Extracellular collagenase isolated from UFPEDA 3421: purification and biochemical characterization.

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 . 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, Ca, Na, K, while full inhibition was given by other tested metalloproteinase inhibitors. Kinetic parameters (K of 27.14 mg/mol, V of 714.29 mg/mol/min, K of 79.9 s and K/K of 2.95 mL/mg/s) and thermodynamic parameters (E of 65.224 kJ/mol, ΔH of 62.75 kJ/mol, ΔS of 1.96 J/mol, ΔG of 62.16 kJ/mol, ΔG of 8.18 kJ/mol and ΔG 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.