Enzymatic method for preparing 4-o-beta-d-galactopyranosyl-d-xylose

FIELD: biotechnology, biochemistry, enzymes.

SUBSTANCE: invention relates to a method for preparing 4-O-β-D-galactopyranosyl-D-xylose used for in vivo assay of lactase activity in human intestine. Method involves interaction of D-xylose with β-D-galactopyranoside for 2-48 h in a solution at pH 5-9, at temperature that changes from the mixture freezing point to 45°C, and the following addition of 10-1000 U of β-D-galactosidase per 1 g of β-D-galactopyranoside. Reaction is terminated by inactivation of enzyme followed by isolation and crystallization of fraction containing 4-O-β-D-galactopyranosyl-D-xylose in the crystallization mixture chosen from acetone/methanol = (5-1):(20-1) and acetone/water = (5-1):(20-1). Invention provides increasing content of 4-O-β-D-galactopyranosyl-D-xylose in the final mixture.

EFFECT: improved preparing method.

42 cl, 2 tbl, 9 ex

 

The present invention relates to a method for obtaining compounds, especially disaccharides used to determine the activity of lactase in the intestine without blood withdrawal.

The scope of the invention

Failure or low activity of intestinal lactase, which leads to lack of ability or complete inability to digest lactose, is a rare inborn metabolic defect, but a common syndrome in adult patients. However, most mammals there is a noticeable reduction in the activity of lactase after weaning from the breast. A person whose ancestors depended on a significant consumption of milk or dairy products for a long time, this reduction is not so frequent. On the other hand, in infants, not yet weaned, failure or low activity of intestinal lactase is quite rare.

The determination of the activity of lactase in the intestine is of great importance in Pediatrics and gastroenterology. Such analysis can be performed directly on a sample of mucous membrane or indirectly by the level of sugar in the blood or breath hydrogen after administration to the subject a dose of lactase.

The lack of direct measurement is that the analysis is complicated and expensive m is Todd, as for taking tissue samples and their subsequent analysis of the necessary special tools and highly qualified staff, not counting the fact that surgery is unpleasant or partly dangerous for the patient.

Other methods of determining the lactase is based on the affinity of disaccharides to lactase and on the fact that these disaccharides are the substrate of lactase and under the action of the enzyme into certain monosaccharides, which are easily absorbed and are excreted in the urine.

In the patent Spain ES-P-9001680 describes the obtaining of disaccharide 4-O-β-galactopyranosyl-D-xylose of formula (I)

to determine the activity of lactase in the intestine. The specified disaccharide is administered orally, acts as a substrate lactase and, therefore, is hydrolyzed in the intestinal tract with the formation of xylose and galactose, and xylose is absorbed and excreted in the urine, where it is determined by a simple colorimetric method. The amount of xylose output from the urine correlates with the level of lactase in the intestine.

In addition, in patent Spain ES-P-9001680 basically described a method of obtaining 4-O-β-galactopyranosyl-D-xylose, which includes the synthesis of benzyl-β-D-xylopyranoside and the following sequence of reactions involving selective protection of functional groups, glycosylation and removal is their protective groups. A multi-stage process, as well as the use of expensive reagents, such as triplet silver at the stage of glycosylation, as well as chromatographic columns on the stage of purification of intermediates and final product, making the process too costly and time-consuming to implement in preparative scale.

Patent Spain ES-P-9502185 and ES-P-9701156 described enzymatic methods for producing mixtures of disaccharides of galactopyranosides that contain the disaccharide (I) and its regioisomers of 2-O-β-D-galactopyranosyl-D-xylose and 3-O-β-D-galactopyranosyl-D-xylose, respectively, which have the following formula:

The methods described in the patents Spain ES-P-9502185 and ES-P-9701156 allow you to get in one stage, after chromatographic purification of the mixture of 2-, 3 - and 4-O-β-D-galactopyranosyl-D-xylose, which are used as substrates and, therefore, can be used to determine the enzymatic activity of lactase in the intestine. Although these methods are feasible in the presence of appropriate substrates and enzymes, their implementation on an industrial scale is associated with a number of problems, such as determining the most suitable ratios of regimeof, the reproducibility of the production of a product specified with the compared with regimeof and identification of potential impurities.

On the other hand, article Gorin and others, The Synthesis of β-Galacto - and β-Glucopyranosyi Disaccharides by Sporobolomyces singularis. Can. J. Chem., 42, 2307-2319 (1964)described a method of microbiological synthesis of a number of disaccharides, including 2-O-β-D-galactopyranosyl-D-xylose and 3-O-β-D-galactopyranosyl-D-xylose. However, in this publication, there is described the use of various synthetic disaccharides.

The object of the invention

As a first object the present invention proposes a solution to the problems mentioned in the prior art.

As another object the present invention proposes an improved method, which includes any enzymatic reaction of D-xylose with β-D-galactopyranoside substrate and subsequent phase separation and purification, which allows to increase the content of 4-O-β-D-galactopyranosyl-D-xylose in the final mixture resulting from the enzymatic reaction, compared with the content of 2 - and 3-O-β-D-galactopyranosyl-D-xylose, and 4-D-β-D-galactopyranosyl-D-xylose can be distinguished from the final mixture by a simple method.

The next object of the invention is 4-O-β-D-galactopyranosyl-D-xylose, which can be obtained by the above method, as well as compositions that include the specified 4-O-β-D-galactopyranosyl-D-xylose.

Another object of the invention javljaetsja the use of 4-O-β-D-galactopyranose the Il-D-xylose to obtain compositions and solutions used in the analysis of in vivo activity of lactase in the intestine.

Description of the invention

These goals are achieved by the present invention through an enzymatic process for the preparation of 4-O-β-D-galactopyranosyl-D-xylose, which includes:

the first stage receiving a first reaction mixture containing 2-20 wt.% D-xylose, 0.5 to 5 wt.% β-D-galactopyranoside substrate, 75-97,5 wt.% the reaction medium that includes a buffer solution with a pH from 5.0 to 9.0; adding to the first reaction mixture from 10 to 1000 units of enzyme β-D-galactosidase 1 g β-D-galactopyranoside and obtaining a second reaction mixture;

the second stage in which the second reaction mixture is introduced into the reaction at a temperature of from a temperature above the freezing temperature of the second reaction mixture to 45°C for from 2 to 48 h, with the aim of obtaining disaccharides;

the third stage in which the reaction is stopped during the formation of the required number of disaccharides, and a processing method selected from the following methods: inactivation β-D-galactosidase by freezing the second reaction mixture at a temperature of from -20°C to -170°C, inactivation β-D-galactosidase by heating the second reaction mixture at a temperature of from 95°to 110°; and the Department β-D-galactosidase from the second reaction mixture by ultrafiltration with the doctrine a third reaction mixture;

the fourth phase, which aglionby fragment β-D-galactopyranoside substrate used in the first stage, is separated from the third reaction mixture by extraction or filtration to obtain a fourth reaction mixture;

the fifth stage, including the allocation fractions containing 4-O-β-D-galactopyranosyl-D-xylose, and the allocation method chosen from the following two methods:

adding to the fourth reaction mixture of celite, solid phase extraction solvent and elution from the column first eluent, and

adding to the fourth reaction mixture of activated charcoal, filtration and elution with a second eluent;

and a sixth stage in which the fractions containing 4-O-β-D-galactopyranosyl-D-xylose, crystallized from a mixture of solvents, which is chosen from compounds: acetone/methanol in a ratio of from 5:1 to 20:1 acetone/water in a ratio of from 5:1 to 20:1.

The content of D-xylose according to the invention in the second reaction mixture is preferably 7.5 wt.%, content β-D-galactopyranoside 1.5 wt.%, and β-D-galactosidase is added in the amount of 100 units per 1 g β-D-galactopyranoside.

The reaction medium optionally may also contain at least one co-solvent selected from a number of dimethylsulfoxide, dimethylformamide, dioxane and their mixtures, prefer the LNO in the amount of 20% of the reaction medium. In one embodiment of the invention, the reaction medium means a buffer solution with a pH of 7.

To ensure voproizvodit reaction is usually carried out at a constant temperature. In one embodiment of the method according to the invention the temperature of the reaction mixture is above the freezing temperature of the second reaction mixture below 40°C. In another embodiment of the invention the reaction is performed at room temperature, which allows to obtain high outputs without cooling the second reaction mixture. Furthermore, the reaction can be conducted at a temperature of -5°37°C. the Reaction is preferably carried out at a temperature below 0°but above the freezing temperature of the second reaction mixture.

According to the invention β-D-galactopyranoside the substrate is preferably chosen from ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and lactose. Enzyme β-D-galactosidase can be a β-D-galactosidase from E. coli or β-D-galactosidase from Kluyveramyces lactis (such as MAXILACT). When using as a substrate Gal-ONP as a reaction product is formed ortho-NITROPHENOL, which is separated by extraction with ethyl acetate, if the reaction is stopped by heating, or it is separated by conventional filtration, if the reaction is stopped by cooling.

At the third stage of the method, if the reaction austan liveout freezing the second reaction mixture, the temperature is preferably -75°C. on the other hand, if the third stage of the reaction is stopped by heating the second reaction mixture, the temperature is preferably 100°C.

At the fifth stage 4-O-β-D-galactopyranosyl-D-xylose is recovered from the reaction mixture using several alternative methods.

In the first alternative method of the fourth reaction mixture to remove water, thus receive the residue containing disaccharides. The remainder will acetimidoyl will get perceivance derived 4-O-β-D-galactopyranosyl-D-xylose, which is separated by chromatography on a column of silica gel. Acetylation is usually carried out with acetic anhydride in pyridine, and the deacetylation perceiving derived spend when catalyzed by sodium methoxide in methanol.

In the second alternative method, the fourth reaction mixture elute from the column first eluent, which is chosen from a number of mixtures of water with methanol, ethanol or isopropanol, preferably a mixture of water/isopropanol containing isopropanol from 1% to 10% (vol./vol.), preferably 2% (vol./vol.).

Elution is carried out at a filtration through a column of filler chosen from the crosslinked polymers of dextran such as Sephadex, acrylamide polymers, such as Biogel, intense activity is consistent coal or a mixture of activated carbon/celite, you get a fraction containing 4-O-β-D-galactopyranosyl-D-xylose.

Before applying to the column fourth reaction mixture is preferably concentrated. In the third alternative method to the fourth reaction mixture is added celite, the mixture was concentrated to dryness and the residue is subjected to extraction with an organic solvent in the Soxhlet extraction apparatus, and then elute from the column with filler. As a solvent it is preferable to use ethyl acetate. Column select column filled with crosslinked polymers of dextran such as Sephadex, acrylamide polymers, such as Biogel, activated carbon or a mixture of activated carbon/celite. Preferably use a column with activated carbon/telicom, and coal inactivate the addition of hydrochloric acid.

The advantage of the third alternate method is that almost all of the xylose mainly when it is used in large excess is removed before application to the column, which can significantly reduce the amount of filler and the first eluent. Another advantage of the third method is the absolute selectivity of ethyl acetate extraction of the solid phase, resulting in the liquid phase is missing disaccharides and contains mainly xylose and Gal is chose.

In the fourth alternative method for the selection of the product at the fifth stage to the fourth reaction mixture was added activated carbon (instead of using the column filler for separating aglycone from the substrate at the fourth stage). This 4-O-β-D-galactopyranosyl-D-xylose is adsorbed on charcoal and then elute the second eluent. Specified elution is preferably carried out sequential washing of the sorbent and in a stepwise concentration gradient isopropanol/water: in the first stage, the content of isopropanol is from 1% to 3%, in the second stage from 3% to 5%, and in the third stage, from 5% to 7%. The preferred concentration isopropanol are 2%, 4% and 6% respectively. After concentration of the extract and crystallization from acetone/water get cleaned 4-O-β-D-galactopyranosyl-D-xylose.

In the fourth alternative method as the substrate preferably using ortho-nitrophenyl-β-D galactopyranoside.

The fourth alternative method has many advantages, for example, there is no need to heat the second reaction mixture to 100°With the purpose of stopping the reaction, there is no need to separate the aglycone from the substrate by the method of extraction in the fourth stage. Similarly eliminates the concentration of the fourth reactionnaire and therefore, it is not the caramelization. In addition, decreases the amount of activated carbon required to fill the column, and the total number of eluents, there is no need to use celite.

In the sixth stage of the method according to the invention the fractions containing 4-O-β-D-galactopyranosyl-D-xylose, crystallized from a mixture of solvents which is selected from a range of acetone/methanol in a ratio of from 5:1 to 20:1, acetone /water in a ratio of from 5:1 to 20:1, preferably in the ratio of 10:1.

In addition, the invention relates to 4-O-β-D-galactopyranosyl-D-xylose obtained in the above manner, and to compositions and saline or water solutions, which include 4-O-β-D-galactopyranosyl-D-xylose obtained this way, and 4-O-β-D-galactopyranosyl-D-xylose to obtain compositions and solutions that are designed to determine in vivo the content of lactase in the human gut.

In such compositions and solutions of 4-O-β-D-galactopyranosyl-D-xylose is used in a mixture with a pharmaceutically acceptable amount of at least one additional component selected from conventional pharmaceutically acceptable stabilizers, protective agents, flavourings, lactose, gelling agents, plasticizers and preservatives.

4-O-β-D-Galactopyranosyl-D-Xylo is a or its containing compositions or solutions administered orally by the way, while xylose, which appears in the urine, determined by a spectrophotometric method. This method is used as a specific, standard and simple diagnostic analysis the failure activity of lactase without blood withdrawal.

Embodiments of the inventions

The invention is illustrated by the following examples, which are described in more detail some of the above characteristics of the method.

Example 1

The influence of the temperature of the reaction was determined by the following method:

to samples of the reaction mixture containing D-xylose (125 mg, 500 mmol) and ortho-nitrophenyl-β-D galactopyranoside (25 mg, 50 mmol) 1.75 ml of buffer solution (0.05 M KN2PO4/K2HPO4, pH 7, 1 mm MgCl2, 5 mm mercaptoethanol), added the following number of units β-galactosidase from E. coli and incubated at the indicated temperatures.

The temperature of the reaction mixture (°)The amount of enzyme (units)
451,6
371,6
251,6
510
-520

Increasing the amount of enzyme necessary to compensate for the reduction of the reaction rate when the matter is some temperature. The data show that the reaction can be conducted at temperatures below the freezing temperature of water due to freezing point depression effect generated in the reaction medium due to the high concentration of sugar in the samples.

In each sample at each stage of the process was determined by the ratio of 4-, 2 - and 3-O-β-D-galactopyranosyl-D-xylose by gas chromatography on the chromatograph equipped with a flame ionization detector and capillary column SE-54 (0.15 mm ×1.5 m, the thickness of the layer of the liquid phase is 0.3 μm, the flow rate of nitrogen of 1 ml/min). Chromatography was performed under the following temperature conditions:

The initial temperature160°
Start time2 min
The rate of temperature change5°C/min
The final temperature250°

Trimethylsilyl derivatives of the analyzed samples was obtained by the following method:

Aliquot (10 μl) were frozen at -170°and freeze-dried to obtain a dry residue. Then to the dry residue was added pyridine (25 μl)containing the internal standard benzyl-β-D-xylopyranoside (10 mm) and N-trimethylsilylimidazole (25 μl)and heated at 60°C for 30 minutes According to CNAE disaccharides are characterized by the following retention times:

benzyl-β-D-xylopyranoside 12,04 min

2-O-β-D-galactopyranosyl-D-xylose 18,46 and 19,50 min

3-O-β-D-galactopyranosyl-D-xylose 18,30 min

4-O-β-D-galactopyranosyl-D-xylose 20,35 and 20,50 min

Table 1 presents the number of 4-O-β-D-galactopyranosyl-D-xylose (compound I) to the total number of regioisomers (2 - and 3-O-β-D-galactopyranosyl-D-xylose) in terms of maximum yield disaccharides

Table 1
Temperature (°)Priblizitelno reaction time (min)The ratio of compound I/compound II+III
459068:32
3715071:29
2518079:21
527080:20
-512083:17

Their table 1 shows that at lower reaction temperature, an increase in the content of 4-O-β-D-galactopyranosyl-D-xylose.

Example 2

The influence of pH on the course of the reaction was determined by the following method:

received samples of the reaction mixture containing Gal-ONP (25 mg, 50 mmol), D-xylose (125 mg, 500 mmol), galactosidase from E. coli (1,6%) in 1.6 ml of buffer solution (50 mm potassium phosphate, the N 8,5, 7 and 5, 1 mm MgCl2, 5 mm mercaptoethanol), and incubated at 37°C.

The course of the reaction was analyzed in the same way as described in example 1.

Table 2 shows the number of 4-O-β-D-galactopyranosyl-D-xylose (compound I) to the total number of regioisomers, 2 - and 3-O-β-D-galactopyranosyl-D-xylose

Table 2
PHPriblizitelno reaction time (min)The ratio of compound I/compound II+III
8,56068:32
715071:29
518081:19

From table 2 it follows that when carrying out the reaction in an alkaline medium (pH 8.5) content of product I is lower than in a neutral environment (pH 7), and the maximum content is achieved by conducting the reaction in an acidic environment (pH 5).

Example 3

For the synthesis of 4-O-β-D-galactopyranosyl-D-xylose 6 g of ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and 25 g D-xylose dissolved in 330 ml of buffer solution (0.05 M KN2PO4/K2HPO4, pH 7, 1 mm MgCl2, 5 mm mercaptoethanol), was added 2 mg (640 units) β-galactosidase from E. coli and incubated at 30°when mixing on an orbital stirrer (agitator) to practice the Eski full consumption Gal-ONP (approximately 4 hours). Monitoring the progress of reactions was performed by TLC (eluent: isopropanol/NH3(30%)/N2O, 7,5:0,5:2,5) with the following values of Rf:

Rf(Gal-ONP)0,58
Rf(D-xylose)0,47
Rf(4-O-β-D-galactopyranosyl-D-xylose)0,17
Rf(2-O-β-D-galactopyranosyl-D-xylose +
3-O-β-D-galactopyranosyl-In-xylose)0,26

The reaction was stopped by heating in a water bath at 100°C for 10 min, and then the resulting ortho-NITROPHENOL was extracted with methylene chloride. The aqueous layer was concentrated to dryness and the residue was azetilirovanie in the usual way (acetic anhydride/pyridine, 1:1, at room temperature over night under stirring on a magnetic stirrer). Then the reaction mixture was concentrated, the residue of pyridine and acetic anhydride were removed by the sequential addition and evaporation of toluene. Precipitated precipitated salt was removed by filtration, the filtrate was concentrated to dryness and the residue was purified by chromatography on a column of silica gel (eluent:gradient of hexane/ethyl acetate, 4:1 to 1:1). First from the column was suirable acetylated D-xylose, and the mixture is then acetylated disaccharides. F. the shares, containing a mixture of disaccharides, concentrated, the residue was dissolved in methanol, was added a solution of 1 M MeONa/MeOH and the mixture was stirred until completion of deacetylation (the course of the reaction was controlled by TLC, eluent: isopropanol/NH3/N2About). The mixture was neutralized by adding Amberlite IR-120 (H+) and concentrated to dryness. The mixture of free disaccharides was twice led from the Meon/acetone, was obtained 1.07 g of purified 4-O-β-D-galactopyranosyl-D-xylose, exit 17% in the calculation of the original Gal-ONP. tpl.171-176°C.

1H NMR (D2O): δ 5,17 and 4,58 (2d, 1H, J of 3.8 and 7.8 Hz, H-1α and N-1β), 4,55 and 4.45 (2d, 1H, J 7.8 Hz, H-1'), of 4.05 (dd, 1H, J 5,3 and 11.6 Hz, H-5e), to 3.38 (dd, 1H, J is 10.6 and 11.6 Hz)at 3.25 (dd, 1H, J 7.8 and 9.4 Hz, H-2').

Example 4

Column with activated carbon/telicom received by the following method:

200 g of activated carbon (DARCO G-60) and 200 g of celite was mixed in a dry state and added water to obtain a homogeneous paste. The paste was treated with 150 ml HCl (35%) for the inactivation of activated charcoal to remove traces of iron and alkaline ash and washed with water until neutral. Then the paste was placed in a chromatographic column (5×50 cm) and condensed.

For the synthesis of 4-O-β-D-galactopyranosyl-D-xylose 5 g of ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and 25 g D-xylose dissolved in 330 ml of buffer solution (0.05 M KN2PO 4/K2HPO4, pH 7, 1 mm MgCl2, 5 mm mercaptoethanol), was added 2 mg (640 units) β-galactosidase from E. coli and the resulting solution was incubated at 37°when mixing on an orbital stirrer (agitator) to almost complete consumption Gal-ONP (approximately 2 hours). Then, in accordance with the procedure described in example 3, the reaction was stopped by heating at 100°C for 10 min and the resulting ortho-NITROPHENOL were extracted with ethyl acetate. The aqueous layer was concentrated to approximately 50 ml, filtered through glass wool and passed through a column of activated carbon/telicom. First from the column was suirable water an excess of D-xylose, and then in a gradient of 2%-10% EtOH/H2O was suirable mixture of disaccharides. Fractions enriched regioisomer 4-O-β-D-galactopyranosyl-D-xylose, were combined and concentrated. Then acetone was added until turbidity and the resulting mixture was stirred in the cold. Purified 4-O-β-D-galactopyranosyl-D-xylose was obtained as a crystalline substance (970 mg, yield 19% in the calculation of the original Gal-ONP), the spectral characteristics of which correspond to data obtained for the product described in example 3.

Example 5

Column with activated carbon/telicom received by the following method:

200 g of activated carbon (DARCO G-60) and 200 g of celite mixed in cohomologie and added water to obtain a homogeneous paste. The paste was treated with 150 ml HCl (35%) for the inactivation of activated charcoal to remove traces of iron and alkaline ash and washed with water until neutral. Then the paste was placed in a chromatographic column (5×50 cm) and condensed.

For the synthesis of 4-O-β-D-galactopyranosyl-O-xylose and 5 g of ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and 25 g D-xylose dissolved in 330 ml of buffer solution (0.05 M KN2PO4/K2HPO4, pH 7, 1 mm MgCl2, 5 mm mercaptoethanol), was added 2 mg (640 units) β-galactosidase from E. coli and the resulting solution was incubated at 37°when mixing on an orbital stirrer (agitator) to almost complete consumption Gal-ONP (approximately 2 hours). Then, in accordance with the procedure described in example 3, the reaction was stopped by heating at 100°C for 10 min and the resulting ortho-NITROPHENOL were extracted with ethyl acetate. The aqueous layer was concentrated to approximately 50 ml, filtered through glass wool and passed through a column of activated carbon/telicom.

With the aim of obtaining crystalline 4-O-β-D-galactopyranosyl-D-xylose was first suirable water an excess of D-xylose, and then in a gradient of 2%-10% EtOH/H2About was suirable mixture of disaccharides. Fractions enriched regioisomer 4-O-β-D-galactopyranosyl-D-xylose, were combined, concentrated and restore and in a minimum amount of water. Then dropwise acetone was added until turbidity and the resulting mixture was stirred at room temperature for 2 h After 2 h when analysis by TLC in the supernatant (clear) was found to have a certain amount of 4-O-β-D-galactopyranosyl-D-xylose. To a solution of the newly acetone was added until turbidity and maintained for another 2 hours Finally added the next portion of the acetone and the solution was kept in the refrigerator over night. Then the resulting supernatant was observed minimum number of 4-O-β-D-galactopyranosyl-D-xylose. Crystalline 4-O-β-D-galactopyranosyl-D-xylose was separated by filtration and washed with acetone.

If this had been purified 4-O-β-D-galactopyranosyl-D-xylose (1,557 mg, 30% yield in the calculation of the original Gal-ONP), the spectral characteristics of which correspond to the data obtained for the product described in example 3.

Example 6

Column with activated carbon/telicom received by the following method:

200 g of activated carbon (DARCO G-60) and 200 g of celite was mixed in a dry state and added water to obtain a homogeneous paste. The paste was treated with 150 ml HCl (35%) for the inactivation of activated charcoal to remove traces of iron and alkaline ash and washed with water until neutral. Then the paste was placed in a chromatographic to lonco (5× 50 cm) and condensed.

For the synthesis of 4-O-β-D-galactopyranosyl-D-xylose 5 g of ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and 25 g D-xylose dissolved in 330 ml of buffer solution (0.05 M KN2PO4/K2HPO4, pH 6.8, 1 mm MgCl2, 5 mm mercaptoethanol)was added 70% β-galactosidase from Kluyveramyces lactis (MAXILACT®) and the resulting solution was incubated at 37°when mixing on an orbital stirrer (agitator) to almost complete consumption Gal-ONP (approximately 2 hours). Monitoring the progress of reactions was carried out by TLC (eluent:isopropanol/NH3(30%)/N2Oh, 7,5:0,5:2,5) with the following values of Rf:

Rf(Gal-ONP)0,58
Rf(D-xylose)0,47
Rf(4-O-β-D-galactopyranosyl-D-xylose)0,17
Rf(2-O-β-D-galactopyranosyl-D-xylose +
3-O-β-D-galactopyranosyl-D-xylose)0,26

In accordance with the procedure described in example 4, the reaction was stopped by heating at 100°C for 10 min, the resulting ortho-NITROPHENOL were extracted with ethyl acetate and the reaction mixture was filtered to remove residues of the enzyme. The aqueous layer was concentrated in vacuum to a volume when listello 45 ml and passed through a column of activated carbon/telicom. First from the column was suirable water an excess of D-xylose, and then in a gradient of 2%-10% EtOH/H2About was suirable mixture of disaccharides. Fractions enriched regioisomer 4-O-β-D-galactopyranosyl-D-xylose, were combined and concentrated. Then acetone was added until turbidity and the resulting mixture was stirred while cooling. Crystalline 4-O-β-D-galactopyranosyl-D-xylose was separated by filtration on a porous filter. If this had been purified 4-O-β-D-galactopyranosyl-D-xylose (817 mg, yield 16% in the calculation of the original Gal-ONP).

Example 7

Column with activated carbon/telicom received by the following method:

200 g of activated carbon (DARCO G-60) and 200 g of celite was mixed in a dry state and added water to obtain a homogeneous paste. The paste was treated with 150 ml HCl (35%) for the inactivation of activated charcoal to remove traces of iron and alkaline ash and washed with water until neutral. Then the paste was placed in a chromatographic column (5×50 cm) and condensed.

For the synthesis of 4-O-β-D-galactopyranosyl-D-xylose 5 g of ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and 25 g D-xylose dissolved in 330 ml of buffer solution (0.05 M KN2PO4/K2HPO4, pH 7, 1 mm MgCl25 mm mercaptoethanol)was added 80% β-galactosidase from E. coli and the resulting solution was incubated at 37°With at first is mesheanii on an orbital stirrer (agitator) within 24 hours Monitoring the progress of reactions was carried out by TLC (eluent:isopropanol/NH3(30%)/N2O, 7,5:0,5:2,5) with the following values of Rf:

Rf(Gal-ONP)0,58
Rf(D-xylose)0,47
Rf(4-O-β-D-galactopyranosyl-D-xylose)0,17
Rf(2-O-β-D-galactopyranosyl-D-xylose +
3-O-β-D-galactopyranosyl-D-xylose)0,26

In accordance with the procedure described in example 3, the reaction was stopped by heating at 100°C for 10 min, the resulting ortho-NITROPHENOL were extracted with ethyl acetate and the reaction mixture was filtered to remove residues of the enzyme. The aqueous layer was concentrated in vacuum to a volume of approximately 70 ml and the concentrated solution was passed through a column of activated carbon/telicom. First column was suirable 2% isopropanol/water (1.3 l), then 4% isopropanol/water (2.6 liters), collecting the total volume of the eluate of 3.9 liters

Fractions enriched regioisomer 4-O-β-D-galactopyranosyl-D-xylose, were combined, concentrated to a small volume of acetone was added until turbidity and the resulting mixture was stirred in the cold. Crystalline 4-O-β-D-galactopyranosyl-D-xilose is separated by filtration on a porous filter. If this had been purified 4-O-β-D-galactopyranosyl-D-xylose (1,213 mg, yield 24% in the calculation of the original Gal-ONP).

Example 8

Column with activated carbon/telicom received by the following method:

24 g of activated carbon (DARCO G-60) and 24 g of celite was mixed in a dry state and added water to obtain a homogeneous paste. The paste was treated with 18 ml of HCl (35%) for the inactivation of activated charcoal to remove traces of iron and alkaline ash and washed with water until neutral. Then the paste was placed in a chromatographic column and condensed.

For the synthesis of 4-O-β-D-galactopyranosyl-D-xylose 5 g of ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and 25 g D-xylose dissolved in 330 ml of buffer solution (0.05 M KN2PO4/K2HPO4, pH 7, 1 mm MgCb, 5 mm mercaptoethanol)was added 80% β-galactosidase from E. coli and the resulting solution was incubated at 37°under stirring by a mechanical stirrer to practically complete consumption Gal-ONP (24 hours). Monitoring the progress of reactions was carried out by TLC (eluent: isopropanol/NH3(30%)/N2O, 7,5:0,5:2,5) in the same way as described in example 7. In accordance with the procedure described in example 3, the reaction was stopped by heating at 100°C for 10 min, cooled, the resulting ortho-NITROPHENOL were extracted with ethyl acetate. To the water solution add the Yali celite (40 g) and the mixture was concentrated to dryness. The solid residue was extracted with ethyl acetate (500 ml) in a Soxhlet extraction apparatus, equipped with a pulp liner. After 23 h the remainder in the form of a solid substance was washed with water (3×40 ml) and the aqueous extract was passed through a column of activated carbon/telicom. First column was suirable 2% isopropanol/water, then 4% isopropanol/water, collecting a total volume of 400 ml. eluate Fractions enriched regioisomer 4-O-β-D-galactopyranosyl-D-xylose, were combined, concentrated to dryness. The residue was led from acetone/water in the same way as described in example 7, was received of 0.44 g of the pure disaccharide in the form of a crystalline substance.

Example 9

For the synthesis of 4-O-β-D-galactopyranosyl-D-xylose 4.12 g of ortho-nitrophenyl-β-D-galactopyranoside (Gal-ONP) and 20.6 g of D-xylose dissolved in 272 ml of buffer solution (0.05 M KN2PO4/K2HPO4, pH 7, 1 mm MgCl2, 5 mm mercaptoethanol), added 66% β-galactosidase from E. coli and the resulting solution was incubated at 37°under stirring by a mechanical stirrer to practically complete consumption Gal-ONP (21 hours). The reaction was stopped by cooling to 0°and ortho-NITROPHENOL was separated by filtration. Then to the filtrate was added 60 g aktivirovannogo coal and the resulting mixture was stirred for 30 minutes At analyzing the supernatant by TLC was Asano, that in solution there is no disaccharides, since these compounds adsorbed on charcoal. The mixture was filtered, the activated carbon was washed with water (400 ml), 2% isopropanol (100 ml), 4% isopropanol (200 ml) and 6% isopropanol (200 ml). The fractions containing 4-O-β-D-galactopyranosyl-D-xylose, concentrated and the residue (2.38 g) was led from acetone/water, was obtained 1.55 g of solid, which was led again from the same solvent mixture in the same way as described in example 7, it was received 1,32 g (32%) of the pure disaccharide.

1. The enzymatic method of obtaining 4-O-β-D-galactopyranosyl-D-xylose, which includes

the first stage receiving a first reaction mixture containing 2-20 wt.% D-xylose, 0.5 to 5 wt.% β-D-galactopyranoside substrate, 75-97,5 wt.% the reaction medium, which is a buffer solution with a pH from 5.0 to 9.0;

adding to the first reaction mixture from 10 to 1000 units of enzyme β-D-galactosidase 1 g β-D-galactopyranoside, and obtaining a second reaction mixture;

the second stage in which the second reaction mixture is introduced into the reaction at a temperature from values above the freezing temperature of the second reaction mixture to 45°C for from 2 to 48 h, with the receipt of disaccharides;

the third stage in which the reaction octanal who live in the formation of the required number of disaccharides, and the way to handle choose between inactivation β-D-galactosidase by freezing the second reaction mixture at a temperature between -20 -170°and inactivation β-D-galactosidase by heating the second reaction mixture at a temperature of from 95 to 110°; and the Department β-D-galactosidase from the second reaction mixture by ultrafiltration, to obtain a third reaction mixture;

the fourth phase, which aglionby fragment β-D-galactopyranoside substrate used in the first stage, is separated from the third reaction mixture by extraction or filtration to obtain a fourth reaction mixture;

the fifth stage, including the allocation fractions containing 4-O-β-D-galactopyranosyl-D-xylose, and the fifth stage select from the following two methods:

adding to the fourth reaction mixture of celite followed by solid phase extraction with a solvent and elution from the column first eluent; and

direct addition to the fourth reaction mixture of activated charcoal followed by filtration and elution with a second eluent; and then

the sixth stage in which the fractions containing 4-O-β-D-galactopyranosyl-D-xylose, crystallized from a mixture of solvents, which is chosen from compounds: acetone/methanol in a ratio of from 5:1 to 20:1, and acetone/water in the ratio of the drop from 5:1 to 20:1.

2. The method according to claim 1, characterized in that the fourth reaction mixture is concentrated before elution from the column.

3. The method according to claim 1, characterized in that the ratio in a mixture of acetone/methanol is 10:1.

4. The method according to claim 1, characterized in that the ratio in a mixture of acetone/water is 10:1.

5. The method according to claim 1, wherein the first eluent is a mixture of water/isopropanol, which contains from 1 to 10% (vol./about.) isopropanol.

6. The method according to claim 1, characterized in that the mixture of water/isopropanol containing 2% (vol./about.) isopropanol.

7. The method according to claim 1, characterized in that the fifth stage involves adding celite to the fourth reaction mixture and concentration to dryness, the subsequent extraction type solid-liquid organic solvent in the Soxhlet extraction apparatus, provided with a liner made of a material that is compatible with the specified solvent and elution first eluent from the column selected from columns filled with cross-linked dextrans, columns, filled polymeric acrylamide, columns filled with activated carbon or columns filled with activated carbon/telicom.

8. The method according to claim 7, characterized in that the solvent means ethyl acetate.

9. The method according to claim 7, characterized in that the solvent is used in an amount of from 10 ml to 25 ml per 1 g of the original D-xylose.

10. The method according to the .7, characterized in that the celite is used in an amount of from 1 to 2 g per 1 g of the original D-xylose.

11. The method according to claim 7, characterized in that the column contains activated charcoal/celite, where coal inactivate addition of 35% hydrochloric acid.

12. The method according to claim 11, characterized in that the celite is used in an amount of from 0.5 to 2 g per 1 g of the original D-xylose.

13. The method according to claim 11, characterized in that the activated carbon is used in an amount of from 0.5 to 2 g per 1 g of the original D-xylose.

14. The method according to claim 7, characterized in that said first eluent is used in an amount of 5 to 25 ml per 1 g of the original D-xylose.

15. The method according to claim 11, wherein hydrochloric acid is used in an amount of from 0.5 to 1.5 ml per 1 g of the original D-xylose.

16. The method according to claim 1, characterized in that, at the fifth stage of the fourth reaction mixture is applied onto activated carbon at least in the second eluent, and 4-O-β-D-galactopyranosyl-D-xylose is adsorbed on charcoal, and the second eluent, which is water, is replaced by a mixture of water/isopropanol with increasing content of isopropanol.

17. The method according to item 16, wherein in the first stage of elution of the content of isopropanol is from 1 to 3%, in the second stage from 3 to 5%, and in the third stage, from 5 to 7%.

18. The method according to item 16, characterized in that the asset is qualified coal is used in an amount of from 2 to 4 g per 1 g of the original D-xylose.

19. The method according to item 16, wherein the second eluent is used in total, including 30 to 50 ml per 1 g of the original D-xylose.

20. The method according to claim 1 or 16, characterized in that the reaction is stopped by cooling the second reaction mixture at 0°C.

21. The method according to claim 1 or 16, characterized in that the fourth reaction mixture receiving compartment aliceooa fragment from β-D-galactopyranoside substrate filtering.

22. The method according to claim 20, characterized in that the fourth reaction mixture receiving compartment aliceooa fragment from β-D-galactopyranoside substrate filtering.

23. The method according to claim 1, characterized in that the content of D-xylose in the second reaction mixture is 7.5 wt.%.

24. The method according to claim 1, characterized in that the content β-D-galactopyranoside in the second reaction mixture is 1.5 wt.%.

25. The method according to claim 1, characterized in that at 1 g β-D-galactopyranoside added 20 units β-D-galactosidase.

26. The method according to claim 1, characterized in that use reaction medium additionally includes a co-solvent selected from a number of dimethylsulfoxide, dimethylformamide, dioxane and mixtures thereof.

27. The method according to p, characterized in that the reaction medium comprises 20 wt.% co-solvent.

28. The method according to claim 1, characterized in that the reaction is carried out at a constant pace is the atur.

29. The method according to claim 1 or 28, characterized in that the temperature of the reaction mixture ranges from -5 to 40°C.

30. The method according to claim 1 or 28, characterized in that the temperature of the reaction mixture has a value above the freezing temperature of the second mixture and below 0°C.

31. The method according to claim 1, characterized in that the temperature of the reaction mixture is -5°C.

32. The method according to clause 29, wherein the temperature of the reaction mixture is -5°C.

33. The method according to item 30, wherein the temperature of the reaction mixture is -5°C.

34. The method according to claim 1, characterized in that the temperature of the reaction mixture corresponds to room temperature.

35. The method according to clause 29, wherein the temperature of the reaction mixture corresponds to room temperature.

36. The method according to claims 1, 27 or 28, characterized in that as the reaction medium using a buffer solution with a pH of 7.

37. The method according to claim 1, characterized in that in the third stage, the reaction is stopped by freezing the second reaction mixture at a temperature of -78°C.

38. The method according to claim 1, characterized in that in the third stage, the reaction is stopped by heating the second reaction mixture to a temperature of 100°C.

39. The method according to claim 1, characterized in that in the third stage, the reaction is stopped by the Department β-D-galactosidase by ultrafiltration.

40. JV the property according to claim 1, characterized in that as β-D-galactopyranoside substrate using ortho-nitrophenyl-β-D galactopyranoside or lactose.

41. The method according to claim 1, characterized in that the enzyme β-D-galactosidase use β-D-galactosidase from E. coli.

42. The method according to claim 1, characterized in that the enzyme β-D-galactosidase use β-D-galactosidase from Kluyveramyces lactis.



 

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