Extracellular collagenase isolated from Streptomyces antibioticus UFPEDA 3421: purification and biochemical...

Collagenases are proteases able to degrade native and denatured collagen, with broad applications such as leather, food, and pharmaceutical industries. The aim of this research was to purify and characterize a collagenase from Streptomyces antibioticus. In the present work, the coffee ground substrate provided conditions to obtaining high collagenase activity (377.5 U/mL) using anion-exchange DEAE-Sephadex G50 chromatographic protocol. SDS-PAGE revealed the metallo-collagenase with a single band of 41.28 kDa and was able to hydrolyzed type I and type V collagen producing bioactive peptides that delayed the coagulation time. The enzyme activity showed stability across a range of pH (6.0-11) and temperature (30-55 °C) with optima at pH 7.0 and 60 °C, respectively. Activators include Mg⁺², Ca⁺², Na⁺, K⁺, while full inhibition was given by other tested metalloproteinase inhibitors. Kinetic parameters (K_m of 27.14 mg/mol, V_max of 714.29 mg/mol/min, K_cat of 79.9 s^-1 and K_cat/K_m of 2.95 mL/mg/s) and thermodynamic parameters (E_a of 65.224 kJ/mol, ΔH of 62.75 kJ/mol, ΔS of 1.96 J/mol, ΔG of 62.16 kJ/mol, ΔG_E-S of 8.18 kJ/mol and ΔG_E-T of -2.64 kJ/mol) were also defined. Coffee grounds showed to be an interesting source to obtaining a collagenase able to produce bioactive peptides with anticoagulant activity.