Mikrobiol. Z. 2017; 79(2):33-47. Ukrainian.
Streptomyces sp. 12 Protease: Purification and Properties
Nidialkova N.A.1, Varbanets L.D.1, Schepelevich V.V.2, Zelena P.P.2,
Yumina Yu.М.2, Garkavaya E.G.3, Troshina L.О.3
1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine
2Taras Shevchenko National University of Kyiv
60 Volodymyrska Str., Kyiv, 01601, Ukraine
3National Aviation University
1 Kosmonavta Komarova Av., Kyiv, 03058, Ukraine
Aim. To optimize the cultivation conditions of Streptomyces sp. 12 for providing a maximum synthesis of proteases, to obtain a purifed preparation of the protease, to investigate its substrate specifcity, physico-chemical properties and functional groups of the active site. Methods. Optimization of cultivation conditions using single-factor experiments were carried out on the base medium of the following composition (g/l): K2HPO4 – 1; MgSO4∙7H2O – 1; NaCl – 1; (NH4)2SO4 – 2; CaCO3 – 2; starch – 10; microelements salt solution (FeSO4∙7H2O – 1, MnCl2∙4H2O – 1, ZnSO4∙7H2O – 1) – 1 ml. Total caseinolytic (proteolytic) activity was quantitatively determined on tyrosine, which is formed by hydrolysis of casein under the action of the investigated enzyme. To isolate and purify the enzyme it is used precipitation by (NH4)2SO4 (90 % saturation), chromatography on neutral and charged TSK-gels. Results. It is established that optimal sources of carbon, nitrogen and minerals in the nutritious medium are corn meal, (NH4)2SO4 and CaCl2 respectively. The growth of Streptomyces sp. 12 was carried out in submerged conditions during 3 days in 200 ml of the optimized nutritious medium at initial value of pH 7.0, temperature 37 °С, 220 rpm. The specifc proteolytic activity was 62.7 U∙mg of protein-1, which is 4.8-fold higher than in the control (base medium). Using fractionation by sulphate ammonium, gel-fltration and ion-exchange chromatography on TSK-gels – Toyopearl HW-55, DEAE 650(M) and Sepharose 6B, Streptomyces sp. 12 protease with specifc activity 538 U∙mg of protein-1 and yields 35.8 % was obtained. Mr of Streptomyces sp. 12 protease is ~35.6 кДа. It is shown that enzyme displays highest activity toward collagen and less activity toward casein, albumin and gelatin. Inhibition by group-specifc reagents revealed that the tested protease is metalloprotease with optimal conditions of action on collagen at pH 8.0 and temperature 50 °С. Conclusions. The obtained Streptomyces sp. 12 protease was characterized by specifc proteolytic activity 538 U∙mg of protein-1 that 4.8-fold higher than activity in the supernatant of culture liquid.
Key words: protease, Streptomyces, optimization of nutrient medium, cultivation, purifcation, molecular weight.
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