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.
A method is proposed for isolating the main components (melittin, apamin, and phospholipase A 2 from the venom of the bee Apis mellifera using HPLC, and the synthesis of two sorbents for fractionating these peptides is described.Bee venom is widely used as a medicament in various diseases. [1]. However, to create highly specific and effective agents it is desirable to use the components of the venom in a highly purified state. In view of this, researchers are faced with the task of developing the simplest, fastest, and cheapest methods of fractionating the venom [2].It is known that the venom of the bee Apis mellifera is a complex mixture of proteins and peptides exhibiting the most diverse biological activities. A number of proteins and peptides have been studied in detail, and the primary structures of the phospholipase A 2, melittin, apamin, and the MCD peptide have been determined [2][3][4][5][6][7][8][9][10][11]. More recently, new methods have been developed for isolating the components of bee venom, since these components are of interest for pharmacology [1]. Purification of the majority of the components is complicated by high surface activity, low molecular mass, and the presence of a large amount of complex substances [3,[11][12][13][14]. All this is leading to the use of different and incompatible methods of isolation by different groups of researchers.Our aim was to improve the methods of fractionation, with a substantial increase in the yields of tlie components of the venom, and a decrease in the number of stages of the isolation process.According to the results of HPLC (Fig. 1), whole bee venom contains substances differing greatly in hydrophobic properties. In the first stage, therefore, we used reversed-phase adsorbents based on silica gel. To check these presuppositions, we carried out the separation of bee venom on the sorbent LiChroprep RP-8 in a stepwise gradient of isopropanol (Fig. 2).Five fractions were obtained which were characterized by the use of HPLC on a Nucleosil 100-5 C18 column. A disadvantage of this sorbent is that the low-molecular-mass colored substances of bee venom are irreversibly sorbed on the gel, which decreases the capacity and impairs the hydrodynamic properties of the column. This sorbent enables products to be obtained in a highly purified state, but a large amount of sorbent with a def'mite capacity stable in various buffer systems is necessary for semipreparative separation. For this purpose we used Polikhrom-1, but the capacity and hydrodynamic parameters of this gel proved to be inadequate for these purposes. We then synthesized a reversed-phase sorbent based on Silokhrom-80 by the direct modification of the silanol groups with octadecyltrichlorosilane in acetone at 50°C for 18 h.The fractionation of the venom of the bee Apis mellifera with the aid of this sorbent gave fractions containing large amounts of impurities. In all probability, this was due to the fact that only 50% of the silanol groups had been modified by the octadecylsilane. The unmodified silanol g...
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