The accumulation dynamics of cellulolytic enzymes in culture media of the basidiomycete fungi Panus tigrinus, Pleurotus ostreatus, Fomes fomentarus, and the micromycete Aspergillus terreus were studied during a long incubation period. It was found that A. terreus was the most active producer of cellulolytic enzymes among the studied fungi. Two protein fractions with cellulase activity were isolated using gel filtration and ion-exchange chromatography. PAAG electrophoresis showed that fraction-I consisted of four components; fraction-II, an electrophoretically homogeneous protein.It is known that cellulolytic enzymes are produced by various microorganisms belonging to various taxonomic groups [1,2]. Cellulases have broad applications and are used mainly in food, textile, chemical, medical, pharmaceutical, paper, and wood processing industries. They are also added to animal feed and to agricultural process wastes [3][4][5][6].The goal of our work was to study the ability of wood-destroying fungi to produce highly active cellulases and to produce active preparations of cellulolytic enzymes.The accumulation dynamics of cellulolytic enzymes in culture media of the fungi Panus tigrinus, Pleurotus ostreatus, Fomes fomentarus, and Aspergillus terreus were determined during different periods of the growth and development, from 7 to 14 days.The results show that A. terreus was the best producer of cellulase compared with the other fungi. The maximum endoglucanase activity was observed 10 days from the start of growth. Therefore, 10-day culture liquid of A. terreus was first filtered through glass wool and then through a millipore filter (0.2 µm pore size) to separate the supernatant from the cell culture in order to isolate enzyme components of the cellulase complex. Cellulase activity in solution was determined by the Wood and Bhat method [7] using Na-CMC (1%) as substrate. The solution (220 mL) contained 106.1 units of total cellulase activity ( Table 1).The protein concentration determined by the Lowry method [8] indicated 154 mg of protein in the collected culture medium from A. terreus. The specific activity of the cellulolytic enzyme in the culture medium was 0.688 U/mg of protein.Then the culture medium was concentrated in a rotary evaporator to 17 mL. The total cellulase activity and protein concentration in the concentrated enzyme solution was 83.38 U and 108.56 mg, respectively. Analysis of the proteins by SDS-PAAG electrophoresis showed nine protein bands with molecular weights from 17 to 120 kDa in the concentrated enzyme solution.
Partially purified enzyme preparation with specific activities of 153.7 U/mg for α-amylase and 0.15 U/mg for protease was produced by selective adsorption on starch. Enzymes were purified until homogeneous electrophoretically by gel-filtration over HW-55 TSK-gel with specific activities of 245 U/mg for α-amylase and 1.44 U/mg for protease. The optimum temperature and pH for purified α-amylase activity are 40-50°C and pH 6.0. The effects of various metal ions on the activity and stability of the enzyme were studied.We have studied the local bacterial strain Bacillus subtilis-150, which produces active α-amylase and neutral protease and the conditions for active fermentation of the strain cultivated on the optimal nutrient medium [1, 2]. The strain B. subtilis-150 differed from the previously studied strain B. subtilis-7A in certain cultural signatures and α-amylase activity [3].Various methods for isolating highly purified and homogeneous forms of bacterial amylase have been reported [4,5]. Fractional precipitation by (NH 4 ) 2 SO 4 for which 60% saturation by (NH 4 ) 2 SO 4 produced an enzyme preparation with specific activity 31 U/mg, where the yield by activity was 66.5% [3], was used earlier to prepare α-amylase preparation from B. subtilis-7A.Herein we report the isolation and investigation of the physical chemical and catalytic properties of purified α-amylase from B. subtilis-150. We precipitated the protein fraction from culture liquid (CL) using acetone, ethanol, and isopropanol. A high yield by activity (82%) was obtained for precipitation of proteins by isopropanol in a 1:1 ratio. α-Amylase was less sensitive to the action of isopropanol and precipitated almost completely whereas the less stable protease was partially precipitated. The resulting enzyme preparation had specific activity 285.3 U/mg (Table 1).Successful use of various natural polysaccharides for purification of amylolytic enzymes has been reported [6,7]. The most suitable sorbent for specific binding of α-amylase was soluble starch, which is very cheap and available. α-Amylase was sorbed on soluble potato starch at -20°C in the presence of ethanol and calcium acetate for 30 min. After the sorption of enzymes on starch was complete, the enzyme-substrate complex was split by increasing the temperature to 40°C and incubating for 60 min to hydrolyze the starch. The α-amylase was released and transferred into buffer with a certain amount of associated protease. We showed previously that α-amylase and protease of this strain have different thermal stabilities [1]. Therefore, the α-amylase activity was retained during incubation for 30 min at 60°C whereas that of protease under the same conditions decreased by more than 80%. Thus, biospecific chromatography over starch was able to produce partially purified α-amylase preparation with a specific activity of 153.7 U/mg.Gel-filtration of partially purified α-amylase preparation was performed over a column (20 × 600 mm) packed with HW-55 TSK-gel (Toyopearl, Japan). The results showed that the eluted ...
Журнал включен в Перечень рецензируемых научных изданий ВАК
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