The method of producing ovomucoid
(57) Abstract:The use of biotechnology. The inventive method of producing ovomucoid protein from whole eggs, including deposition of related proteins with polyethylene glycol, the concentration of ovomucoid also polyethylene glycol and clean ovomucoid acetone at pH 3.8 and 4.8. The use of the proposed method eliminates the use of highly toxic reagent - trichloroacetic acid. Consequently, the proposed method becomes cleaner. Furthermore, the method allows more than 10 times to reduce the consumption of acetone compared with the method of the prototype due to the concentration of ovomucoid polyethylene glycol. table 1. The invention relates to the field of biotechnology and relates to a method of obtaining ovomucoid (Ω) of the whole protein eggs (CBA).The invention can most effectively be used in technological processes of isolation of individual proteins from protein eggs.Known methods for producing ovomucoid by processing CBA trichloroacetic acid or its salts (1,2). This is mainly deposited related proteins, and ovomucoid remains the kami of these methods are the use of highly toxic substances - trichloroacetic acid and high consumption of organic solvents.A method of obtaining Ω, we have chosen for the prototype (3) providing for the deposition of the main mass of related proteins, CBA, except OM, a mixture of 0.5 M trichloroacetic acid and acetone at a ratio of 1:1.5 at pH 3.5. Further purification OHM spend double precipitation with acetone at a volume ratio solution Ω acetone is 1:2,5.The disadvantage of the prototype method is the use of toxic trichloramines acid.Another disadvantage of the known method is the use of large quantities of acetone.The present invention is to develop a more environmentally friendly method of producing ovomucoid due to the exclusion of trichloroacetic acid and reduction of consumption of organic solvents.The solution of this problem is achieved by the fact that instead of trichloroacetic acid precipitation associated proteins performed with polyethylene glycol (PEG) concentration of ovomucoid in the supernatant also spend glycol, and cleaning OHM carried out with acetone.The essence of the invention lies in the fact that when the concentration of the polymer (peg, 1 tbsp.) 8,0-16,0 weight. The specified concentration of PEG corresponds to the optimal value at which a 50.0-85.0 per cent associated protein enters the sediment, while the main part of the OM remains in the supernatant. The use of PEG with MM less than 1.0 CD is not effective because of the large flow rate of the polymer, and the use of PEG with MM more than 20,0 KD leads to high viscosity systems. After the complete dissolution of PEG (1-2 hours) system CBA PEG incubated at room temperature for several hours. The precipitate proteins separated by centrifugation and discarded. To the supernatant was added with stirring PEG to achieve its concentration in the system 18,0-28,0% weight. (Peg, 2 tbsp.). When the concentration of the PEG below 18.0 weight. ovomucoid deposited not completely, and increasing the concentration of PEG above 28,0 weight. does not increase the number of deposited OHMS. After complete dissolution of the PEG system is maintained at room temperature for several hours, then decanted upper phase, leaving a viscous liquid phase, containing ovomucoid.Phase containing Ω diluted with distilled water, adjusted with acetic acid to a pH of 3.8 to 4.8, lighten the resulting solution by centrifugation (T-24, 1500 g, 10 min) and mixed with acetone at shootorials for urine to cold at 4-6 degrees.With. then separated from the sediment OM centrifugation. The residue extracted twice with distilled water, supernatant are combined and freeze-dried. The result is the product of ovomucoid with purity 95,0-96,0% Yield 50,0 80,0%
Below is a specific example of implementation of the proposed method.Example 1.Conduct preliminary preparation of CBA, filtering homogenized egg whites through the coarse fabric filter. To 250 ml of prepared protein with stirring, add the dry PEG with 4,5 MM CD in the amount of 44.1 g, which is of 15.0 wt%. (Spah, 1 CL). The mixture is stirred for 0.5 hour to dissolve the PEG, then aged 12 hours at room temperature. The precipitate proteins separated by centrifugation (T-24, 1500 g, 10 min) and discard. The supernatant is again added under stirring PEG in the number of 25.8 g, which is 23, the weight. (SPAG, 2 tbsp.). After complete dissolution of the PEG, the mixture is left for 12 hours at room temperature. The upper phase is separated and discarded, and the lower, which is a liquid viscous mass is then diluted with double distilled water. To the solution was added acetic acid to pH 4.5, centrifuged and add 115 ml of acetone in a volumetric aspect] is called) and extracted twice with distilled water, the extracts are combined and freeze-dried.The release of the drug Ω is 2.1 g (77%). Purity according to chromatographic analysis 96,0%
Other examples of the method are presented in table. 1.The use of the proposed method eliminates the use of highly toxic reagent trichloroacetic acid.Consequently, the proposed method becomes cleaner.Furthermore, the method allows more than 10 times to reduce the consumption of acetone compared with the method of the prototype due to the concentration of ovomucoid polyethylene glycol.Literature
1. Lineweaver C. H. Murray W. J. Bil.Chem. 171, 565 (1947).2. E. Frederica, H. F. deutsch, J. Biol.Chem. 181, N 2, p. 499-510 (1949).3. C. H. lapuk, Questions of medical chemistry, 1966 T. 12, vol. 6, pp. 636-638. 1 Way to get ovomucoid protein from whole eggs by deposition associated proteins and purification of ovomucoid presidenial acetone, characterized in that the deposition of proteins is performed with polyethylene glycol (mol.m. 1 20 KD when the concentration of glycol in the mixture 8 to 16 wt. then spend the concentration of ovomucoid polyethylene glycol when its concentration in the mixture 18 of 28 wt.
FIELD: biotechnology, biochemistry.
SUBSTANCE: invention relates to extracts prepared from vegetable somatic embryos for the cell-free translation system and/or the coupled transcription-translation system. Method involves preparing embryonic callus from the primary material and the embryonic suspension culture. After induction of the secondary somatic embryogenesis extract is prepared from somatic embryos. Based on the extract the diagnostic system is developed for detection of biologically active compounds. Invention provides overcoming the species limitations and strain specificity and to attain the high effectiveness of the cell-free translation system and the coupled transcription-translation system also.
EFFECT: improved preparing method, valuable biological and biochemical properties of system.
49 cl, 5 dwg, 2 tbl, 9 ex