RESUMO
An actinobacteria strain was isolated from an olive waste mill and tested for protease production on skimmed milk media. The strain identification was achieved through both 16 S rDNA sequencing and phenotypic characterization. The enzyme was purified using the ammonium sulfate/t-butanol three-phase partitioning (TPP) method, followed by characterization to investigate the effect of pH, temperature, and various chemical agents. Subsequently, the enzyme was assessed for its milk coagulation activity. The strain belonging to the Streptomyces genera, exhibits significant phylogenetic and phenotypic differences from the aligned species, suggesting its novelty as a new strain. The enzyme was best separated in the TPP aqueous phase with a 5.35 fold and 56.25% yield. Optimal activity was observed at pH 9.0 and 60 °C, with more than half of the activity retained within the pH range of 7-10 over one hour. The protease demonstrated complete stability between 30 and 60 °C. While metallic ions enhanced enzyme activity, EDTA acted as an inhibitor. The enzyme displayed resistance to H2O2, SDS, Tween 80, and Triton X-100. Notably, it was activated in organic solvents (ethyl acetate, petroleum ether, and xylene), maintaining > 75% of its original activity in butanol, ethanol, and methanol. Additionally, the enzyme yielded high milk coagulant activity of 11,478 SU/mL. The new Streptomyces sp. protease revealed high activity and stability under a wide range of biochemical conditions. Its use in the dairy industry appears particularly promising. Further industrial process investigations will be valuable in determining potential uses for this enzyme.
RESUMO
Complications associated with Type 1 diabetes (T1D) have complex origins that revolve around chronic hyperglycemia; these complications involve hemostasis disorders, coagulopathies, and vascular damage. Our study aims to develop innovative approaches to minimize these complications and to compare the outcomes of the new approach with those of traditional methods. To achieve our objective, we designed novel nanoparticles comprising covalent organic frameworks (nCOF) loaded with insulin, termed nCOF/Insulin, and compared it to subcutaneous insulin to elucidate the influence of insulin delivery methods on various parameters, including bleeding time, coagulation factors, platelet counts, cortisol plasma levels, lipid profiles, and oxidative stress parameters. Traditional subcutaneous insulin injections exacerbated hemostasis disorder and vascular injuries in streptozotocin (STZ)-induced diabetic rats through increasing plasma triglycerides and lipid peroxidation. Conversely, oral delivery of nCOF/Insulin ameliorated hemostatic disorders and restored the endothelial oxidant/antioxidant balance by reducing lipid peroxidation and enhancing the lipid profile. Our study pioneers the understanding of how STZ-induced diabetes disrupts bleeding time, induces a hypercoagulable state, and causes vascular damage through lipid peroxidation. Additionally, it provides the first evidence for the involvement of subcutaneous insulin treatment in exacerbating vascular and hemostasis disorders in type 1 diabetes (T1D). Introducing an innovative oral insulin delivery via the nCOF approach represents a potential paradigm shift in diabetes management and patient care and promises to improve treatment strategies for type 1 Diabetes.