JPH0587238B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing galactosaminooligosaccharides. More specifically, the present invention relates to the production of galactosaminooligopentasaccharides (GOS5), galactosaminoligohexasaccharides (GOS6),
The present invention relates to a method for very efficiently producing galactosaminooligosaccharides having a relatively high degree of polymerization. The present invention also relates to a method for continuously producing the oligosaccharide using an ultrafiltration membrane. (Prior art) Polygalactosamine (Japanese Patent Publication No. 56-12639, Japanese Patent Publication No. 1-41160) is a type of Deuteromycota.
Galactosamine produced by Paecilomycess sp. In recent years, it has become known that polysaccharides or their oligosaccharides produced by microorganisms, plants, or animals have various physiological activities, and interest in polysaccharides and their oligosaccharides has increased. It has also been confirmed that chitin, chitosan, and their oligosaccharides, which are known as polysaccharides similar to polygalactosamine, have antitumor activity. Furthermore, polygalactosamine itself was found to have similar physiological activity (Kouki ISHITANI et al.
al., J. Pharmacobio-Dyn, 11 , 58-65 (1988),
There is also growing interest in the physiological activity of oligosaccharides. Polygalactosamine and galactosaminooligosaccharides may be useful for uses other than physiological activity, and in particular, oligomers are expected to exhibit properties that polymers do not have in terms of application and action.
Attention has been paid. Galactosaminooligosaccharides are obtained by hydrolyzing polygalactosamine. As decomposition methods, methods using acids or alkalis and methods using hydrolytic enzymes are known, but the yield of galactosaminooligosaccharides with a high degree of polymerization is very low. In particular, when polygalactosamine is hydrolyzed with hydrochloric acid, as a result of random decomposition, the amount of oligosaccharides obtained is in the order of mono-galactosamine, di-galactosamine, tri-galactosamine, tetra-galactosamine, and penta-galactosamine, The higher the degree of polymerization, the lower the yield, and the yield of oligosaccharides with a higher degree of polymerization than pentamer was extremely poor. Another drawback was that the work was dangerous because highly concentrated hydrochloric acid was used at high temperatures. On the other hand, the method using a polygalactosamine degrading enzyme has the great advantage of being able to carry out the reaction at room temperature, which is not found in the hydrolysis methods using acids and alkalis. However, most of the galactosamino-oligosaccharides obtained by conventionally known enzymatic decomposition methods have a low degree of polymerization, similar to hydrolysis with acids and alkalis, and galactosamino-oligosaccharides with a relatively high degree of polymerization. The yield of sugar was low. (Problems to be Solved by the Invention) Galactosaminooligosaccharides with a relatively high degree of polymerization are expected to have various physiological activities, but conventional methods using acids or alkalis or using hydrolytic enzymes have relatively low levels of bioactivity. The yield of galactosaminooligosaccharides with a high degree of polymerization is poor, and there is no method for efficiently obtaining the oligosaccharides, which has narrowed the scope of research and application thereof. (Means for Solving the Problems) The present invention was made to solve the above-mentioned problems at once, and was made for the purpose of efficiently producing galactosaminooligosaccharides having a relatively high degree of polymerization. It is something that Conventionally, enzymatic hydrolysis has been used as a method for obtaining galactosaminooligosaccharides, and polygalactosamine-degrading enzymes include, for example, Pseudomonas sp. H881 strain (FERM P-8955), Bacillus sp.
sp.A-4 (FERM P-10473), Bacillus sp.E-
1 (FERM P-10581) and the like are known. The present inventors have carefully studied the substrate specificity, reaction conditions, etc. of each enzyme, and found that
It has been found that by setting the pH to a value lower than the optimum pH of each enzyme, the production of oligosaccharides with a low degree of polymerization can be suppressed and galactosaminooligosaccharides with a relatively high degree of polymerization can be efficiently obtained. For example, Figure 1 shows the CM-Sephadex column chromatography pattern when the reaction pH was set at 6.0 and 4.0.
Comparing the production rate of galactosaminooligosaccharides, the PH
At PH 6.0, most galactosaminooligosaccharides have a low degree of polymerization, whereas at PH4.0, galactosaminooligosaccharides with a relatively high degree of polymerization are efficiently obtained. Based on this knowledge, the present invention aims to improve the pH of the enzyme reaction solution.
By setting the PH to a low PH of 3.0 to 4.5, which is outside the optimum PH of polygalactosamine degrading enzyme, we have completed a method for efficiently obtaining galactosaminooligosaccharides with a relatively high degree of polymerization. In addition, especially from an industrial standpoint, the reaction solution is passed through an ultrafiltration membrane to remove low-molecular products from the reaction system, undecomposed polygalactosamine and enzymes are returned to the reaction system, and polygalactosamine is replenished into the reaction system. By doing so, we were able to complete a continuous reaction system. The polygalactosamine used as a raw material in the method of the present invention may be prepared by a conventional method, and examples of its preparation are as follows. Galactosamine-producing bacterium Paecilomyces I-1
(FERMBP-1180) with 5% sucrose and 1 peptone
%, calcium chloride 0.6%, and pH 7.0 for 5 days at 27°C under aerobic conditions.The culture solution was centrifuged, and the supernatant after removing the bacterial cells was ultrafiltered while heating. Concentrate with a membrane (molecular weight cutoff 100,000), add salt to this concentrate, adjust the pH to 8.5, precipitate it, collect this precipitate, dissolve it in acid, and repeat the operation to adjust the pH to 8.5 again. High purity polygalactosamine can be obtained. Furthermore, the polygalactosamine-degrading enzyme used in the present invention is produced by a microorganism belonging to the genus Pseudomonas or the genus Bacillus. As a culture medium for polygalactosamine-degrading enzyme-producing bacteria, either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances, and other nutrients. For example, when using the enzyme of Pseudomonas sp. An example can be given. Culture temperature is 20~40℃, preferably 30~
38℃ range, culture starting pH 6-8, preferably 7
If shaken or stirred in the vicinity for 35 to 72 hours, polygalactosamine-degrading enzyme can be obtained in the culture solution. Then, the polygalactosamine degrading enzyme is isolated and purified as necessary. For example, the crude enzyme is separated from the culture filtrate by ethanol precipitation, dissolved in an aqueous solvent, subjected to Sephadex G-50 gel filtration, CM-Sephadex C-25 ion exchange chromatography, phenyl-Sepharose CL-
A purified polygalactosamine degrading enzyme is obtained by treatment such as 4B hydrophobic chromatography. When the polygalactosamine degrading enzyme thus obtained is allowed to act on a polygalactosamine solution at a pH lower than the enzyme's optimum pH, particularly at pH 3.0 to 4.5, it is possible to efficiently obtain oligogalactosamine with a relatively high degree of polymerization. I can do it. To give an example of the reaction, first, polygalactosamine is dissolved in a low concentration of acid. Examples of acids include organic acids such as acetic acid and formic acid;
Inorganic acids except sulfuric acid can be widely used. The pH of the polygalactosamine solution thus obtained is lower than 6.0,
For example, after adjusting the temperature to 3.0 to 4.5, the polygalactosamine degrading enzyme prepared above is added and enzymatic decomposition is performed at a temperature of around 30°C. Furthermore, according to the present invention, low-molecular products are taken out of the reaction system by passing the reaction solution through an ultrafiltration membrane (on the other hand, undecomposed polygalactosamine and high-molecular substances partially decomposed by it are removed by enzymes). (The reaction system was then refilled with the raw material polygalactosamine), adsorbed onto an ion exchange resin, and sequentially eluted with a solvent with an appropriate concentration gradient for comparison. Each galactosaminooligosaccharide fraction with a high degree of polymerization can be obtained continuously. This continuous method is particularly excellent as an industrial production method. As the ultrafiltration membrane, commercially available ones such as polyacrylonitrile type and polyether sulfonic acid type can be used as appropriate.Module types include tubular module, hollow module, pleated module, and spiral module. etc. are selected and used as appropriate. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 Preparation of polygalactosamine 600 g of glucose, 60 g of polypeptone, CaC ₂ .
125 g of 2H ₂ O was dissolved in 17 g of tap water, the pH was adjusted to 7.0 with concentrated NaOH solution, and the mixture was transferred to a 30-volume jar fermenter. Pressure and heat sterilization (121° C., 20 minutes) was performed by injecting steam into this medium solution. After cooling, add 150 ml of a medium of the same composition (glucose 3%, polypeptone 0.3%, CaC ₂ 0.5%, PH 7.0) to a 500 ml Erlenmeyer flask at 26°C.
Peecilomyces I-1 cultured with shaking for 4 days
(FERM BP-1180) was aseptically inoculated at a volume ratio of about 10%. After inoculation, the cells were cultured for 5 days at 27°C, aeration rate of 0.5 VVM, and stirring rate of 200 RPM. After completion of the culture, the culture was filtered through a filter cloth to obtain a culture filtrate 17. By passing this culture filtrate through an ultrafiltration membrane with a molecular weight cutoff of 150,000 (UF membrane tubular module F type manufactured by Mitsubishi Rayon Engineering Co., Ltd.) while heating the culture filtrate to 50°C to 60°C, the low molecular fraction was removed. It was concentrated until the amount of removed liquid was about 3. Furthermore, bacterial cell residue and heat-denatured proteins were removed by centrifugation at approximately 14,000×G. After centrifugation, approximately 750 g of table salt (approx.
25% concentration), stir, and after dissolving, add concentrated NaOH
The pH was adjusted to 7.0-8.5. After allowing the salted-out product to stand overnight, it was collected on Saran cloth (copolymer of vinylidene chloride and vinyl chloride, trade name). Furthermore, by spraying a large amount of alkaline water (pH 7.0 or higher) over the salted-out material, excess salt, neutral sugars, and other impurities mixed in the culture were washed away. Next, about 3 times the volume of 0.1M hydrochloric acid solution was added to the salted out product washed with water and dissolved. Concentrate this lysate.
A NaOH solution was added to adjust the pH to 8.5, which is the isoelectric point of polygalactosamine. After leaving it overnight to precipitate ten analytical precipitates, the precipitates were collected on Saran cloth in the same manner as above and washed with a large amount of tap water. This washed product was dissolved once again in 0.1M hydrochloric acid, subjected to isoelectric precipitation, and purified by repeated washing with water. After sterilizing this purified precipitate at 121℃ for 15 minutes,
By freeze-drying, 7 g of purified powder of PF-102 containing polygalactosamine as a main component (about 99% purity as polygalactosamine) was obtained. Depending on the purpose, some of the above purified powder may be added to 0.1M.
It can also be dissolved in hydrochloric acid and fractionated using an ultrafiltration membrane with a molecular weight cutoff of 300,000 (Molecular Sieve Membrane Type XM300 manufactured by Amicon) to separate those with an average molecular weight of 160,000 to 300,000 and those with an average molecular weight of 300,000 or more. can. Example 2 Preparation of polygalactosamine degrading enzyme Pseudomonas sp.H881, FERM P-8955
was inoculated into 100 ml of a seed medium having a composition of 0.5% glucose, 0.05% yeast extract, and 0.05% polypeptone in a 500 ml Erlenmeyer flask, and cultured at 30°C for 20 hours. The obtained seed culture was inoculated into the enzyme production medium obtained in Example 1 containing 0.25% polygalactosamine (PF-102), 0.25% glucose, 0.05% yeast extract, and 0.05% polypeptone in 30 jar fermenters. Incubate the bacteria at 30℃, aeration volume 1VVM, and stirring number.
Cultured at 200 RPM for 48 hours. The resulting culture is centrifuged (14,000 RPM) to remove bacterial cells, and chilled ethanol is added to the resulting culture filtrate to a concentration of 60% to precipitate proteins.The precipitated proteins are centrifuged to remove them from the solution. separated. The obtained protein was adsorbed onto a CM-Sephadex C-25 column (2.5 x 60 cm) equilibrated with 0.1 molar acetate buffer (PH5.0), and 0 to 0.5
Elution was performed using the same buffer with a molar saline gradient. The eluted enzyme activity fraction was collected and concentrated using an ultrafiltration device (molecular weight cut off: 10,000). Next, 2
Sephadex G- was prepared as a solution in 0.1 molar acetate buffer (PH6.0) containing molar salt and equilibrated with the same buffer.
Chromatography was performed on 50 columns (5 x 90 cm). Next, the salt concentration in the active fraction was increased to 4 molar and adsorbed onto a phenyl-Sepharose CL-4B column (2.5 x 20 cm) equilibrated with the same solution.
50 mg of purified polygalactosamine-degrading enzyme (yield 23.1%, specific activity 52 μg GaIN/min/mg protien) eluted with 0.1 molar acetate buffer with a reverse concentration gradient of sodium chloride.
I got it. Example 3 Preparation of galactosaminooligosaccharides 20 g of polygalactosamine was dissolved in 0.1 molar acetic acid of 5, adjusted to pH 4.0 using sodium hydroxide, and the polygalactosamine degrading enzyme solution was dissolved at 0.2 units/ml (1 unit is galactosamine per minute
Add so that the enzyme titer yields 1 μmol).
The reaction was carried out at 30°C. After 30 minutes, heat to inactivate the enzyme and stop the reaction, and then immediately pour the reaction solution into
CM-Sephadex column (210mm ID x 30cm x
3) and applied a concentration gradient of NaC, each galactosaminooligosaccharide was sequentially eluted and fractionated. Each image was desalted, concentrated, and lyophilized to obtain di-galactosamine, tri-galactosamine, tetra-galactosamine, penta-galactosamine, hexa-galactosamine, hepta-galactosamine, octa-galactosamine, and nona-galactosamine. The yield of galactosaminooligosaccharides in the method of the present invention is shown in Table 2 in comparison with the conventional method (PH6.0, Table 1). The amount of polygalactosamine used for decomposition was 20 g in both cases.

【table】

【table】

[Table] As is clear from the results in Tables 1 and 2, according to the method of the present invention, the yield of galactosaminooligosaccharides with a relatively high degree of polymerization is significantly increased compared to the conventional method. I understand that. Example 4 Preparation of galactosaminooligosaccharide (continuous system) 200 g of polygalactosamine was dissolved in 0.1 molar acetic acid of 50, adjusted to pH 4.0 using sodium hydroxide, and the polygalactosamine degrading enzyme solution was dissolved at 0.2 units/ml. (1 unit has an enzyme titer of producing 1 μmol of galactosamine per minute) and the reaction was carried out at 30°C. The reaction solution has a molecular weight cutoff.
Using a 30,000 ultrafiltration membrane (DC-2 type hollow fiber HIP-30 manufactured by Amicon Far East Limited), the galactosaminooligosaccharide produced by continuous decomposition was taken out of the reaction system. A 0.1% solution of polygalactosamine dissolved in 0.1 molar acetic acid, pH 4.0, was added to the reaction system at a rate of 10 ml/min. The galactosaminooligosaccharide taken out of the reaction system was immediately transferred to a CM-Sephadex column (210 mm ID
x 30 cm x 3) and applied a concentration gradient of NaC, each galactosaminooligosaccharide was sequentially eluted and fractionated. After desalting and concentrating each fraction, galactosaminooligosaccharides were obtained by freeze-drying. The amount of each galactosaminooligosaccharide produced 2 hours after the start of the reaction was 12 g of di-galactosamine,
The contents were 36 g of tri-galactosamine, 9.4 g of tetra-galactosamine, 13.8 g of penta-galactosamine, 18.0 g of hexa-galactosamine, 11.6 g of hepta-galactosamine, 6.0 g of octa-galactosamine, and 4.0 g of nona-galactosamine. (Effects of the invention) According to the present invention, by newly adopting a very simple process of controlling the pH of the enzyme reaction solution, various galactosaminooligosaccharides with a relatively high degree of polymerization can be produced in very high yields. can be manufactured at a high rate. Furthermore, if ultrafiltration membrane treatment is performed, continuous production becomes possible, so this method is particularly suitable as an industrial production method.

[Brief explanation of the drawing]

FIG. 1 is a CM-Sephadex column chromatography pattern at pH 6 and 4. In the figure, ~ represents the peaks of mono-~nona-galactosamine, respectively.

Claims (1)

[Claims] 1. When producing galactosaminooligosaccharides by allowing a polygalactosamine-degrading enzyme to act on polygalactosamine, the pH of the reaction solution is adjusted to the optimum pH of the enzyme.
1. A method for producing galactosaminooligosaccharides, which is characterized by obtaining galactosaminooligosaccharides with a specific high degree of polymerization by setting the pH to be lower than that of PH. 2. The method for producing galactosaminooligosaccharide according to claim 1, characterized in that the pH of the reaction solution is set to 3 to 4.5. 3. The galactosamino-oligosaccharide produced using an ultrafiltration membrane is taken out of the reaction system, the reaction solution containing undegraded polygalactosamine and enzyme is returned to the reaction system, and polygalactosamine is replenished into the reaction system. 3. The method for producing a galactosaminooligosaccharide according to claim 1 or 2, characterized in that a galactosaminooligosaccharide having a high degree of polymerization is obtained.