JP2003088394A - Method for producing organic hydrolyzate and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for selectively and efficiently producing a polymeric organic substance partial hydrolyzate in an industrial scale. SOLUTION: This method for producing the polymeric organic substance partial hydrolyzate is characterized by simultaneously carrying out the hydrolysis of the polymeric organic substance in the presence of a hydrolytic enzyme and the purification of the obtained polymeric organic substance partial hydrolyzate by an electrophoresis using an ultrafiltration membrane and a dialysis membrane.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for simultaneously performing partial hydrolysis and purification of a high molecular weight organic substance, that is, a method for simultaneously producing and purifying a low molecular weight organic substance.

[0002]

2. Description of the Related Art For example, chitosan oligosaccharide, which is a partial decomposition product of chitosan which is one of organic substances, is desired to be developed for use in food additives, cosmetics, pharmaceuticals and the like.

In particular, higher chitosan oligosaccharides having 5 or more sugars have recently been found to have various physiological activities such as antibacterial activity, antitumor activity and immunostimulatory activity.
About 10,000 chitosan is expected to be applied to adhesives and paints, and its added value and demand are increasing.

However, a method for efficiently producing such higher chitosan oligosaccharides having 5 or more sugars and higher chitin oligosaccharides has not been established yet, and in particular, higher chitosan oligosaccharides having 7 or more sugars and higher chitin oligosaccharides have been established. Is difficult to prepare, and is currently not commercially available.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method and an apparatus for producing an organic substance having a low degree of polymerization selectively, efficiently and on an industrial scale.

[0006]

The present invention includes the following items 1 to 1.
Regarding 1

Item 1. Partial decomposition of high molecular weight organic compounds characterized by simultaneous partial decomposition of high molecular weight organic compounds using hydrolases and purification of the obtained partial decomposition products of high molecular weight organic compounds by electrodialysis using an ultrafiltration membrane Method of manufacturing things.

Item 2. By partially decomposing the polymer organic matter in a reaction tank having a polymer organic matter and a hydrolase capable of partially decomposing the organic matter, and simultaneously performing electrodialysis, the partial decomposition product is passed from the reaction vessel through an ultrafiltration membrane. Item 2. The method according to Item 1, wherein the method is introduced into a separation tank.

Item 3. Item 3. The method according to Item 2, wherein the reaction tank and the separation tank are present in an internal space partitioned by a dialysis membrane, and the reaction tank and the separation tank are partitioned by an ultrafiltration membrane.

Item 4. A method for producing a partially decomposed organic substance, comprising supplying the high molecular weight organic compound to a reaction tank and separating the obtained partially decomposed organic polymer into a separation tank simultaneously or sequentially.

Item 5. High molecular weight organic substances are chitin, chitosan,
Item 1 characterized by being a charged macromolecule including polysaccharides including pectin, peptidoglycan, glycosaminoglycan and proteoglycan, proteins and nucleic acids
The method according to any one of to 4.

Item 6. High molecular weight organic substances are chitin, chitosan,
Item 6. The method according to Item 5, which is a polysaccharide containing pectin, peptidoglycan, glycosaminoglycan and proteoglycan.

Item 7. Item 7. The method according to Item 6, wherein the high molecular weight organic substance is chitosan.

Item 8. Item 1 to 4 wherein the hydrolase is at least one selected from the group consisting of chitinase, chitosanase, cellulase, lysozyme, pectinase, pectinesterase, glucosidase, glucosaminidase, proteoglycanase, protease and nuclease.
The method described in any one of.

Item 9. Item 5. The method according to any one of Items 1 to 4, wherein the hydrolase is an enzyme having a chitosan degrading activity.

Item 10. An electrodialysis apparatus comprising a reaction tank equipped with a hydrolyzing enzyme of a high molecular weight organic compound, a separation tank for separating a partially decomposed product of the high molecular weight organic material, and an electrode tank.

Item 11. Item 11. The apparatus according to Item 10, wherein the reaction tank and the separation tank are separated by an ultrafiltration membrane, and the reaction tank and the electrode tank and the separation tank and the electrode tank are separated by a dialysis membrane.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION The high molecular weight organic substance in the present specification includes a charged polymer such as a polysaccharide such as chitin, chitosan, pectin, peptidoglycan, glycosaminoglycan, proteoglycan, protein and nucleic acid. Chitin excludes those with 0% deacetylation. The molecular weight of the high molecular weight organic substance is 10 to 5,000,000, preferably 10,000 to 100.
The molecular weight of the partially decomposed product is 100 to 50,000, preferably 1,000 to 20,000.

As described above, the partially decomposed product of chitosan is known to have antibacterial properties, antitumor properties, immunostimulatory activity, etc., and the partially decomposed product of pectin has antibacterial action, intestinal action, etc. It is known that the partially decomposed product has a blood pressure lowering action, a calcium absorption promoting action, a high sweetness and the like as a peptide.

The hydrolase used for the partial decomposition of the high molecular weight organic substance is not particularly limited and is appropriately selected depending on the type of the high molecular weight organic substance. For example, for the partial decomposition of chitin and chitosan, chitinase, chitosanase, lysozyme,
Cellulase, pectin, pectinesterase, etc., peptidoglycan, glucosidase, etc., glycosaminoglycan, glucosaminidase, etc.
Proteoglycans, proteases and the like can be used for proteoglycans, proteases and the like for proteins, and nucleases and the like for nucleic acids.

The method of using the enzyme in the present invention is not particularly limited, but it may be simply added to a solution or suspension of a high molecular weight organic substance for reaction, and immobilized on a membrane or a particulate solid for use. can do.

The apparatus 1 used in the present invention is an electrodialysis apparatus 1 equipped with a reaction tank 2, a separation tank 3 and an electrode tank 4 as illustrated in FIG. 1, the reaction tank 2 and the separation tank 3 is ultrafiltration membrane 5
The reaction tank 2 and the electrode tank 4, and the separation tank 3 and the electrode tank 4 are each partitioned by a dialysis membrane 6.

In the reaction tank 2, a high molecular weight organic substance reacts with a hydrolase. The reaction tank 2 is, depending on the reaction conditions, etc.,
The stirring can be performed using a stirrer 7, a stirrer such as a liquid feeding device, and the temperature can be kept constant at a predetermined temperature by using a constant temperature device such as a heater or an incubator.

In the separation tank 3, the partially decomposed product of the high molecular weight organic substance that has passed through the ultrafiltration membrane 5 is separated from the high molecular weight organic substance, hydrolase and the like.

The ultrafiltration membrane 5 can be used with any membrane as long as it does not permeate enzymes such as chitosanase and high molecular weight organic substances and can pass through partially decomposed products such as desired chitosan oligosaccharides produced. be able to. The ultrafiltration membrane includes flat membrane type, multistage flat membrane type, hollow fiber type and the like, and any type can be used.

Further, by providing a plurality of ultrafiltration membranes having different pore sizes or using a separation tank through such a plurality of ultrafiltration membranes, the partial decomposition products having various molecular weights can be separated. You can also get a picture.

The electrode tank 4 is equipped with an electrode 9 and is separated from the reaction tank 2 and the separation tank 3 by a dialysis membrane 6. A known electrode can be used as the electrode 9, and the electrode 9 is not particularly limited, but for example, a platinum electrode or the like can be used. Due to the presence of the electrode tank 4, salts move to the electrode tank 4 through the dialysis membrane 6,
The partially decomposed product present in the separation tank 3 can be purified (concentrated).

The dialysis membrane 6 may be of any type as long as it does not allow the partial decomposition product of the high molecular weight organic material to pass therethrough and allows an electric current to flow, and a known material such as an ion exchange membrane or a reverse osmosis membrane can be used. For example, various polymers such as polypropylene and polyvinyl alcohol can be used for the ion exchange membrane, and polyamide and the like can be used for the reverse osmosis membrane.

By circulating the electrolytic solution in the electrode tank 4 using the pump 11, it is possible to keep the temperature, salt concentration, etc. of the electrode tank constant.

A high-molecular organic material solution and an enzyme are placed in the reaction tank 2, and a hydrolysis reaction is carried out at a predetermined reaction temperature. The reaction temperature and reaction pH are not particularly limited as long as they are the temperature and pH at which the enzyme reaction occurs, but the optimum temperature and optimum of the enzyme used
pH is preferred.

Specifically, the reaction temperature is, for example, 10 to 60 ° C, preferably 20 to 50 ° C. Further, the pH is exemplified as 3 to 8,
4.5 to 7 is preferable. The conditions for electrodialysis are not particularly limited,
Those skilled in the art can appropriately select conditions such as current and voltage.

The enzyme reaction can be carried out in water, and in order to stabilize the pH, phosphate buffer, acetate buffer, Tris-
It is preferable to use various buffer solutions such as a hydrochloric acid buffer solution and a borate buffer solution. The concentration of the buffer solution is preferably 0.01 to 2M, 0.0
5 to 1M is more preferable.

In the reaction vessel, 0.1-10%, preferably 0.2-1
A predetermined amount of a chitosan solution dissolved in a buffer solution so as to have a concentration of 0.1% and an enzyme such as chitosanase at 0.1 to 5 U / ml, preferably 0.5 to 3 U / ml are added.

A stirring device such as a stirrer may be used so that the reaction is promoted. The stirring speed may be 100 to 2000 rpm, preferably 250 to 1000 rpm.

As shown in FIG. 2, for example, in the case of chitosan having a positive charge, chitosan and an enzyme (chitosanase) are reacted in a reaction tank, and a partially decomposed product (chitosan oligosaccharide) permeates through an ultrafiltration membrane. By doing so, it moves to the separation tank on the negative electrode side. At this time, the enzyme and the high molecular weight organic substance that has not been partially decomposed do not pass through the ultrafiltration membrane and remain in the reaction tank.

The method of taking out the partially decomposed product of the obtained high molecular weight organic matter from the separation tank and the method of purifying the taken out partially decomposed product are not particularly limited, and known methods can be used.

Further, by continuously adding an aqueous solution of a high molecular weight organic substance to a reaction tank or by adding an organic substance in a solid state such as a powder state, production and purification of a partially decomposed product are continuously conducted. Alternatively, it can be performed sequentially. The reaction may be carried out batchwise or continuously.

[0038]

EXAMPLES Examples will be given below to explain the present invention in more detail.

Example 1 Enzyme reaction conditions were 0.1 M acetate buffer (pH 5.3), substrate concentration
0.5%, each of the reaction tank, separation tank and electrode tank is filled with 50 ml of buffer solution, the reaction temperature is 35 ° C., the stirring speed in the reaction tank is 500 rpm
Reaction and separation by electrodialysis were performed. The enzyme is Bacillus p
umils BN262-derived chitosanase (Wako Pure Chemical Industries, Ltd.)
Was used for the reaction with a specific activity of the enzyme of 40 U / mg and a final concentration of 33 μg / ml.

Ultrafiltration membrane P0200 (Advantech Co., cut off molecular weight 20,000), reverse osmosis composite membrane NTR-7400 (Nitto Denko)
15V using the residual chlorine allowable limit of 100ppm or less)
Electrodialysis at a constant voltage was performed while causing an enzymatic reaction. After 48 hours of reaction time, 2 ml was taken from 50 ml of the solution in the separation tank, and the average molecular weight was measured. The average molecular weight of chitosan is Shodex GPC
Measurement and analysis were carried out by an apparatus in which a multi-angle light scattering detector was connected to SYSTEM-21. The tested chitosan was crustacean-derived chitosan 7B (deacetylation degree 70%) (Funakoshi Co., Ltd.)
Was used. The sample for molecular weight measurement was measured using a 0.45 μm membrane filter.

Chitosan molecular weight measurement conditions HPLC apparatus; pump Shodex WP-03 Differential refractive index detector Shodex RI-71S multi-angle light scattering detector Wyatt Dawn DSP Water-based GPC column Shodex Ohpak SB-800MHQ + SB-800R × 2 Measurement conditions Column temperature 40 ℃ Eluent 1% acetic acid + 0.05M sodium acetate Flow rate 0.4ml / min Analysis software; ASTRA for Windows (registered trademark) Version 4.
20. After performing enzymatic decomposition and electrodialysis with 0.5% chitosan 7B solution,
The results of the differential refractive index detection by HPLC and the measurement of the average molecular weight are shown in FIGS. Chitosan 7B had an average molecular weight of about 200,000 before decomposition, but it was degraded to a low molecular weight by enzymatic decomposition, and a low molecular weight chitosan was purified in a wide range from about 20,000 to several hundreds (yield 50.0 %).

200 ml of ethanol was added to 50 ml of the separated liquid, and the mixture was stirred and then centrifuged at 3,000 G. The precipitate was dried and weighed to obtain 0.43 g of precipitate. As a result of measuring the molecular weight, the precipitate due to ethanol has a molecular weight of 2,000.
The average molecular weight was 9,000 (Figs. 5 and 6). The yield was 20.8%.

Example 2 0.5% chitosan 10B (deacetylation degree 100%) (Funakoshi
7 and 8 show the results of detection of the differential refractive index by HPLC and measurement of the average molecular weight after enzymatic decomposition and electrodialysis were carried out under the same conditions as in Example 1 using a solution of Co., Ltd. Chitosan 10B
Although the average molecular weight before decomposition was about 150,000, the decomposed products produced in the separation tank showed a distribution of molecular weights of 300 to 3,000, and chitosan oligosaccharides with an average molecular weight of around 1,000 were obtained. The yield was 34.9%.

Example 3 Chitosan was extracted using Absidia butleri HUT1001 strain (preserved strain of Hiroshima University) to prepare a chitosan oligosaccharide. Pre-culture was performed using YPD agar medium (1% yeast extract, 2% polypeptone, 2% glucose, 2% agar) at 24 ° C. When spores were fully established, add 5 ml of 0.01% tween 80 solution and mix well. did. Then Miracross (made by CALBIOCHEM)
And filtered to prepare a spore suspension.

The main culture was carried out in two 500 ml shake flasks, each containing a basal medium (2% glucose, 0.1% yeast extract, 1% polypeptone, 0.1% dipotassium phosphate, 0.05% magnesium sulfate heptahydrate, 0.5%). Dispense 200 ml of ammonium sulfate, 0.01% calcium chloride dihydrate, pH 4.5), sterilize, and then use Absidi
Spores of a butleri HUT1001 strain were inoculated at 1 × 10 ⁴ cells / ml and cultured at 28 ° C., amplitude 3 cm, shaking speed 140 rpm for 72 hours. The cultured cells were filtered with a glass filter (2G2), washed with distilled water, and freeze-dried.

Chitosan was extracted from the cells by freeze-drying and then adding 100 ml of 2% sodium hydroxide solution to the cells at 115 ° C.
After 60 minutes of autoclaving, the solution was hot and filtered through a glass filter (2G3), washed with hot water, and further washed with distilled water until the filtrate became neutral. 50 ml of a 2% acetic acid solution was added to the alkali insoluble matter of the glass filter, and the mixture was stirred with a magnetic stirrer at room temperature for 24 hours, and then centrifuged (1000 G, 15 minutes). 1 more on this glass filter
00 ml of 2% acetic acid solution was added, extraction was performed at room temperature for 60 minutes, and centrifugation was performed. The two supernatants were combined, 0.1N sodium hydroxide solution was added dropwise to adjust the pH to 8.5, and the resulting precipitate was centrifuged (1000G, 15 minutes), washed with water, centrifuged and freeze-dried. After that, the result of weighing was 360 mg of chitosan.

A 0.5% chitosan solution was prepared from the obtained chitosan, and this was used to enzymatically decompose it under the same conditions as in Example 1,
The results of differential refractive index detection by HPLC and measurement of the average molecular weight after electrodialysis are shown in FIGS. Absidia
Chitosan extracted from butleri had an average molecular weight of about 130,000 before degradation, but the degradation products produced in the separation tank showed a distribution of molecular weights of 500 to 7,000, and chitosan oligosaccharides with an average molecular weight of around 1,000 were obtained. It was The yield was 43.1%.

[0048]

According to the method of the present invention, the enzymatic reaction and the electrodialysis are carried out at the same time, whereby the hydrolysis of the polymer substance and the purification of the partially decomposed product obtained can be carried out at the same time.

Further, by using an enzyme, the reaction can be easily controlled, and the reaction can be stopped at a desired point.
Even if the reaction is carried out in a buffer or the like, the separation tank can be desalted, so that the partially decomposed product can be purified at the same time.

That is, according to the method of the present invention,
Since a partially decomposed product of a high molecular weight organic compound can be obtained selectively and at low cost, it is not necessary to use an expensive method such as gel chromatography, and the desired product can be produced on an industrial scale.

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic configuration of an apparatus for producing an organic matter decomposition product of the present invention.

FIG. 2 is a schematic diagram showing hydrolysis and separation / purification of a polymer organic substance according to the present invention.

FIG. 3 is a diagram showing chromatograms separated by HPLC in Example 1. The vertical axis represents the differential refractive index, and the horizontal axis represents the developing solution volume (ml).

FIG. 4 is a diagram showing a result of analyzing a molecular weight distribution in Example 1. The vertical axis represents the molecular weight distribution and the horizontal axis represents the developing solution volume (ml).

FIG. 5 is a diagram showing a chromatogram obtained by separating an ethanol-precipitated product by HPLC in Example 1. The vertical axis represents the differential refractive index, and the horizontal axis represents the developing solution volume (ml).

FIG. 6 is a diagram showing the results of analyzing the molecular weight distribution of the ethanol-precipitated product in Example 1. The vertical axis represents the molecular weight distribution and the horizontal axis represents the developing solution volume (ml).

7 is a diagram showing chromatograms separated by HPLC in Example 2. FIG. The vertical axis represents the differential refractive index, and the horizontal axis represents the developing solution volume (ml).

FIG. 8 is a diagram showing a result of analyzing a molecular weight distribution in Example 2. The vertical axis represents the molecular weight distribution and the horizontal axis represents the developing solution volume (ml).

9 is a diagram showing chromatograms separated by HPLC in Example 3. FIG. The vertical axis represents the differential refractive index, and the horizontal axis represents the developing solution volume (ml).

FIG. 10 is a diagram showing the results of analyzing the molecular weight distribution in Example 3. The vertical axis represents the molecular weight distribution and the horizontal axis represents the developing solution volume (ml).

[Explanation of symbols]

1 Electrodialysis device 2 reaction tank 3 separation tanks 4 electrode tank 5 ultrafiltration membrane 6 dialysis membrane 7 stirrer 8 starter bar 9 electrodes 10 power 11 pumps

Continued front page    F-term (reference) 4B064 AF04 AF11 AF23 AG01 CA21                       CB01 CC21 CD16 CD19 CD20                       CE06 DA01 DA10                 4D006 GA06 GA17 HA01 HA41 KA31                       KA52 KA57 KB30 KD30 MA01                       MA03 MA12 MC23 MC33 MC54                       PB13 PB52 PB53 PB70 PC11                       PC41 PC80                 4D061 DA10 DB18 DC11 DC12 EA09                       EB01 EB04 EB13 EB18 EB19                       EB30 FA09

Claims (11)

[Claims] 1. The method is characterized in that the partial decomposition of a high molecular weight organic compound using a hydrolase and the purification of the obtained partial decomposition product of a high molecular weight organic compound by electrodialysis using an ultrafiltration membrane are performed simultaneously. Method for producing partially decomposed high molecular weight organic matter. 2. A partial decomposition product of a high molecular weight organic compound and a hydrolase capable of partially decomposing the organic compound is electrolyzed at the same time by partially decomposing the high molecular weight organic compound from the reaction tank. The method according to claim 1, wherein the method is conducted through a filtration membrane to a separation tank. 3. The reaction tank and the separation tank are present in an internal space partitioned by a dialysis membrane, and the reaction tank and the separation tank are partitioned by an ultrafiltration membrane. The method described. 4. A method for producing a partially decomposed organic matter, comprising supplying the high molecular weight organic matter to a reaction tank and separating the obtained partially decomposed organic matter into a separation tank simultaneously or sequentially. . 5. The high molecular weight organic substance is a charged high molecular substance including a polysaccharide containing chitin, chitosan, pectin, peptidoglycan, glycosaminoglycan and proteoglycan, a protein and a nucleic acid.
The method according to any one of to 4. 6. The method according to claim 5, wherein the high molecular weight organic substance is a polysaccharide containing chitin, chitosan, pectin, peptidoglycan, glycosaminoglycan and proteoglycan. 7. The method according to claim 6, wherein the high molecular weight organic substance is chitosan. 8. The hydrolase is chitinase, chitosanase, cellulase, lysozyme, pectinase, pectinesterase, glucosidase, glucosaminidase,
5. At least one selected from the group consisting of proteoglycanase, protease and nuclease.
The method described in any one of. 9. The method according to claim 1, wherein the hydrolase is an enzyme having a chitosan degrading activity. 10. A reaction tank equipped with a hydrolyzing enzyme of a high molecular weight organic compound, a separation tank for separating a partially decomposed high molecular weight organic compound,
And an electrodialysis device equipped with an electrode tank. 11. The apparatus according to claim 10, wherein the reaction tank and the separation tank are separated by an ultrafiltration membrane, and the reaction tank and the electrode tank and the separation tank and the electrode tank are separated by a dialysis membrane.