Purification and Characterization of a Thermostable Alkaline Collagenase from Thermoactinomyces Sp. E-21 Strain

ABSTRACT

A thermophilic Thermoactinomyces 21 E that produced a highly thermostable alkaline collagenase was isolated from Bulgarian thermal regions. The enzyme was purified by ultrafiltration and gel filtration chromatography with a 101 fold increasing in specific activity and 58% recovery. The enzyme preparation exhibited activity over a broad pH and temperature range of pH 4,0–13,0 and 25°–90°C, respectively. Optimum activity was observed at pH 9,0–9,5 and temperature 60°–65°C. Thermostability was enhanced in the presence of 10 mM calcium ions. 50% of the original activity remained after incubation at 85° C for 30 min. The collagenase has a relative molecular mass of 50 000 as determined by SDS—PAGE electrophoresis and gel-filtration chromatography. The enzyme activity was markedly inhibited by Fe²⁺ and Cu²⁺, whereas Mn²⁺, Sr²⁺, Cd²⁺, Ba²⁺, Zn²⁺, Ni²⁺ exhibited low inhibitory effect. Mg²⁺, Ca²⁺, Co²⁺ slightly enhanced the collagenase activity. The purified thermostable collagenase was capable of hydrolysing many soluble and insoluble substrates—collagens and synthetic peptides.