Kitosan nanolifleri üzerine β-galaktosidaz immobilizasyonu

Enzymes are biocatalysts which offer a wide variety of specific functions. Large amounts of enzymes are used in native forms at industrial scale. Use of free enzyme means one-time application with no chance of recovery and reusability. Additionally free enzymes show low stability in harsh processing environments. They are sensitive to changing processing conditions such as pH and temperature change. Immobilized enzymes serves some opportunities for recovery and multiple use of enzymes providing higher stability and selectivity. Enzyme immobilization is defined as the attachment of a free enzyme on a carrier (supporting) surface which restricts or prevents the mobility of the enzyme. Immobilization technique was started to be used in 1916. The studies showed that immobilization facilitates higher stability in challenging processing conditions. In the following years, the performance of immobilized enzyme was studied to improve it. It was discovered that performance of the immobilized enzyme is highly dependent on the surface characteristics and binding methods. Afterwards, novel carrier materials and methods were studied. Inorganic or organic, hydrophilic or hydrophobic, porous or nonporous features of the carrier has altered the interaction of the enzyme with the surface. It affected the activity and the stability of the immobilized enzyme. With development of nanofibers it was found that nanomaterials has a promising potential as carrier surfaces due to their high surface area. The change of some biological and chemical characters in nanoscale compared to the macro or micro scales, pointed out that nanomaterials nano-materials can be utilized in broad range of applications. Nanotechnology involves production of materials, devices or a system in the range of 1 to 100 nm. Nanosheets, nanoparticles, nanotubes, nanofibers are examples of nanostructures. Among these, nanofibers has attracted the most attention due to their high surface to volume ratio, simple and cost-effective production, easily modifiable surface properties. The most preferred method is electrospinning for production of nanofibers. Electrospinning system consists of a high voltage power supply, pump, syringe and a collector plate. The polymer is dissolved in a suitable solvent and located on the pump in a syringe. The power supply gives a charge to the system which creates an electrical field between the capillary of the syringe and the collector plate. With the pump the solution is fed with a definite rate to the electrical field and collected on the collector plate. In this study nanofibers were fabricated using electrospinning, which is a simple and cost effective technique. The polymer was preferred as chitosan which is a non-toxic, biocompatible, biofunctional material. Since the type of solvent changes the solubility of polymer hence structure of the nanofiber, acetic acid, trifluoroacetic acid (TFA) and dicloromethane (DCM) were studied. The best solubility was observed for the solution of TFA and DCM. A solution of TFA and DCM was prepared in the ratio of 70:30. Chitosan was dissolved in TFA-DCM solution at 3% concentration. Chitosan nanofibers were fabricated at 25 kV with feed rate of 0.4 ml/h. Distance of the plate from needle was changed between 10 and 15 cm. β-galactosidase was preferred for immobilization, since it is one of the mostly used enzymes in food industry. It is used for degradation of lactose into glucose and galactose. Unlike chitosan, chitosan nanofibers are water soluble. Because of that, chitosan nanofibers reqiure to be functionalized to become insoluble and active by a coupling agent. Glutaraldehyde (GA) was used as functionalizing agent at concentration of 4%. Chitosan nanofibers were treated in GA and with GA vapor. After functionalization, chitosan nanofibers were incubated in enzyme solutions with different enzyme concentrations. The enzyme immobilization efficiency, reusability and thermal stability were measured by simply measuring the activity of enzyme solutions by use of a spectrophotometer. Assays were conducted by comparing the activities of free and immobilized β-galactosidase. The activity was determined by use of a substrate o-Nitrophenyl ß- D – Galactopyranoside (ONPG) which gives a colorful product after hydrolzed by β-galactosidase. A successful enzyme loading efficiency was obtained up to 69±20%. The immobilized and the free β-galactosidase showed highest activity at 50 °C. The activities at 50 °C were considered as 100% activity to observe the percental change in activity clearly. The activities of the immobilized and free enzyme decreased when the temperature was increased up to 90°C. At 70°C the activity of the free enzyme decreased sharply to 31±0.01% as the activity of the immobilized enzyme was lowered to 73.5±0.1%. After 10 cycles of use of immobilized β-Galactosidase at 40°C, 68±26% of the enzyme activity was retained. These results was found to be promising when compared with the data from literature. The reusability of chitosan nanofibers can be improved by production of magnetic chitosan nanofibers in further studies. It should be taken into consideration that nanomaterials have different chemical and physical characteristics compared to their macro and micro size. The results of intake of nanofibers into the body is not known for certain. For use of nanofibers in food applications, toxicological studies and regulations are required.