First report on Chlorella vulgaris collagenase production and purification by aqueous two-phase system

Abstract

Collagen is triple helical structured protein, which is undegradable by most proteases and reacts only with collagenases. These enzymes are applied and important in several commercial segments and the interest for new collagenases has only been increasing. Microalgae are not a very prominent source of enzymes but are extremely commercially attractive forms of life due to photosynthetic growth. Yet, the literature is absent on reports about algae collagenases and this work is the first report on Chlorella vulgaris collagenolytic enzyme production, purification and characterization. C. vulgaris was cultivated in autotrophic and mixotrophic conditions and the biomass used to obtain the cell extract. The autotrophic extract displayed a higher collagenolytic activity and was used for ATPS experimental design. Through 24-full factorial design, high collagenolytic activity was observed at pH 8.0, PEG molar mass of 1500 g/mol, PEG concentration of 12.5% (w/w) and phosphate buffer concentration of 15% (w/w). In these conditions, a purification factor of 10.79 was obtained for a partition coefficient of 23.74 with the collagenase retained in top PEG rich phase. Purified collagenase optimum activity was observed under pH 9 and 37 °C, displayed less gelatinolytic and caseinolytic activity, with higher collagenolytic activity with azocoll than crude extract, attesting this enzyme is a true collagenase. The collagenolytic activity was inhibited by EDTA, an indicative of a metallocollagenase.