Adsorption chromatographic purification of α-amylase Bacillus subtilis on super-cross-linked polystyrene sorbents
Abstract
The paper presents the results of studying the process of sorption of Bacillus subtilis α-amylase on super-cross-linked polystyrene sorbents MN-200, MN-202 (non-ionic) and MN-500 (sulfocathionite). We are considering the possibility of obtaining a reversible-dissociating complex of α-amylase with these sorbents. As a raw material for the obtaining of α-amylase a technical enzyme preparation amylosubtilin G3X prepared by fermentation of Bacillus subtilis was used.
Optimal conditions of the process of sorption of α-amylase on the sorbents studied were selected. The sorption isotherms of α-amylase on the MN-200, MN-202 and MN-500 sorbents have the BET isotherm form, which indicates the orderliness of the sorption centers and the statistical distribution of the protein molecules on the sorbent grain. The highest selectivity is possessed by sulfonate cation exchanger MN-500 (Kd=169 ± 0.5 ml / g). The eluent for desorption of α-amylase was selected: an ammonia solution (pH 10-11) for MN-500 and an ammonia solution (pH 10-11) with the addition of an organic solvent (ethanol) for MN-200 and MN-202.
The kinetic-dynamic analysis of the α-amylase sorption process on the MN-200, MN-202 and MN-500 sorbents according to the regularization parameter λ showed that the regular regime is achieved only by sorption on the sulfonate cation exchanger MN-500 (λ=0.44). For MN-500, the highest yield at the desorption stage of α-amylase was obtained, and the concentrating effect was observed.
Thus, MN-500 sulfonate cation exchanger can be recommended for preliminary chromatographic purification of Bacillus subtilis α-amylase in the production of a complex preparation combining the properties of the enzyme preparation and the enterosorbent
Downloads
References
2. Rachkova N.S., Khavkin A.I., Trudnyi patsient, 2012, Vol. 10, No 2-3, pp. 41-44.
3. Zinchenko L.N., Kravtsova A.G., Oreshina I.A., Vestnik Pridnestrovskogo universiteta. Ser.: Mediko-biologicheskie i khimicheskie nauki, 2015, No 2(50), pp. 32-37
4. Tsyurupa M.P. Diss. d-r. chem. nauk. Moscow, 1985, 427 p.
5. Tsarenko O.V. Diss. kand. biol. nauk. St. Petersburg, 2000, 125 p.
6. Gracheva I.M. Tekhnologiya fermentnykh preparatov. Moscow, Agropromizdat Publ., 1987, 335 p.
7. Grigor'eva E.P. Diss. kand. biol. nauk. St. Petersburg, 2001, 142 p.
8. Krasovitskaya I.A. «Molodaya farmatsiya – potentsial budushchego», Farmatsiya, Sbornik materialov VI Vserossiiskoi nauchnoi konferentsii studentov i aspirantov s mezhdunarodnym uchastiem, April 25-26, 2016, St. Petersburg, 2016, pp. 351-353
9. Shkutina I.V., Stoyanova O.F., Selemenev V.F., Butyrskaya E.V. et al., Khimiko-farmatsevticheskii zhurnal, 2009, Vol. 43, No 9, pp. 50-52.
10. State pharmacopeia RF XIII Edition. Moscow, 2015, Vol. 1, 1468 p.
11. Rukhlyadeva A.P., Polygalina G.V. Metody opredeleniya aktivnosti gidroliticheskikh fermentov. Moscow, Legkaya i pishchevaya pr-t' Publ., 1981, 288 p.
12. Sal'nikova S.A. Diss. kand. farmatsevt. nauk. St. Petersburg, 2014, 147 p.
