Выделение, очистка и биохимические свойства кератинолитического фермента, секретируемого Penicillium Citrinum

Разработан метод выделения и очистки кератинолитического фермента, секретируемого культурой Penicillium citrinum, с помощью которого получен электрофоретически гомогенный энзимный препарат с молекулярной массой 21 кДа и степенью очистки в 64 раза. Изучены физико–химические и биохимические свойства полученного фермента, позволяющие отнести его к разряду нейтральных термолабильных кератинолитических сериновых протеиназ.

Purification, clearining and some properties keratinolytic enzyme, secreting by Penicillium Citrinum

The application of microorganisms keratinolytic enzymes is in different fields were keratins, should be hydrolyzed, such as medicine, cosmetics, detergent and leather industry, waste bioconversion. The aim of this study was the development of pure keratinase obtaining technology using the deuteromyces. In previous studies we shown the culture of Penicillum citrinum was perspective for keratinase production because excreted keratinolytic enzymes in culture media and was possessed of the keratinolytic activity highest level among another studied deuteromyces during the submerged cultivation in the presence of hair as the source of carbon. The method of isolation and purification of keratinolytic enzyme, secreting by P. citrinum, was developed, concluding consequent using of gel filtration chromatography and affinity chromatography, and allowing to receive electrophoretically homogeneous enzyme preparation with a molecular mass 21 kDa, the degree of enzyme clearining 64 times and keratinolytic activity 580 ± 51 μM/mgхh. The optimal pH and temperature for enzyme were 7,4 and 30 ⁰C, respectively. PSMF and DTT inhibited the keratinase, while EDTA didn’t influence on enzyme activity. Studying the properties of the enzyme allows consider it belonged to the neutral termolable keratinolytic serine proteinases. The enzyme was active upon the keratins of different origin and some soluble proteins, which allow consider it perspective not only as keratinase, but also as proteinase. At the same time collagen and dipeptides, specific for it (Ley-Gly, Gly-Gly), couldn’t be hydrolyzed by the enzyme, and this is an advantage for the application of the keratinase for the cosmetic and pharmaceutical purposes or in the leather industry were collagen should not be attack. Thus, the protease obtaining technology from P. citrinum was developed.