Purification and characterization of a new thermophilic collagenase from Nocardiopsis dassonvillei NRC2aza and its application in wound healing.

A thermostable metallo-collagenase enzyme (150 kDa), recently identified in a newly isolated actinomycestes strain (Nocardiopsis dassonvillei NRC2aza), has been purified from natural source, characterized to have application in wound healing. A simple 3 step purification procedure gave an increase of purity by 6.23 fold with a specific activity of 387.2 U mg-1. The enzyme activity showed stability across a range of pH (7.0-8.5) and temperature (40-55 °C) with optima at pH 8.0 and 60 °C, respectively. Activators include Mg+2, Ca+2, Zn+2, Na+, K+ and Ba+2, while Mn+2, Co+2, Ni+2and Ag+ ions gave partial inhibition. Full inhibition was given by other tested ions and metalloproteinase inhibitors. Broad substrate specificity was demonstrated including activity against a native collagen. The Km and Vmax of the enzyme using azocollagen were 5.5 mg/ml and 1280 U, respectively. The purified collagenase enhanced wound closure in vitro and in vivo and the repair process was dose dependent. Topical application of the purified collagenase (either of 25 or 50 U) to cutaneous wounds significantly accelerated the rate of wound healing and the formation of granulation tissue. Hence, the purified collagenase has a great potential as a therapeutic agent in wound care and collagen related diseases.