Producing 2s canola protein using ion exchange

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing 2S canola protein, involving solubilisation of canola proteins from canola oil seed flour using 0.25-0.35M sodium chloride solution with pH 5-6 to form a canola protein solution. The canola protein solution is separated from the residual canola oil seed flour. The canola protein solution is brought into contact with a cation-exchange medium at pH 5-6 to bind 2S canola protein to the cation-exchange medium in preference to other canola proteins. The bound 2S canola protein is separated from unbound canola proteins and impurities, and the bound 2S canola protein is extracted from the cation-exchange medium using 0.55-0.7M aqueous sodium chloride solution.

EFFECT: method enables to obtain 2S canola protein with purity of 99,9%.

6 cl, 9 tbl, 2 ex

 

The technical field to which the invention relates.

The present invention provides a method for the production of 28-canola protein in a clean, mostly, including the use of ion-exchange chromatography.

Prior art

Isolates canola protein having a protein content of at least 100% of the mass. (N×6,25), calculated on dry basis (d.b.), you can get flour from oilseeds canola in the manner described in conjunction examined patent applications U.S. No. 10/137391 with filing date may 3, 2002 (publication of the patent application U.S. No. 20030125526 A1) and No. 10/476230 with a filing date of June 9, 2004 (publication of the patent application U.S. No. 20040254353 A1) and in the corresponding PCT publication no WO 02/089597, the assignee of which is the author of this application and which are included in the list of references to this application. The method is a multistage process involving the extraction of flour from oilseeds canola salt solution; separating the resulting aqueous protein solution from residual meal from oilseeds; increasing the protein concentration of an aqueous solution of at least about 200 g/l while maintaining the ionic strength of the solution basically constant by using a selective membrane technique; thinning the obtained concentrated protein solution into chilled water for mandatory UN paid the formation formation of protein micelles; the deposition of protein micelles by settling with obtaining an amorphous, sticky, gelatinous, like gluten, the protein micellar mass (PMM) and the extraction of supernatant protein micellar mass having a protein content of at least about 100 wt%. when defining a method kildala (N×6,25). In the context of describing the protein content is determined in terms of dry weight. Retrieved RMP may be subjected to drying.

In one embodiment of the method described above supernatant from the stage of settling RMP is processed with the aim of obtaining protein isolate containing dried protein wet PMM and supernatant. This procedure can be carried out by pre-concentrating the supernatant using ultrafiltration membranes, mixing the concentrated supernatant with the wet PMM and drying the mixture. The resulting protein isolate, canola has a high degree of purity of at least about 90% of the mass. protein (N x 6,25), preferably at least about 100 wt%. protein (N x 6,25).

In another embodiment of the method described above supernatant from the stage of settling RMP is processed to extract the protein from the supernatant. This procedure may be performed by pre-concentrating the supernatant using ultrafiltration member and drying of the concentrate. The resulting protein isolate, canola has a high degree of purity of at least about 90% of the mass. protein (N x 6,25), preferably at least about 100 wt%. protein (N x 6,25).

The methods described in the aforementioned patent applications in the United States are essentially periodic ways. In conjunction examined patent application U.S. No. 10/298678 with a filing date of November 19, 2002 (publication of the patent application U.S. No. 20040039174 A1) and the corresponding published International application number WO 03/043439, the assignee of which is the author of this application and which is included in the list of references to this application, describes a continuous method for the production of protein isolates canola. In accordance with this method, the flour from oilseeds canola continuously mixed with a saline solution; the mixture is transported via a pipeline with the simultaneous extraction of protein from flour from oilseeds canola with the formation of the aqueous protein solution; aqueous protein solution is continuously separated from the residual meal from oilseeds canola; aqueous protein solution is continuously passed through a selective membrane with the aim of increasing the protein content of aqueous protein solution, at least about 200 g/l while maintaining the ionic strength of the solution basically constant; the resulting concentrated Belko the initial solution is continuously mixed with chilled water so, to initiate the formation of protein micelles, and the protein micelles are continuously deposited by sedimentation, while the supernatant merges continuously up until the settling tank will not accumulate the required number of RMP. RMP is diverted from the reservoir tank and can be drying. RMP has a protein content of at least about 90% of the mass. in the determination of nitrogen by Kjeldahl method (N×6,25), preferably at least about 100 wt%. (N×6,25).

As described in the aforementioned patent applications U.S. No. 10/137391 and 10/471230, merged with sludge supernatant can be subjected to the treatment to extract the protein extracts of canola.

It is known that the seeds of the canola contain from about 10% to 30% of the mass. proteins, and several different protein components canola has already been identified. These proteins vary in different sedimentation coefficients (S). These known and identified proteins include the 12S globulin, known as cruciferin, the 7S globulin and 2S albumin, known as napin.

Canola also known as canola or oilseed rape.

In conjunction examined patent applications U.S. No. 10/413371 with a filing date of August 25, 2003 (publication of the patent application U.S. No. 20040034204) and No. 10/510766 with a filing date of April 15, 2003, and the corresponding published International application number WO 03/08876, the assignee to whom who is the author of this application and which are included in the list of references to this application, describes the composition of canola protein extracts obtained from the RMP, and canola protein extracts isolated from the supernatant. The canola protein extracts isolated from the supernatant contains mainly protein 2S, along with smaller amounts of 7S protein and trace amount of protein 12S. Protein 2S is a low molecular weight albumin. The product of the RMP contains mainly protein 7S, along with those present in relatively small quantities proteins 2S and 12S. Proteins 7S and 12S are globulins with a higher molecular weight, and the 7S protein molecule is half the protein molecules 12S.

As described in the aforementioned applications, the canola protein extracts isolated from supernatant, the protein has a profile that includes:

from about 60% to 95% of the mass. protein 2S,

from about 5% to 40% of the mass. protein 7S and

from 0 to about 5% of the mass. protein 12S, preferably

from about 70% to 95% of the mass. protein 2S,

from about 5% to 30% of the mass. protein 7S and

from 0 to about 2% of the mass. protein 12S.

The canola protein extracts obtained from the RMP has a protein profile which includes;

from about 60% to 98% of the mass. protein 7S,

from about 1% to 15% of the mass. protein 12S

from 0 to about 25% of the mass. protein 2S, preferably

from about 88% to 98% of the mass. protein 7S,

from about 1% to 10% of the mass. protein 12S

from 0 to about 6 wt%. protein 2S.

It is established that : the Lenna of supernatant protein isolate, canola, consisting predominantly of 2S protein, shows some applications of functional properties, superior functional properties selected from RMP protein isolate, canola, consisting mainly of protein 7S. In the methods described in the aforementioned application, to obtain the isolate canola protein from the supernatant must pass through the stages of education RMP and ensure the supernatant, i.e. essentially through fractionation of proteins from canola.

In the patent application U.S. No. 11/038086 with a filing date of July 21, 2005 (publication of the patent application U.S. No. US 2005-0181112 A1), the assignee of which is the author of this application and which is included in the list of references to this application (WO 2005/067729), describes the way in which the supernatant from the precipitation of the RMP before or after membrane processing is subjected to heat treatment with the aim of deposition of 7S protein and the protein solution enriched protein 2S. The remaining solution may be spray-dried to obtain 2S protein in dry form.

Protein 2S, containing the minimum relative proportion of proteins 7S and 12S, has better solubility than raw protein 2S (including at acid pH values), and is able to provide improved transparency of aqueous solutions and non-alcoholic drinks and beverages for athletes, giving transparent obog is on protein drinks.

The invention

The present invention uses an alternative method involving ion exchange to obtain, mostly pure 2S canola protein, not containing mostly 7S and 12S proteins canola.

In charged ion-exchange chromatography ion-exchange medium is used to bind oppositely charged molecules, while still charged and uncharged materials this environment is not delayed. This makes ion exchange chromatography is a useful tool for purification of charged molecules such as proteins. Two major classes of proteins canola have completely different isoelectric points. Isoelectric point globulin 7S/12S is in the range from 6 to 7, while for albumin 2S this value is approximately 11. This distinction is used in the present invention for the separation of proteins from each other by ion-exchange chromatography.

The invention provides ion-exchange process in which a protein 2S is captured in the associating it with a cation exchange medium, while other proteins and impurities are washed away in the free (unbound) form. Then 2S protein is extracted from the cation-exchange environment by influencing the cation-exchange medium salt solution with acceptable high salt concentration.

According to one aspect of the present invention, provides the I method of production, basically, pure 28-canola protein, including solubilization of proteins of canola meal from oilseeds canola with the formation of a solution of canola protein; separating the protein solution from the residual canola meal from oilseeds canola; the contacting of the solution of canola protein from the cation exchange medium under conditions in which cation exchange environment binds preferably 25-canola protein and not other proteins canola; Department associated 28-canola protein from the unbound proteins canola and impurities and the allocation of the associated 28-canola protein from the cation exchange environment.

Detailed description of the invention

As mentioned above, ion-exchange chromatography is subjected to a solution of proteins canola order preferred binding 28-canola protein with ion exchange medium and the subsequent extraction 28-canola protein in a clean, mainly the form of ion-exchange environment.

The procedure may be performed in any convenient manner. In one preferred aspect of the invention an aqueous canola protein solution is in contact with the cation-exchange medium at a pH of about 5 to 6, in which both classes of proteins are positively charged. The salt concentration is used to limit binding to a lesser extent positively charged 7S/12S canola protein, as well as impurities such as sinepin.

An aqueous solution of proteins canola most conveniently paragraph shall be provided by extraction of flour from oilseeds canola. Extraction is carried out aqueous salt solution of the desired salt concentration and at a pH effective to provide the preferred binding 28 protein with cation exchange environment. In most cases, the salt solution has a salt concentration from about 0.25 M to 0,35M NaCl, and pH aqueous canola protein is about 5 to 6.

Extraction of flour from oilseeds canola can be carried out at pH values outside the desired range, and then set the pH of the solution canola protein in the pH range of about 5 to 6 using any suitable acid or base, if necessary.

Alternatively, the protein can be extracted from the flour from oilseeds canola salt solution with a lower salt concentration and then adding additional salt to the desired concentration. However, it is preferred to perform the extraction of the salt solution with a salt concentration required for ion exchange, the solution of the extract has a form that allows to apply it directly on the cation-exchange environment for the Department of 2S canola protein immediately after receipt. This leaves very little time for oxidation reactions or binding of phenolic compounds with protein.

The solution of the extract proteins canola is applied to cation-exchange environment, which can be of any udobnaia, for example, the shape of the granules of resin or membrane adsorber. In membrane adsorber ion-exchange groups are associated with microporous membranes. Using membranes instead of filled resin columns allows the use of higher flow rate and to accelerate the process.

Contact solution extract of canola protein from the cation exchange medium under appropriate pH and salt level leads to adsorption preferably the 2S protein, in contrast to the less positively charged 7S/12S proteins. After separation of the cation-exchange medium from a solution of the extract proteins canola 28 protein can be removed from the cation-exchange environment by bringing it into contact with an aqueous salt solution having a higher concentration of salt than the salt-containing aqueous solution of proteins canola, for example, from approximately 0,55M to 0,70M NaCl.

Suirvey solution 28 protein has a high concentration of salt and before drying of the protein desalinated any suitable way, such as diafiltrate. This procedure ensures the production of high-purity isolate 28-canola protein not containing mainly 78/128 protein having a protein content of at least about 100 wt%. (N×6,25), calculated on dry basis (d.b.).

7S/12S-canola proteins can be extracted from the extract of canola protein after its contact with the ion exchange medium in nedina mirovnom in contrast to the Department of 2S protein isoelectric precipitation or deposition heating.

Information confirming the possibility of carrying out the invention

Example 1

This example shows how to get mostly clean 2 S-protein canola using a cation exchange column.

(a) Extracting protein

Series 15% (wt./about.) extraction of flour from oilseeds canola was carried out as conventional, using 150 ml of saline solution of 22.5 g of flour. The samples were mixed for 30 minutes at room temperature using a magnetic pen. In each case, the extract was separated from the spent flour by centrifugation at 10200 g for 10 minutes, and then OSVETLENIE by successive filtration through a paper filter with a pore size of 25 μm and filtering syringe with a pore size of 0.45 μm. The protein content in the clarified extract was determined by analytical method LECO (using the analyzer for the determination of nitrogen by the method of burning - LECO FP-528), and the protein profile - exclusion (based on separation by size) chromatography (SEC) HPLC (liquid chromatography high resolution). In different experiments, the concentration of salt in the salt solution was varied from 0,26M to M NaCl.

Manipulation of the concentration of salt in the extraction has had some influence on the protein content and protein profile of the source clarified extract (table 1 below). The increased concentration of the Sol is preferably within the protein yield and extraction 28-protein, but it had a negative impact on the separation of proteins, which will be seen below.

Table 1
Analysis of the extracts obtained at different levels of salt
The concentration of NaCl (M)pHProtein concentration (%)The peak area (%) 2S-protein by HPLC-gram
0,26the ceiling of 5.602,62n.d.*
0,295,672,71n.d.*
0,305,61n.d.*36,9
0,355,612,8237,4
n.d.* = not determined

(b) Chromatography

The samples were subjected to cation exchange (CIEX) chromatography using a column of SP Sepharose XL (20 ml), working together with GradiFrac system LPLC (Pharmacia Biotech) with header peaks. When analyzing each series of samples 10 ml of clarified extract was applied N. the column through the sample loop in the system. When applied concentrations of salt in the salt solution of 2S protein was associated with the column, while the 7S and 12S proteins canola and other impurities passed through the column in the free (unbound) form. Unbound material was located, after which the column was applied salt solution with a higher salt concentration than in the extract, to retrieve the associated 2S protein. Apply the salt concentration was regulated as the separation processes in order to achieve better separation of proteins and to ensure full extraction of the bound material. Applied program analysis samples GradiFrac shown in the following table 2.

0
Table 2
The program GradiFrac for CIEX-extract 2S protein from extract
Volume (ml)The concentration of NaCl in the buffer (M)The flow rate of buffer (ml/min)The maximum amount of the collected fractions (ml)Function
0Extraction50Trim column
to 19.9Extraction50
20Valve for injection of the sample is opena) introducing sample
Extraction525b) linking 2S
20Valve for injection of the sample is closedC) elution 7S/12S
50Extraction525g) elution of impurities
119,9
120Elution525Elution 2S
194,9Elution525
1951,005Purification column
254,91,0050
255Extraction50Trim column
335Extraction50

Note: as the development of the method, there had been some minor changes to the levels.

Note: once it was established that at the stage of elution allocated the entire 28-protein purification column 1M NaCl between analyses were not conducted.

Erwerbende fractions from all of the processes of division in each day of the experiments were combined and frozen at -60°C, except for the product from the process 20-26, which was cooled for subsequent desalting the next day. Table 3 below shows the salt concentration used for extraction and elution in various separation processes.

Table 3
The salt concentration is rementeria for extraction and elution of the 2S protein in various separation processes
A series of samplesThe concentration of NaCl in extraction (M)The concentration of NaCl for elution 2S (M)
10,300,55
20,300,60
3-80,350,65
9-130,290,65
14-260,260,65

The salt concentration used for the extraction/separation of proteins and elution of 2S protein, finely adjusted as the process of separation. In the first separation process used the salt concentration was 0,30M/0,55M. The unbound material was collected in the form of an overlapping pair of peaks, the first peak was found to contain almost all 7S/12S proteins, a small amount of unbound 2S protein and most of the impurities present in the extract, except for the part sinapine. The second peak in the pair, which was slightly elongated and protrudes from the column contained, it was found that a significant number sinapine and very neznicitelnosti protein and other impurities. Elution 0,55M NaCl did not result in the allocation of all the 2S protein from the column as a distinct peak 2S protein was obtained during purification column 1M NaCl solution.

In the second separation process, the level of salt for elution was increased to 0,60M in order to improve the release of 2S protein. This time the peak of the 2S protein was found to have decreased during the purification column. In the third process of dividing the level of NaCl on phase elution was increased to 0,65M, and this level has been found to effectively eliminate peak, previously visible in the purification column. The initial level of salt in the third separation process was increased to 0,35M NaCl, hoping to bring the two free peak. The distance between the paired peaks decreased, but the couple still preserved. So, carrying out the separation at such high levels of salt given the increase in the number of 2S protein that is not associated with the column and detected in the unbound material.

Subsequently, the initial level of salt was reduced to 0,29M, then to 0,26M, which has consistently led to lower level of losses 2S protein from unbound material (table 4 below). There were concerns that the lower the initial level of salt will lead to binding of some part 7S/12S proteins or sinapine with the column, which would be highly undesirable, because it would complicate the stage elution of 2S protein, However, observations have shown, when 0,26M NaCl only protein associated with the column, was the 2S protein.

Table 4
The content of the 2S protein in the total number of unrelated material in terms of the salt concentration in the extract
The concentration of NaCl (M)The peak area 2S (estimated)
0,2666769
0,29115540
0,30n.d.*
0,35224545
n.d.* = not determined

(C) Desalting lirovannomu 2S protein

Frozen samples lirovannomu 2S protein was placed in the refrigerator overnight to thaw. The next day, still frozen containers were placed in a warm water bath in which the sample was heated until complete melting of the ice crystals. Further, all of the thawed samples were filtered through a paper filter with a pore size of 25 μm and were combined into one large sample. The resulting solution of 2S protein was obessolivanija by concentration and diafiltration (DF) on ultrafiltration member the Noah installing Vivaflow 5000 MWCO Hydrosart. The total amount collected 2S-protein fractions amounted to approximately 1500 ml of the combined solution of 2S protein was concentrated to 25-30 ml, and then thereto was added 300 ml of water for diafiltration. The sample was re-concentrated to 25-30 ml) and to it was added the next 300 ml of water, after which the sample was again concentrated. As can be seen from the results given in table 5 below, appeared to be particularly effective carrying out desalting in two stages up to about 10 volumes of diafiltration.

Table 5
Reductions in the salt content of diafiltrate
Sample 2SConductivity (MSM)pHProtein concentration (%)
United GradiFrac fraction49,6of 5.830,31
After adding water to DF1of 5.685,28n.d.*
After adding water DF20,9434,92n.d.*
The floor is secured retentate n.d.*n.d.*of 5.84
n.d.* = not determined

The resulting retentate were subjected to freeze-drying.

(g) the Finished product

The color of the finished dry product was carried out using chromameter Minolta CR-310; was prepared, the solution for color evaluation of the product in the wet state. For this dry protein (0.5 g) was mixed with water (10 ml) using a vortex mixer. Then the sample was centrifugals at 7800 g for 10 minutes (mostly to remove air), and the protein content in the supernatant was determined by the LECO analyzer. Aliquot amount (8 ml) of the supernatant were transferred to a small chemical beaker and to it was added an amount of water sufficient to bring the protein content up to 5%. After photographing the sample aliquot number was used to analyze the protein profile (SEC HPLC). Some samples were also diluted with water to 3.5% protein; they were also photographed. The protein content in the dry powder was determined by the LECO analyzer, but this amount of sample was not sufficient for analysis on moisture content. Therefore, the protein was expressed only in terms of wet mass.

In this study, the total number of goto is on product amounted to 1.63, The protein powder in terms of wet mass was 105,82% wt./mass. (N×6,25). If we Express this in terms of dry weight, as the standard requires, in this case, the protein content will be even higher. Chromatographic analysis registrationpage product showed that 96.1 per cent of the peak area detected at 280 nm, refers to the 2S protein, and 3.8% of the peak area to pronabio. Therefore, 99.9% of the peak area is studied protein. Proteins 7S and 12S was not found.

Color evaluation of dry product are shown in table 6.

Table 6
Color dry 28-protein, isolated from the extract by cation exchange
SampleL*a*b*
2S83,05-2,6115,49

Color registrationpage water wet product showed a greenish hue, and the transparency of the solution was excellent. This suggests that in the complete absence 7S/12S-proteins transparency of the solution should remain practically stable under most conditions.

The product yield was quite high them. Representative output is quite difficult to calculate, because the conditions of the separation were changed many times, and, as you know, there was loss of 2S protein, in particular, during the initial separation processes due to incomplete binding resin, and incomplete elution. The final separation was blueraven all the 2S protein, but still remained a small amount of non-protein column. As mentioned above, the decrease in the initial concentration of salt can help to solve the problem, provided that it will not lead to binding of other species with the column. If you take that out of 260 ml (26×10 ml injection) clarified extract is extracted 1.63 g 28-protein, by extrapolation it is possible to calculate that out of 1000 l of clarified extract can be obtained 6.3 kg 2S protein.

Example 2

This example illustrates the use of a cation-exchange membrane to obtain, mostly pure 2S canola protein.

(a) Extracting protein

10% (wt./about.) extraction 30 g flour from oilseeds canola was performed using 300 ml of 0.3 m NaCl by combining the flour with the salt solution and mixing the samples for 30 minutes at room temperature using a magnetic pen. The extract was then separated from the spent flour by centrifugation at 10200 g for 10 minutes, then OSVETLENIE by th the sequential filtration through a paper filter with a pore size of 25 μm and the filter disks with pore size of 1 μm and 0.45 μm. The protein profile of the extract was determined by SEC HPLC.

The concentration of 0,26M NaCl identified in example 1 as the best choice of the level of salt to the extraction solution. This level of salt was first tested in preliminary experiments with membrane adsorber (data not shown), but, as has been established, a small number 7S-/ - 12S-proteins and some sinapine was associated with the membrane. The increase in salt content in the extraction solution to 0,3M NaCl limited binding 7S-/ - 12S-proteins. Analysis of the protein profile 0,3M NaCl extract showed that 64.6 per cent of the area of protein peaks belonged to 7S-/ - 12S-proteins and 35.4% 2S protein.

(b) Ion exchange

Ion-exchange separation was carried out using two placed sequentially membrane systems with strongly acidic cationic adsorbent Sartobind S75 (Sartorius AG, göttingen, Germany). To promote various solutions through the membrane installation was used peristaltic pump. The scheme of separation of the samples shown in table 7.

Table 7
The scheme of separation of samples to highlight the 2S protein from the extract using cation-exchange membrane adsorber
Stage SolutionVolume(ml)Flow rate (ml/min)
Trim0,3M NaCl4020
Download sampleThe clarified extract1020
Rinse membrane0,3M NaCl5020
Elution 2S protein0,67M NaCl3020

Approximately 32 of the separation process were completed within two days. In each of these days erwerbende 2S-protein fractions from all of the processes of division were combined and frozen until further demineralization. Protein profiles of unbound materials/swabs and buervenich fractions was determined by SEC HPLC.

It is established that the loss of 2S protein from the unbound fraction were low (7.7% of the area of protein peaks) and accounted for, in all probability, the system is overloaded. Erwerbende fraction almost all (99.6% of the area of protein peaks) consisted of 2S protein. In the first day of the processes of separation in quality is TBE buffer for elution were used 0,65M NaCl, and at the end of the day was spent cleaning of membrane adsorbers with 1M NaCl (40 ml). Analysis (SEC HPLC) were obtained using 1M NaCl of the eluate showed the presence of a small amount of 2S protein, which was not blueraven 0,65M NaCl. On the second day in separation as a buffer for elution were used 0,67M NaCl. Cleaning of the membranes 1M NaCl did not lead to the release of the 2S protein, which indicates that 0,67M NaCl is a sufficient concentration to retrieve all related 2S protein.

(C) Desalting selected 2S protein

Suirvey 2S protein was obessolivanija by concentration and diafiltration (DF) on the ultrafiltration membrane plant Vivaflow 5000 MWCO Hydrosart. The amount collected all the 2S protein fractions amounted to approximately 1000 ml of It was concentrated to 25-30 ml, and then thereto was added 300 ml of water for diafiltration. The sample was re-concentrated to 25-30 ml, and again it was added for a further 400 ml of water, after which the sample is again concentrated. Retentate obtained after the second stage of diafiltration, were subjected to freeze-drying.

Conductivity (electrical conductivity) of different samples was measured with a conductivity meter. The purpose of diafiltrate was reduced conductivity of the sample to below 1 MSM (millisiemens). For analysis of protein profile of permeate p is Kanalas SEC HPLC.

Two stages of diafiltration effectively reduced the conductivity of the sample 2S protein (table 8). In permeate from ultrafiltration or diafiltration protein was not detected.

Table 8
Reductions in the salt content of diafiltrate
A sample of the 2S proteinConductivity (MSM)
The eluate49,8
After adding water to DF17,56
After adding water to DF20,79
The resulting retentaten.d.*
n.d.* = not determined

(g) the Finished product

The color of the finished dry product was carried out using chromameter Minolta CR-310; was also prepared solution for color evaluation of the product in the wet state. For this dry protein (0.6 g) was mixed with water (10 ml) using a vortex mixer. Then the sample was centrifugals at 7800 g for 10 minutes, and using the LECO analyzer was determined by the protein content in the supernatant. Aliquot amount (8 ml) of the supernatant were transferred to a small the Oh chemical beaker and to it was added the amount of water sufficient to bring the protein content up to 5%. Then the specimen was photographed, and aliquot volume of sample was used for analysis of protein profile (SEC HPLC). Some samples were also diluted with water to 3.5% protein, and done their pictures. The protein content in the dry powder was determined by the LECO analyzer, but a specified number of sample was insufficient for analysis for moisture content. Therefore, the protein was expressed only in terms of wet mass.

In this study, the total number of the finished product was 1,54, the Degree of purity of the product was very high in protein content in the powder 101,99% wt./mass. (N×6,25) in terms of wet mass. As mentioned above, the specified amount of the sample was insufficient for analysis for moisture content, so the protein could not be expressed in terms of dry weight. Chromatographic analysis registrationpage product showed that 99.85% of the peak area detected at 280 nm, refers to the 2S protein. No 7S-protein, no-12S-protein was not detected.

Color evaluation of the dry product obtained by the use of membrane 2S-adsorber shown in tabl.

Table 9
Color dry what about the 2S protein, isolated from the extract by cation exchange using membrane adsorber
SampleL*a*b*
2S84,21-2,4015,09

Color in the wet state and transparency of the solution product, registrationentry water, resembled the 2S protein, obtained by column chromatography. This suggests that in the complete absence 7S-/ - 12S-proteins transparency of the solution should remain practically stable under most conditions.

Summary of invention

Briefly, the present invention provides a method for extracting high-purity 2S canola protein using ion exchange chromatography. Possible modifications within the scope of the present invention.

1. Method for the production of the 2S canola protein, which is characterized by:
the solubilization of proteins of canola meal from oilseeds canola using sodium chloride brine having a salt concentration of 0,25M 0,35M and pH 5 to 6 with the formation of a solution of protein canola,
separation of the protein solution from the residual canola meal from oilseeds canola,
the contacting of the solution of canola protein from the cation exchange medium p from 5 to 6 for binding to cation exchange medium, preferably the 2S canola protein, and not other proteins canola,
Department associated 2S canola protein from the unbound proteins canola and impurities and
removing a linked 2S canola protein from the cation-exchange medium with an aqueous solution of sodium chloride with a salt concentration of from 0.55 to 0.7 M.

2. The method according to claim 1, characterized in that the cation-exchange medium used in the form of pellets of a resin or membrane adsorber.

3. The method according to claim 2, characterized in that the cation-exchange medium is a membrane adsorber, in which cation exchange groups are associated with microporous membranes.

4. The method according to claim 1, characterized in that the selected 2S canola protein is collected in the form of an aqueous solution of sodium chloride.

5. The method according to claim 4, characterized in that an aqueous solution of sodium chloride 2S-protein absoluut and 2S-protein dried.

6. The method according to any one of claims 1 to 5, characterized in that the unbound proteins canola and impurities are separated from the associated 2S protein by washing the cation exchange environment.



 

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12 cl, 11 dwg, 9 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: described is expression in plants of the mutant polypeptide of the CDK (CKI) inhibitor, having dominant-negative antagonistic activity towards wild-type CKI proteins.

EFFECT: high plant vigour, root mass and size of seeds, enhancement of plant growth at an early stage.

68 cl, 4 dwg, 3 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: constructed are recombinant plasmid DNA pAUTL-GFP bearing a hybrid gene coding green fluorescent protein (GFP) and a signal sequence of autolysin from Chlamydomonas reinhardtii. The produced plasmid is used to transform an Agrobacterium strain. A suspension of the transformed agrobacterial cells in a logarithmic growth stage is incubated with cells of Chlorella microalgae for 14-16 hours A protein secretion level is evaluated by measuring fluorescence of a culture environment.

EFFECT: new compounds show effective biological properties.

2 cl, 1 dwg, 1 tbl, 2 ex

FIELD: agriculture.

SUBSTANCE: plant material is treated with mutagen or is transformed with a polynucleotide structure, containing a sequence of nucleic acid coding mutant polypeptide of large subunit of acetohydroxyacidsynthase (AHASL).

EFFECT: plants acquire tolerance to wide range of herbicides.

74 cl, 2 dwg, 6 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to producing versions of group I Poaceae (holy grass) allergen, also can be used either for specific immunotherapy (hyposensitisation) of patients with grass pollen allergy, or for preventive immunotherapy of grass pollen allergies. The produced versions are characterised by Cys41 Ser, Cys57Ser, Cys69Ser, Cys72Ser, Cys77Ser, Cys83Ser and Cysl39Ser substitutes in a Phi p1 mature protein sequence. Also, a structure of the allergen versions can be presented with no fragments relevant to amino acid residues 1-6, 1-30, 92-104, 115-119, 175-185 and 213-220 or 1-6, 115-119 and 213-220 as a part of a primary sequence of Phi p1 mature protein.

EFFECT: invention allows producing a version of group I Poaceae allergen characterised lower IgE responsiveness as compared with common wild allergen and substantially maintained responsiveness to T-lymphocytes.

8 cl, 9 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: nucleus fractions are extracted from plant cells, from which histone cromatin proteins are extracted using ion-exchange chromatography with amberlite IRC-50 in discontinuous gradient of guanidine hydrochloride.

EFFECT: method enables profound study of molecular-genetic mechanisms of cell nucleus structures and the role of protein components in their organisation.

3 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: biologically active peptide WAMP-1 having the following amino acid sequence: Ala1-Gln2-Arg3-Cys4-Gly5-Asp6-Gln7-Ala8-Arg9-Gly10-Ala11-Lys12-Cys13-Pro14-Asn15-Cys16-Leu17-Cys18-Cys19-Gly20-Lys21-Tyr22-Gly23-Phe24-Cys25-Gly26-Ser27-Gly28-Asp29-Ala30-Tyr31-Cys32-Gly33-Ala34-Gly35-Ser36-Cys37-Gln38-Ser39-Gln40-Cys41-Arg42-Gly43-X, where X-Cys44 or Cys44-Arg45, expresses antimicrobial action.

EFFECT: peptide can be used for protection of plants and foodstuff against pathogenic fungi and bacteria.

3 dwg, 2 tbl, 4 ex

FIELD: genetic engineering.

SUBSTANCE: in a genetically transformed plant, activity of protein Rpi-blb2 is increased.

EFFECT: resistance of the plant to Oomycetes pathogens.

13 cl, 31 dwg, 8 tbl, 17 ex

FIELD: agriculture.

SUBSTANCE: in plant expression is increased for at least one protein Bax inhibitor - 1 (BI1) in at least one vegetable tissue. At the same time expression in leaves epidermis is left mainly invariable. Plants are transformed by recombinant expression cassettes and vectors, which include sequence of nucleic acids that codes BI-protein under control of tissue-specific promoter, which mainly does not have activity in epidermis of leaves.

EFFECT: transformation provides for arrangement or improvement of resistance to at least one biotic or abiotic stress factor in plants, preferably to vegetable pathogens.

13 cl, 16 dwg, 7 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel intermediate compounds of the given formula or acid-addition salts or solvates thereof, where R1 is -(CO)NH2, -CH2NH2 or -CN; R2=R3=H or R2 and R3 together form a cyclic boronate or a boric acid ester; X is an auxiliary group consisting of i) a five- or six-member heterocyclic aromatic ring containing at least one N atom, which is part of an imino group, where said N atom forms a bonding site for a cyclohexapetide ring and ii) tetrazolyl, for which the nitrogen forms a bonding site for a cyclohexapeptide ring. The present invention also relates to a method of producing caspofungin using said intermediate compounds.

.

EFFECT: improved method.

11 cl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes the chromatographic method for treatment of insulin, insulin analogue or derivative of insulin from a mixture containing an akin admixture using a non-linear gradient.

EFFECT: possibility to produce better resolution during separation and higher purity of a target product.

14 cl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: invention describes the chromatographic method for treatment of insulin, insulin analogue or derivative of insulin from a mixture containing an akin admixture using a non-linear gradient.

EFFECT: possibility to produce better resolution during separation and higher purity of a target product.

14 cl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: residue A0 (waste produced during industrial production of a gamma-globulin fraction from abortive blood at the first stage of fractioning) is extracted by 0.05 M sodium-acetate buffer. The produced extract is centrifuged to produce a supernatant. The produced supernatant is chromatographed, and rechromatography is carried out on DEAE cellulose. At the same time the ballast proteins are eluated with 0.05 M sodium-acetate buffer with the following eluation of ESB-containing fraction with a linear gradient of molarity of sodium-acetate buffer from 0.05 M to 0.25 M. Collection of fractions eluated in the range of 0.075 M - 0.15 M of sodium acetate with further concentration, dialysis against distilled water and lyophilisation. The dried fraction is dissolved in 0.015 M sodium-acetate buffer and applied onto KM-cellulose balanced with the same buffer (0.015 M sodium-acetate buffer) with the following performance of the stepped eluation, concentration of the fraction, containing ESB and a-1-acid glycoprotein (α- 1- AGP) and produced with eluation of 0.15 M sodium-acetate buffer. Concentration of this fraction. Dialysis against 0.01 M tris HCl-buffer containing 0.15 M NaCl with subsequent application of the produced fraction onto a column with an immunosorbent IgG anti - α- 1- AGP-sepharose. At the same time the fraction, which is not bound with immunosorbent, is dialysed and lyophilised with subsequent performance of gel-penetrant HPLC on the column with T8K-gel in 0.1 M potassium-phosphate buffer, containing 0.1 M potassium chloride and 5 mM of trilon B. The fraction containing highly pure ESB is dialysed against distilled water and lyophilised.

EFFECT: invention makes it possible to expand arsenal of facilities used in diagnostics of malignant tumors.

1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: residue A0 (waste produced during industrial production of a gamma-globulin fraction from abortive blood at the first stage of fractioning) is extracted by 0.05 M sodium-acetate buffer. The produced extract is centrifuged to produce a supernatant. The produced supernatant is chromatographed, and rechromatography is carried out on DEAE cellulose. At the same time the ballast proteins are eluated with 0.05 M sodium-acetate buffer with the following eluation of ESB-containing fraction with a linear gradient of molarity of sodium-acetate buffer from 0.05 M to 0.25 M. Collection of fractions eluated in the range of 0.075 M - 0.15 M of sodium acetate with further concentration, dialysis against distilled water and lyophilisation. The dried fraction is dissolved in 0.015 M sodium-acetate buffer and applied onto KM-cellulose balanced with the same buffer (0.015 M sodium-acetate buffer) with the following performance of the stepped eluation, concentration of the fraction, containing ESB and a-1-acid glycoprotein (α- 1- AGP) and produced with eluation of 0.15 M sodium-acetate buffer. Concentration of this fraction. Dialysis against 0.01 M tris HCl-buffer containing 0.15 M NaCl with subsequent application of the produced fraction onto a column with an immunosorbent IgG anti - α- 1- AGP-sepharose. At the same time the fraction, which is not bound with immunosorbent, is dialysed and lyophilised with subsequent performance of gel-penetrant HPLC on the column with T8K-gel in 0.1 M potassium-phosphate buffer, containing 0.1 M potassium chloride and 5 mM of trilon B. The fraction containing highly pure ESB is dialysed against distilled water and lyophilised.

EFFECT: invention makes it possible to expand arsenal of facilities used in diagnostics of malignant tumors.

1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method includes conservation of cells in presence of buffered 80-90% glycerine with the following removal of cell shells with 3% triton X-100, extraction with growing concentrations of salts: 0.14 M, 0.35 M; 2 M NaCl, 6 M guanidine hydrochloride with 0.1% b-mercaptoethanol, extraction of positively charged proteins from above fractions with the help of ion-exchange chromatography with amberlite IRC-50 in the continuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% in 0.1 M potassium-phosphate buffer pH 6.8 and detection of inhibiting activity in them relative to tripsin.

EFFECT: invention may be used to analyse molecular-genetic mechanisms of procaryote cell structure generation and role of protein components in their organisation, and also features of genome remodelling.

9 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing, separating and purifying the human granulocyte colony stimulating factor (hG-CSF). Cells of a recombinant producer strain are obtained and the culture fluid is then concentrated. Bacterial cells are broken down while separating inclusion bodies via double treatment with high pressure. Protein impurities are removed. The inclusion bodies are dissolved and protein is recovered. Protein renaturation is carried out for 70-90 hours while diluting the protein solution to 400 ml. The obtained solution is deposited on an anion-exchange chromatographic column with diameter of 300 mm with linear rate of deposition of 600-1000 ml/min. The hG-CSF is then concentrated successively on chromatographic columns with anion-exchange or cation-exchange sorbents. The hG-CSF is then purified on a cation exchanger in fraction recycling conditions and stabilised by dialysis.

EFFECT: method enables to obtain, in one cycle, a stable preparation of hG-CSF in amount of 15-20 g with high homogeneity on HPLC and high hemostimulating activity.

5 cl, 1 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing, separating and purifying the human granulocyte colony stimulating factor (hG-CSF). Cells of a recombinant producer strain are obtained and the culture fluid is then concentrated. Bacterial cells are broken down while separating inclusion bodies via double treatment with high pressure. Protein impurities are removed. The inclusion bodies are dissolved and protein is recovered. Protein renaturation is carried out for 70-90 hours while diluting the protein solution to 400 ml. The obtained solution is deposited on an anion-exchange chromatographic column with diameter of 300 mm with linear rate of deposition of 600-1000 ml/min. The hG-CSF is then concentrated successively on chromatographic columns with anion-exchange or cation-exchange sorbents. The hG-CSF is then purified on a cation exchanger in fraction recycling conditions and stabilised by dialysis.

EFFECT: method enables to obtain, in one cycle, a stable preparation of hG-CSF in amount of 15-20 g with high homogeneity on HPLC and high hemostimulating activity.

5 cl, 1 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing, separating and purifying the human granulocyte colony stimulating factor (hG-CSF). Cells of a recombinant producer strain are obtained and the culture fluid is then concentrated. Bacterial cells are broken down while separating inclusion bodies via double treatment with high pressure. Protein impurities are removed. The inclusion bodies are dissolved and protein is recovered. Protein renaturation is carried out for 70-90 hours while diluting the protein solution to 400 ml. The obtained solution is deposited on an anion-exchange chromatographic column with diameter of 300 mm with linear rate of deposition of 600-1000 ml/min. The hG-CSF is then concentrated successively on chromatographic columns with anion-exchange or cation-exchange sorbents. The hG-CSF is then purified on a cation exchanger in fraction recycling conditions and stabilised by dialysis.

EFFECT: method enables to obtain, in one cycle, a stable preparation of hG-CSF in amount of 15-20 g with high homogeneity on HPLC and high hemostimulating activity.

5 cl, 1 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel intermediate compounds of the given formula or acid-addition salts or solvates thereof, where R1 is -(CO)NH2, -CH2NH2 or -CN; R2=R3=H or R2 and R3 together form a cyclic boronate or a boric acid ester; X is an auxiliary group consisting of i) a five- or six-member heterocyclic aromatic ring containing at least one N atom, which is part of an imino group, where said N atom forms a bonding site for a cyclohexapetide ring and ii) tetrazolyl, for which the nitrogen forms a bonding site for a cyclohexapeptide ring. The present invention also relates to a method of producing caspofungin using said intermediate compounds.

.

EFFECT: improved method.

11 cl, 9 ex

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