Dados do Trabalho

Título

TRYPSIN IMMOBILISATION ON GRAPHENE OXIDE: EFFECT OF ENZYME CONCENTRATION ON THE CAPACITY OF IMMOBILIZATION AND PROTEOLYTIC ACTIVITY

Introdução

Trypsin is crucial for digestion, breaking down proteins into peptides for absorption. In the food industry, it reduces milk allergenicity in infant formula. Furthermore, it's used as an anti-inflammatory, biomarker for pancreatitis, pancreatic cancer, and cystic fibrosis. Enzymes are often immobilised in industrial applications to enhance stability, enable reuse, and facilitate reaction recovery. In this study, we report the use of graphene oxide (GO) as a support for trypsin immobilization. This nanomaterial has been attracting attention, especially due to its large specific surface area with different functional surface groups that allow the stacking of biomolecules on their surface without chemical surface modification.

Material e Métodos

The GO was synthesised by the exfoliation and oxidation of graphite flakes. The effect of trypsin concentration was studied: 0.5 mL of the enzyme (2.5; 5.0; 7.5; 10 mg/mL) in buffer phosphate (0.01 M at pH 7.0) was added to 1.5 mL of GO solution (2,0 mg/mL in water) and mixing for 1 hour in an ice bath. The capacity of immobilization (mg of enzyme/mg of GO), immobilization efficiency (%) and relative activity (%) of immobilized trypsin were evaluated against the free enzyme. The number of cycles of reuse was also determined. Casein from bovine milk was used as a substrate for enzyme activity.

Resultados e Discussão

The increase of trypsin concentration to 10mg/mL resulted in a significant increase in the immobilisation capacity by the graphene oxide which varied from 0.36 mg/mg ± 0.01 to 1.66 mg/mg. Moreover, the amount of immobilized trypsin showed a noteworthy increase from 86.72% ± 0.80 to 99.69% ± 0.29. The high content of immobilized trypsin on GO can be associated with the large surface area of the support associated with the low molecular weight of trypsin (23 kDa). The opposite behaviour is reported for enzyme activity, which reduces by 20% when the trypsin concentration exceeds 5.0 mg/mL. In terms of reusability, immobilised trypsin exhibited the capability of reuse about four times, maintaining about 40% of residual activity.

Conclusão

This study's findings can help improve the development of biocatalysts by understanding the interaction mechanism between trypsin and graphene oxide during enzyme immobilisation in nanomaterials.