SU1028334A1 - Method of producing lysozyme - Google Patents

Abstract

The method of obtaining lysocime, including washing ticks with saline, homogenizing them, repeatedly freezing the homogenate, heating it, processing with sodium phosphate, desalting by gel filtration and then successively extracting with alcohol, a solution of distilled water, distilled water, and isolating the target product, followed by sequential extraction with alcohol, distilled water, and separation of the desired product. In order to increase the activity of lysozyme, pre-mites are exposed to monochromatic polarized light of the visible range with a wavelength of 6328 A, with a density of 0.1250, 50 mW / cm for 0.5-30 min {With one TpexKjpaTjio with an interval between treatments of 2-6 hours, and washing and again with C / J the following treatment is carried out 1-120 hours after exposure monochromatic polarized light.

Description

The invention relates to the biological industry, in particular, to methods for producing biologically active substances from animal raw materials. The known method of obtaining lysozyme from ticks by placing them before isolation in a constant field with a strength of 25-600 Oe for 1-48 h 1 However, the known method is long-lasting. There is also known a method for producing zozyme, including washing the tick with saline, homogenizing them, repeatedly freezing and thawing the homogenate, heating it, treating with sodium phosphate, desalting with gel filtration and then sequential extraction with alcohol, buffer solution, distilled water, and isolating the desired product 2. However, the method does not provide the output of the enzyme sufficiently high activity. The aim of the invention is to increase the activity of lysozyme. The goal is achieved according to the method of obtaining lysozyme, including washing ticks with saline, homogenizing them, repeatedly freezing and thawing the homogenous, heating it, treating with phosphate sodium, desalting by gel filtration by sequential extraction with alcohol, a buffer solution distilled with water and separating the target product, previously ticks are exposed to monochromatic polarized light of the visible range with a wavelength of 6328 A, power 0.125-0.50 mW / cm for 0.5-30 minutes one or three times with an interval between treatments of 2-6 and washing and subsequent processing are carried out 1-120 hours after the last exposure to monochromatic polarized light. Example, Ornithoden papiEEipes ticks with a total mass of 900-1000 mg are placed in a chamber and introduced into zones of influence of a visible laser source with a wavelength of 6328 A, with a capacity of 0.125 mW / cm for 45 minutes once followed by the release of organ lysozyma in 1 h after processing. The mites subjected to laser treatment are washed with 250 ml of physiological solution, 50 ml of alcohol, and then washed three times with physiological saline solution each with 250 ml, homogenized, D1; 1 distilled water is added in such a way that the total volume of the homogenate reaches 90 ml, and adjusted to pH 4.0 with glacial acetic acid. Then the homogenate is frozen 3 times and thawed until the cellular structure of the tissues is completely destroyed. The separation of the accompanying proteins of the tick homogenate is carried out by heating to 3 minutes followed by rapid cooling ,. Disubstituted sodium phosphate was added to the cooled homogenate to a concentration of 0.1 M and the pH was adjusted to 8.5, the precipitated precipitate was separated by centrifugation for 10 minutes at a speed of 9000 rpm. Centrifugals are desalted by gel filtration on Sephadex G-25. The fractions containing a desalted solution of tick tissues are collected and lyophilized. In further studies, the centrifuge is filtered through a sterile cotton-gauze pad, evaporated on a rotor, then filtered and lyophilized. The resulting powder is dissolved in 5 ml of physiological saline, eight times the amount of alcohol is added to precipitate the bactericidal substance, the latter is extracted with acidified acetic acid with 60-degree alcohol, buffer solution j distilled WATER. Then the amorphous powder containing the bactericidal substance is dissolved in 10 m of dilute acetic acid and dried on a lyophilic, after which the powder is dissolved in 10 ml of 2 M acetate buffer containing 5% sodium chloride solution in 1-2 hours the sediment falls out bactericus {1 single substance (lysozyme). The yield of biologically active substance (lysozyme) from 500 mites weighing 1000 mg is 5.6 mg. The activity of the isolated bactericidal substance (lysozyme) is determined by the method of diffusion in agar. The bactericidal substance, in the form of mites, diffused under standard conditions in agar, lyses the membranes of dead M. BysodeiEticicus cells, evenly poured into agar plates. As a result, various zones of lysis are formed, the diameter of which determines the activity of the bactericidal substance. PRI m, e p 2. Take 6 groups of ticks of 500 individuals in a group. Pliers of the first group (control) are not subjected to laser treatment. Ticks of the second and sixth groups are affected by a source of laser radiation with a wavelength of 6328 A and a power of 0.125 mW / cm. Ticks of the 2nd group are affected for 0.25 min, Z.y - min, 4th - 15 min, 5th 30 min and bth - 45 min twice with an interval of 2 hours between treatments. The production of lysozyme from ticks of these groups is carried out after 60 hours. Prize. Take 6 groups of mites of 500 individuals in each group. Ticks of the 1st group (control) are not subjected to laser beam treatment. On ticks of the 2nd to 6th groups, they are affected by a laser source with a power of 0.25 mW / cm. Ticks of the 2nd group are affected by laser radiation S for 0.25, the 3rd 0, 50 min., The 4th one - 14 min., The 5th 30 min. And the 6th - 45 min. Ticks of experimental groups are treated twice with an interval of 2 hours between treatments. The preparation of lysozyme from ticks by the experimental and control groups is carried out in 60 hours. EXAMPLE 4. 6 groups of 500 individuals in a group are taken. Ticks of the 1st group are not treated (control). On ticks 2,3,4,5 and 6th. the groups are affected by a laser source with a power of 0.750 mW / on ticks of the 2nd group for 0.25 minutes, the 3rd is 0.50 minutes, the 4th is 15 minutes, the 6th is 45 minutes, twice at intervals of between 2 treatments and 2 hours, lysozyme is obtained after 60 hours. EXAMPLE 5 6 sizing groups of 50d specimens per group are taken. Ticks of the first group (control) are not subjected to processing. Pincers of groups 2-6 are treated with a laser source with a power of 0.500 mW / cm. Ticks of the 2nd group are treated for 0.25 min, the 3rd one - 0.50 min, the 4th - 15th 5th - 30 min, and the 6th - 45 min twice with an interval of 2 hours between treatments. , the production of lysozyme is carried out after 60 hours. The dependence of the activity and the yield of dry matter of lysozyme on the time and power of laser irradiation (examples 1-5) is presented in the table. 1, Table

Irradiation power 0.125 mW / cm2

The output of the dry island, mg 0.5

5.1 Lysozyme Activity, AU 2860 + 101

The output of the dry island, mg 5.0

5.4 The activity of lysozyme, AU 28601101

The output of the dry island, mg 5.0

5.5 Lysozyme Activity, UD- 3180 ± 106

The output of the dry island, mg 5.0

5.3 Activity. whether the architect, unit 3260 + 123 4950 + 162

5.2

5.3

5.2

5.2

5.4

5.4

5.4

5.3

5.7

5.5

5.4

5.2

S.

5.4

5.2

5.4 4040 + 115 44051147 .62311170 7l90tl73 7700 + 185 4450 ± 129 5375 + 154 6750 ± 145 7990 + 175 7950 + 195 4700 + 183 5800 + 129 7290 ± 193 8920 ± 210 7800 ± 170 Radiation power 0.250 mW / cm 2 Irradiation power 0.500 mW / cm. Irradiation power 0.750-mW / cm; 5300 + 175 6900 ± 186 78501235

Example Take b groups of rio mites 500 individuals in a group. Pincers of the 1st group - control. The affected 2nd groups will be affected by the laser power source; 0.500 mW / cm for 5 minutes twice with an interval of 2–2 hours of treatment. Preparation of lysozyme issupply Preliminary Method Activity 49031175 Lysozyme, BD 3750 + 149

The yield of dry matter, mg 5.2

5.2

Known method

Activity

285 ± 116

28501116 lysozyme, U

Yield. Suchora Island, mg 5.0

5.0 EXAMPLE 7. 8 groups of mites with 500 animals per group are taken. Ticks of the 1st group (control) are not subjected to treatment. Mites 2-8 of the group are affected by a laser source with a wavelength of 6328 A, power O, 25r mW / cm for .5 min. Ticks of the 2nd group are treated once, the 3rd one - twice in 2 hours, the 4th one - twice in 4 hours, the 5th or twice in 6 hours, the 6th - three times in 2 hours, the 7th - three times in 4 hours and 8th — Three times in hours. Obtaining lysozyme is carried out 60 hours after treatment in the manner described in Example 1. EXAMPLE 8 Take 8 groups of mites, 500 animals in each group.

after 0.5 h (2- group), 1.0 h (3-), 60 h (4-), 120 h (5-), 150 h (b), according to the method described in example 1.

In tab. Figure 2 shows the dependence of the activity and dry matter yield of lysozyme on the time elapsed after irradiation of ticks.

table 2

The proposed method

5.4

5.5

5.6

31001180

3150 + 185

2930il50

5.0

5.0

5.0 10370 ± 235 105701297 84561273 Ticks of the 1st group (control) are not subjected to treatment. Ticks of groups 2-8 are exposed to a laser source with a wavelength of 6328 A, a power of 0.500 mW / cm for 5 minutes. Ticks of the 2nd group are treated once, the 3rd one - twice in 2. hours, the 4th one - twice in 4 hours, the 5th one - twice in 6 hours, the 6th one - three times in 2 hours, the 7th group three times in 4 h, 8 of the 8th — three times in one hour. The preparation of lysozyme is carried out 60 hours after tick treatment in the manner described in Example 1. A gabl3 represents. the dependence of the activity and the yield of dry matter on repeated irradiation of ticks.

Table 3

Claims (1)

METHOD FOR PRODUCING LYSOZYM. including tick washing with physiological saline, their homogenization, repeated freezing of the homogenate, its heating, treatment with sodium phosphate, desalination by gel filtration, followed by sequential extraction with alcohol, buffer solution, distilled water and incorporation of the target product, characterized in that, in order to increase lysozyme activity, previously ticks are exposed to monochromatic polarized light of the visible range with a wavelength of 6328 A and a power of 0.1250.50 mW / cm ^ for 0.5-30 minutes § one to three times with an interval between treatments of 2-6 hours, and washing and subsequent treatment is carried out 1-120 hours after exposure to monochromatic polarized light.