catalase on PA 6.6.
Moreover the three-dimensional cross-linked protein layer is visible using SEM-technique. Figure 2 shows pictures of PET and PA fabrics with and without immobilized catalase.
Catalase is an iron containing enzyme, so the protein load can be analyzed quantitatively by atomic absorption spectroscopy (AAS). The results are summarized in Table 1. The data correspond well with the qualitative measurements. 6.2 mg catalase per gram PA 6.6 are immobilized using no cross-linking agent. The load is more than three times higher using DAP or CHMV. Only 1.3 mg catalase are immobilized on 1 g PET. With the cross-linking agent DAP the best load was reached (32.2 mg/g PET). This relates to 64 % of the protein input.
The enzyme catalase catalyzes the degradation of hydrogen peroxide into oxygen and water. Covalent immobilization of enzymes is always accompanied by a decrease of activity because of the loss of mobility. Moreover, covalent bonds impair the ability of the enzyme to form the enzyme-substrate-complex (relative activity = activity/activity of free catalase). A successful enzyme immobilization must include a minimum of activity loss and a maximum of possible reuses. By measuring the enzymatic decomposition of hydrogen peroxide as a function of time, it is possible to calculate the relative activity of the immobilized enzyme in comparison to free catalase. The immobilization products obtained with catalase show even after 20 reuses a significant activity using the cross-linker DAP or CHMV (see Table 1). The catalase loses 80 - 90 % of their activity. Nevertheless, the integral activity after twenty reuses is much higher than the activity of free catalase, which could only be used once in technical processes. Immobilizing catalase on PA 6.6 using DAP as cross-linking agent gave the best results. The integral activity increases to 366 % after twenty reuses.
Low-cost synthetic textile fabrics made of polyester or polyamides are an alternative carrier material for the immobilization of enzymes. With a low preparative expense fabrics with a high protein load and a distinct activity can be produced by irradiating the materials with excimer-UV-lamps in the presence of cross-linking agent. The special construction of fabrics allows an adjustable throughput and a high substrate turn-over.
Moreover conventional immobilization products are mostly offered as granulates or pellets, which must be filtrated after the enzymatic reactions. Fabrics can be removed very quickly from the reactor without any filtration step and any protein residues after the enzymatic reaction.
Catalase can be used e.g. in textile manufacturing after bleaching to decompose residual hydrogen peroxide, which disturbs the following dyeing process. The catalase treatment decreases the amount of needed washing water strongly.
Unfortunately the free enzyme can be used only once, because the treated liquor must be rejected before the next manufacturing batch can be started. Immobilized catalase can be taken out of the treated liquor and is therefore reusable in the next batch. In the investigated case up to 32.2 mg catalase can be immobilized on 1.0 g of the synthetic carrier materials and even in solutions with very high substrate concentrations the photochemical treated fabrics are reusable at least twenty times retaining a high activity.
The investigations in this field of photochemical enzyme immobilization on synthetic polymers are just started and are not limited to textile constructions. Moreover the procedure seems to be transferable to many other enzymes or other proteins of biochemical interest. Confirming this supposition, the new photochemical immobilization technique is able to compete with conventional immobilization procedures and will open widespread applications in biocatalysis in future.