Isolation, purification, and study of the physicochemical properties of xylanase of the Bacillus subtilis 9 strain

L. R. Valiullin^1,2, Rin. S. Mukhammadiev^1,2, Rish. S. Mukhammadiev^1,2, V. I. Egorov², A. P. Glinushkin^2
1Federal Center of Toxicological, Radiation and Biological Safety, Nauchnyi gorodok, 3, Kazan, 420008, Russian Federation
²All-Russian Research Institute of Phytopathology, ul. Institut, vl. 5, r.p. Bol’shie Vyazemy, Odintsovskii r-n, Moskovskaya obl., 143050, Russian Federation

Abstract. The research aimed to isolate and purify xylanase from the culture liquid of the B. subtilis 9 strain, to study its physicochemical properties and to determine the possibility of creating a feed additive. The object of the research was the Bacillus subtilis 9 strain, a xylanase producer capable of releasing an enzyme with an activity of 10.6±0.3 units/mg of culture liquid in 24 hours. Isolation and purification of xylanase included obtaining an enzyme-containing supernatant, precipitation of proteins with ammonium sulfate, dialysis, ion-exchange chromatography on a DEAE-sepharose column, and gel filtration on a Sephadex G-100 column. The enzyme activity was determined by staining the reducing sugars with 3,5-dinitrosalicylic acid. The most complete recovery of xylanase was observed at 60% saturation of the enzyme-containing supernatant with crystalline ammonium sulfate, after 24 h of salt action. Multistage purification resulted in electrophoretically homogeneous xylanase with a purification degree of 23.48, a yield of 58.6%, and a specific activity of 248.9 units/mg of protein. The molecular weight of the native xylanase molecule was determined by gel filtration and electrophoresis in PAGE in the presence of SDS-Na and amounted to 23.0 kDa. The enzyme was a monomer. Testing the action of a wide range of metal ions (KCl, NaCl, MgCl2, ZnCl2, CaCl2, HgCl2, MnCl2, CdCl2, CoCl2, FeCl2, AlCl3, ZnSO4, EDTA) showed the dependence of xylanase activity on the presence of Ca2+ and Mn2+ ions at concentrations of 1 mM and 10 mM, as well as Fe2+ ion at a concentration of 10 mM. Optimal conditions for the action of xylanase were created in a weakly acidic medium (5.0–5.5 pH units) at moderate temperatures (40.5–50.0 C).

Keywords: xylanase; Bacillus subtilis; isolation; purification; physicochemical properties of the enzyme.

Author Details: L. R. Valiullin, Cand. Sc. (Biol.), leading research fellow; Rin. S. Mukhammadiev, Cand. Sc. (Biol.), research fellow (е-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.); Rish. S. Mukhammadiev, Cand. Sc. (Biol.), research fellow; V. I. Egorov, Cand. Sc. (Biol.), research fellow; A. P. Glinushkin, D. Sc. (Agr.), Corresponding member of the RAS, director.

For citation: Valiullin LR, Mukhammadiev RinS, Mukhammadiev RishS, et al. [Isolation, purification, and study of the physicochemical properties of xylanase of the Bacillus subtilis 9 strain] Dostizheniya nauki i tekhniki APK. 2021;35(10):66-71. Russian. doi: 10.53859/02352451_2021_35_10_66.