M.G. Holyavka - Ph.D. (Biol.), The Research Associate of Biophysics and Biotechnology Department, The Voronezh State University, Voronezh, Russia. E-mail: holyavka@rambler.ru T.A. Kovaleva - Dr. Sc. (Biol.), The Voronezh State University, Professor of Biophysics and Biotechnology Department, Voronezh, Russia. E-mail: tamara_kovaleva@inbox.ru E.A. Hrupina - Student, The Voronezh State University, Voronezh,Russia V.G. Artyukhov - Dr. Sc. (Biol.), Professor, The Head of Biophysics and Biotechnology Department, The Voronezh State University, Voronezh, Russia. E-mail: artyukhov@bio.vsu.ru V.N. Kalaev - Dr. Sc. (Biol.), Professor, The Voronezh State University, Voronezh, Russia

Purification technique to a homogeneous condition for inulinase from Helianthus tuberosus tubers and results of research for its physical, chemical and kinetic properties are presented in this article. It is established, that among extracting substances used by us (the distilled water, the acetate buffer (рН 4.5 and 5.5), sodium chloride (5 and 10 %) and sodium hydroxide (0.1 M)) for inulinase from Helianthus tuberosus tubers 10 % of NaCl appeared the most perspective. The enzyme extracting at 37 ºС during 1 hour was an optimum combination of temperature and incubation time. Comparing inulinase activity in Helianthus tuberosus tubers in various seasons, we came to conclusion, that it shows higher catalytic ability in February-April, than in October-December (when inulin concentration in tubers is maximum). It is interesting also, that inulinase activity in the sprouted tubers several times above, than that are at rest. It is revealed, that for inulinase sedimentation it is possible to use acetone and isopropanol in a proportion 1:3 and 1:4. Ethanol is less perspective for this enzyme. Results of experiments showed, that inulinase sedimentation is most expedient to carry out in a range of ammonium sulfate concentration from 35-40 up to 60 %. Application of ammonium sulfate raised extent of inulinase purification only in 1.4 times, therefore we considered as inexpedient a combination of sedimentation stage by acetone and sedimentation by ammonium sulfate stages and left only the first of them. For release of enzymes from low-molecular impurity we used gel chromatography on sephadex G-25. The ion-exchange chromatography on DEAE-cellulose was the following stage of inulinase purification. At first, we carried out an elution by the acetate buffer (рН 4.7) in steps in NaCl concentration range of 0.005-0.2 mol/l. The inulinase fraction left by one peak at salt concentration of 0.025-0.05 mol/l. Further we made experiments on inulinase purification by the method of an ion-exchange chromatography on DEAE- cellulose in a рН gradient 8.0-4.0. As a elution result 4 fractions, possessing of inulinase activity, were received. Calculation of I/S relations for enzymes 4 fractions, received by us, allows to argue, that only 3 are inulinase. We named them conditionally inulinase I, inulinase II and inulinase III. Thus, in our laboratory the technique for allocation and purification to a homogeneous condition for three inulinase fractions (I, II and III) from Helianthus tuberosus was developed: purification extent and exit respectively 55.7 and 5.4 % (I), 28.9 and 3.1 % (II), 21.6 and 1.9 % (III). On electrophoregrams, received by Davies's modified method, we observed the existence of one strip, that testifies to homogeneity of enzymes fractions, which for this reason can be used for the further analysis. It is revealed, that inulinase I has an activity optimum at 48 °С, inulinase II - at 39 °С, and inulinase III - at 44 °С. It is established, that the curve of specific activity dependence from values of substrate рН for inulinase I has a maximum at рН 6.8, for inulinase II at рН 6.2, and for inulinase III - at рН 4.5. It is shown, that optimum of substrate concentration for all three enzymes is the 5×10-4 mol/l. The analysis of kinetic curves for dependence of V(S) showed, that the greatest affinity to inulin shows inulinase I. Proceeding from the received results, it is possible to conclude that all types of inulinases are quite suitable for use in the industrial purposes, therefore the choice of this or that enzyme should be caused, first of all, by conditions of production and features in `the technological line.