RU2095417C1 - METHOD OF PREPARING 1 → 3; 1 → 6 - β-D-GLUCAN SHOWING IMMUNOSTIMULATING ACTIVITY - Google Patents

Abstract

FIELD: biotechnology, medicine. SUBSTANCE: invention relates to preparing translam - 1 → 3; 1 → 6 - β-D-glucan. Biologically active 1 → 3; 1 → 6 - β-D-glucan is obtained by enzymatic transformation of laminarin with endo-1 → 3; 1 → 6 - β-D-glucanase Lo from bivalve mollusk Chlamus albidus at enzyme concentration (0.7-1) x -1 → 3 - β U/ml and a mixture is incubated up to complete enzyme inactivation. Chromatography purification of the end product is carried out on polychrome-1 by successive elution of by-side reaction products with water, 2.5% and 10% an aqueous ethanol. The end product - translam is eluted with 15% an aqueous ethanol. EFFECT: improved method of preparing. 2 tbl

Description

 The invention relates to the pharmaceutical industry, relates to methods for producing active preparations of carbohydrate nature from raw materials of plant origin, in particular, brown algae.

Mixed 1 __ → 3; 1 __ → 6-β-D - glucans among neutral polysaccharides are of the greatest interest, since they have a pronounced effect on organisms of animals and plants in much lower concentrations than other polysaccharides [1 3] Some 1 __ → 3; 1 __ → 6-β-D - glucans are immunostimulants, have radioprotective and antitumor properties [1, 2, 4 6]
As a prototype, a well-known method of obtaining having immunostimulating activity 1 __ → 3; 1 __ → 6-β-D - glucan from the fungus Pseudoplectanias class Diseomycetes [7] which is as follows. The fungal mycelium is extracted with large volumes of water, the extract is concentrated and the substance is precipitated with an equal volume of ethanol. 1 __ → 3; 1 __ → 6-β-D - Glucan is sequentially purified by dialysis, chromatography on DEAE-G-50 Sephadex, dialyzed and dried again. The yield of the substance is 6.5%. The glucan isolated from this fungus has a high molecular weight (about 2.5 • 10 ^-5 ). The ratio 1 __ → 3; and 1 __ → 6 bound residues in the molecules, biological activity and effective doses of 1 __ → 3; 1 __ → 6-β-D - glucan from the fungus Pseudoplectanias class Diseomycetes are close to the corresponding characteristics of the translamin obtained by the new method.

 As was shown [8, 9] as a result of the enzymatic transformation of low molecular weight 1 __ → 3; 1 __ → 6-β-D - glucan of laminarin from the brown alga Laminaria cichorioides was obtained higher molecular weight and having a different structure than the original laminarin, 1 __ → 3; 1 __ → 6-β-D - glucan called translam. Translam has a wide range of biological activities: an immunostimulating effect, stimulating the T-system of animal immunity [8] exhibits an anti-tumor effect against sarcoma-180 [9] Translam is readily soluble in water, with its introduction there are no side effects. These circumstances prompted us to develop a technologically advanced method for producing translam proposed for patenting.

 The objective of the invention is to develop a simple method for producing translam, allowing to produce a sufficiently large amount of the target product.

The problem is solved in that in the method of obtaining translam 1 __ → 3; 1 __ → 6-β-D - glucan having immunostimulating activity by exposure to laminarin endo-1 __ → 3-β-D - glucanase Lo from bivalve mollusk Chlamys albidus and chromatographic purification of the target product, endo-1 __ → 3- β-D - Lo glucanase from Chlamys albidus is used at a concentration of (0.7 1) • 10 ^-2 u / ml, the mixture is incubated until the enzyme is completely inactivated, and the target reaction product is chromatographed on polychrome-1 by sequential elution of the reaction by-products with water , 2.5% aqueous ethanol, and 10% aqueous ethanol, target pro BCCH translam eluted with 15% aqueous ethanol.

Thus, the task is carried out due to the enzymatic method of obtaining the target product and use for separation of the products of the enzymatic reaction of polychrome (PH-1). It was found that the Lo enzyme at a low concentration loses its activity under the conditions of translam production during the day. When carrying out the enzymatic reaction, such a negative property of endo 1 __ → 3-β-D - Lo glucanase as lability is used. The minimum amount of the enzyme was selected (0.7 1) • 10 ^-2 units / ml, under the influence of which, during the day, laminarine is transformed with the maximum yield of the target product, and endo 1 __ → 3-β-D - Lo glucanase time is completely inactivated and the reaction stops. Due to this, there is a saving of the enzyme, the production of which requires expensive reagents, and a reduction in the stages of controlling the exit of translam and stopping the reaction at the desired depth.

 Next, the products of the enzymatic reaction are separated into polychrome-1, using a stepwise gradient of water, aqueous ethanol. Glucose is eluted with water and 2.5% aqueous ethanol and 1 __ → 3 or 1 __ → 3; 1 __ → 6-β-D - gluco-oligosaccharides, 10% aqueous ethanol fraction 1 __ → 3; 1 __ → 6-β-D - glucans (mm 3 8 kDa), inactive in animal immunity, 15% aqueous ethanol translam.

The proposed method is as follows. A solution of laminarine (3 L; 9 10 mg / ml laminarin in 0.1 M NaCl and 0.05 M acetate buffer; pH 5.0 5.5 optimal conditions for the action of the enzyme [10]) are treated endo-1 __ → 3 -β-D - Lu glucanase from Chlamys albidus at a concentration of (0.7 - 1) • 10 ^-2 u / ml (1 unit of enzyme activity is equal to its amount that catalyzes the formation of 1 μM glucose per minute). The mixture is incubated for 23 25 hours at 22 25 ^o C. During this time, under these conditions, the accumulation of translam in the amount of 20 25% of the original laminarin, and the enzyme is completely inactivated. Next, the mixture was separated on a column (7 x 65 cm) with polychrome-1, using elution with water and 2.5-, 10-, 15-th aqueous solutions of ethanol (table. 1). The output of translam is 20 - 25%
The method of producing translam has the following advantages over the known. Isolation of translam from the products of the enzymatic transformation of laminarine is carried out using a hydrophobic sorbent polychrome-1, which has a large capacity. The basis for obtaining biologically active 1 __ → 3; 1 __ → 6-β-D - glucan provides a method for the enzymatic transformation of laminarine. Using in the method the ability of endo-1 __ → 3-β-D - Lu glucanase from Chlamys albidus to inactivate over time eliminates the possibility of hydrolysis of translam (up to its disappearance) due to an overdose of the enzyme; there is no need for such labor-intensive stages as monitoring the progress of the reaction and inactivation of excess enzyme (for which the reaction mixture would need to be heated quickly, for example, which is difficult in case of large volumes).

 The method is illustrated by the following examples.

Example 1. Laminarin (30 g) was dissolved in 3 L of 0.05 M acetate buffer pH 5.0, containing 0.1 M NaCl. Introduce 120 units of endo-1 __ → 3-β-D-glucanase Lo and incubate the mixture at 25 ^o C for a day. The incubation mixture (3 L) is applied to a column (7 x 65 cm) in which 2.5 kg of polychrome-1 is loaded. The elution is carried out first with water and 2.5% aqueous ethanol each time until a negative reaction to sugars (according to the phenol-sulfuric acid method [II]). At the same time, salts, glucose, low molecular weight gluco-oligosaccharides are completely washed out. Then 10 and 15% aqueous ethanol elute, respectively, fraction 1 __ → 3; 1 __ → 6-β-D - glucans (Mm 3 8 kDa), inactive in animal immunity, and translam, each time washing the column to a negative reaction to sugar. The free volume of the column used is 3 l, i.e. after passing 3 l of eluent, sugar begins to come out. Therefore, when eluting and collecting each fraction, the first 3 L of the eluate can be discarded, and the remaining volume that contains sugar can be collected. All collected fractions are freeze-dried. Under these conditions, 93% of glucose and low molecular weight products are formed, the yield of translam is 3%
Polychrome-1 regeneration is carried out sequentially with 50% ethanol (10 L) and water (10 L). After such regeneration, the column is ready for a new cycle. The properties of the sorbent during use do not change, the column parameters do not change either: once you analyze the column and set the parameters, you can use these indicators.

Example 2. The method is carried out as in example 1, but 45 units of the enzyme are added to the laminarine solution. Under these conditions, about 32 glucose and low molecular weight oligosaccharides are formed, 45% of the fraction 1 __ → 3; 1 __ → 6-β-D - glucans (Mm 3 8 kDa), inactive in animal immunity, translam yield 16%
Example 3 (table. 2). The method is carried out as in example 1, but 30 units of the enzyme are added to the laminarine solution. Under these conditions, about 23% of glucose and low molecular weight oligosaccharides are formed, fraction 1 __ → 3; 1 __ → 6-β-D - glucans (mm 3 8 kDa) is 49%; translam yield 23%
Example 4. The method is carried out as in example 1, but 20 units of the enzyme are added to the laminarine solution. Under these conditions, 19% glucose and low molecular weight oligosaccharides are formed, fraction 1 __ → 3; 1 __ → 6-β-D - glucans (mm 3 8 kDa) is 55%; translam yield 20%
Example 5. The method is carried out as in example 1, but 15 units of the enzyme are added to the laminarine solution. Under conditions, 12% glucose and low molecular weight oligosaccharides are formed, fraction 1 __ → 3; 1 __ → 6-β-D - glucans (mm 3 8 kDa) 77% translam output 7

Claims (1)

The method of obtaining 1 _ → 3; 1 _ → 6-β-D-glucan having immunostimulating activity, including a stage of its chromatographic purification, characterized in that laminarin is exposed to endo-1 _ → 3-β-D-glucanase L _о from bivalve mollusk Chlamys albidus taken in concentration (0.7 - 1) • 10 ^{- 2} units / ml, and the reaction mixture is incubated until the enzyme is completely inactivated, after which the reaction products are chromatographed on polychrome 1, with subsequent elution with water, 2.5%, 10 % aqueous ethanol and elution of the target product with 15% aqueous ethanol.

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