Cistatinas recombinantes de cana-de-açúcar : especificidade, produção em biorreator e conservação por liofilização

Abstract

Phytocystatins are inhibitors of cysteine peptidases found naturally in plants. Sugarcane cystatins are distinguished by their high inhibitory activity and possible biotechnological applications. In this work, a comparative panel of the inhibitory activities of CaneCPIs 1, 2, 3, 4, 5, and 6 against relevant human cathepsins and a cathepsin L of the sugarcane weevil, Sphenophorus levis, demonstrated the inhibitory efficiency of these cystatin. From this panel it was possible to observe that CaneCPIs 4 and 5 have potent inhibitory activity with very low Ki values for all cathepsins tested. Due to the insolubility of CaneCPI-4 produced in E. coli, Cterminal mutations were performed to make it more soluble, but were not able to alter solubility of the protein, but rather its inhibitory capacity. CaneCPI-4 was also expressed in Pichia pastoris. An increase in scale was carried out for the production of CaneCPIs 4 and 5 in bioreactor, and CaneCPI-5 was successfully expressed and used in a study that showed its ability to adhesion to dental enamel and protection against acid erosion. CaneCPI-4 was subjected to lyophilization with different stabilizers and it was shown to be stable even without the use of them, proving that this cystatin is quite stable even under stressful conditions for the molecule. Therefore, this study demonstrates the inhibitory potential of CaneCPIs, the possibility of producing them in bioreactor and of being lyophilized, all this information is important for a future industrial production and commercial use of sugarcane cystatin for various biotechnological purposes.