JPH04293493A - Produciton of dextran - Google Patents

Abstract

PURPOSE:To produce high-purity dextran useful in the field of medical treatment, biochemistry, etc., in high yield from an inexpensive raw material. CONSTITUTION:Amylose or a low saccharified reducing thick malt syrup is enzymatically and chemically treated with dextrin dextranase to produce dextran.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention is directed to converting dextran (α-1,6 glucan), which is used in the fields of medicine and biochemistry, into amylose or reduced starch syrup (referring to reduced starch decomposition products). Dextran is produced enzymatically and chemically from (hereinafter the same).

0002

[Prior Art] Dextran has traditionally been produced using sucrose as a raw material for Leuconostoc bacteria.
mesenteroides) or dextransucrase derived therefrom (hereinafter referred to as DSase), and is used in the fields of medicine and biochemistry. DSase produces dextran using only glucose, which is a constituent sugar of sucrose, but at this time, fructose, which is also a constituent sugar, is not used at all for dextran production and is liberated. Because of this mechanism of action, when dextran is produced from sucrose using DSase, the sugar yield is never 50%.
will not exceed. In reality, only a yield of about 37% was obtained.

[0003] Dextrin dextranase (hereinafter referred to as D
It's called Dase. ) about 40 years ago, Gluconobacter oxydans ATCC11, the bacterium that causes stringy beer.
894 strains (Gluconobacter oxyda
ns American Type Culture
re Collection Strain N
o. 11894) and has a degree of polymerization of 3 or more.
It has already been reported that dextran is produced from maltooligosaccharides consisting of 1,4 bonds (Journal
of Biological Chemistry
, Vol. 161, pp. 161-174 (1951)). According to this report, the effect of DDase is on α with a degree of polymerization of 3 or more.
Dextran is produced by transferring glucose residues on the non-reducing terminal side of -1,4-bonded linear malto-oligosaccharides and hydrolysates containing them, such as amylose and starch.

[0004] However, when the degree of polymerization becomes too high, the action of DDase is not observed, and for example, the production of dextran from amylose has not been known until now. In addition, D for reduced starch syrup obtained by hydrogenating starch decomposition products
The action of Dase is also unknown.

[0005]

[Problem to be solved by the invention] The production yield of dextran by DSase is low, so even though dextran is widely acknowledged to be safe, it is expensive and is hardly used in foods. . If dextran can be supplied at low cost, it can be expected to be used as a highly safe polysaccharide for food. To achieve this, it is necessary to produce dextran at higher yields from inexpensive raw materials.

[0006]

[Means for Solving the Problems] Therefore, the present inventor attempted to produce dextran by allowing DDase to act on amylose or reduced starch syrup. DDase
The purification of is described in detail in the patent application filed January 10, 1991 by the present inventor. DDase is Gluconobacter oxydans ATCC11894 strain (Gluco
nobacter oxydans America
an TypeCulture Collection
on strain No. 11894) or Gluconobacter oxydans strain ATCC 11895 (Gl
uconobacter oxydans Ameri
can Type Culture Colle
ction strain No. 11895), and these acetic acid bacteria that contain DDase are not known to be pathogenic.
It is safe to use DDase in food raw material processing. DD
Ase was easily extracted directly from the above-mentioned bacterial cells using an organic solvent, particularly a low polar organic solvent, and further efficiently purified by various chromatography techniques. As a result, a report made about 40 years ago suggested that only a few percent of the amount of enzyme present in bacterial cells could be obtained, but it was possible to obtain a larger amount of the enzyme in a pure manner.

[0007] Amylose used in the present invention is a polymer compound in which D-glucose is linearly polymerized with α-1,4 glucosidic bonds. In particular, those having an average degree of polymerization of about 17 obtained when starch is cleaved with a debranching enzyme are preferably used. Reduced starch syrup refers to starch hydrolyzate, maltooligosaccharide, or a mixture thereof that has been hydrogenated and reduced. In the present invention, the degree of decomposition is preferably about 5 to 10, but there is no particular limit on the degree of hydrogenation, whether the reducing end is partially or completely reduced.

[0008] DDase purified as described above has the following characteristics. 1) Action This enzyme acts on maltooligosaccharides with a degree of polymerization of 3 or higher, including amylose, and produces dextran by transferring glucose residues on the non-reducing terminal side of these substrates. This action progresses until the substance used in the reaction becomes a maltooligosaccharide such as maltose, which has a degree of polymerization of two. This action of DDase is the same even if the glucose located at the reducing end of the maltooligosaccharide has been modified such as hydrogenation. 2) Optimum pH and Stable PH The optimal pH for DDase's action was 4.0 to 4.5, and the pH was stable at 2.5 to 6.0 when left for 30 minutes under various pH conditions. 3) Optimal temperature and stable temperature The optimal operating temperature of DDase was 37 to 45°C, and the temperature was stable at 45°C or lower when left at various temperatures for 30 minutes. Moreover, this enzyme showed activity up to 55°C. 4) Molecular weight The molecular weight of DDase by electrophoresis was approximately 300,000. 5) Titer measurement method Low-saccharide reduced starch syrup was used as the enzyme reaction substrate, and the titer was measured by a conventional method. The titer was defined as 1 U, which is the amount of enzyme required to convert 1 μmol of glucose units into dextran in 1 minute.

[0009] Enzyme-chemical action can be carried out by conventional methods. That is, for example, the enzyme reaction condition is preferably a temperature of 2.
The reaction time is 0 to 45°C and 3 to 72 hours. The DDase used at this time may be purified or crudely purified. That is, for example, crudely purified DDase extracted from bacterial cells using an organic solvent may be used. Furthermore, if a part of the raw material amylose or reduced starch syrup remains unreacted, starch liquefaction type α-amylase or the like may be added to decompose it. If you do this process,
By precipitating only dextran using alcohol, highly pure dextran can be effectively obtained.

0010

[Action] Until now, it has been thought that amylose does not interact with DDase and that dextran is not produced. However, D
The production of dextran by Dase is a result of the transfer of glucose units from the substrate used in the reaction to dextran.
The process proceeds until the molecular weight of the substrate decreases to maltose (degree of polymerization 2). In other words, once it becomes maltose, the transfer reaction to dextran no longer occurs. Therefore, the higher the degree of polymerization of the maltooligosaccharide used in the reaction, the higher the sugar yield in dextran production. for example,
If dextran is produced from maltotetraose (polymerization degree 4), maltose as a reaction residue will be produced in half the amount of the original maltotetraose, so the theoretical sugar yield will be 50%. Amylose (degree of polymerization 1
6 to 17), it is also about 80%. If dextran can be produced from a highly polymerized substance such as amylose, it will be possible to produce dextran at a higher yield than the conventional method of producing dextran using DSase. Furthermore, maltose, which is a reaction residue, causes side reactions such as reacting with other maltose to produce panose, although the reaction rate is extremely low. Therefore, if the reducing end is reduced to maltitol, which is the reaction residue, maltitol will no longer react with maltose or have any reactivity with maltitol, so no by-products will be produced and it will contribute to improving the sugar yield. It will be possible.

Actually, as shown in Table 1, DDase
produced dextran from amylose in fairly high yields. In other words, it has been revealed for the first time that amylose is an excellent substrate for producing dextran. In this case, the value of 70% yield based on sugar is DDas
DD is higher than any maltooligosaccharide used when producing dextran using e.
This is obvious if one considers the action of ase and the fact that amylose has the highest degree of polymerization among the raw materials that can be supplied.

Further, when the yields from various oligosaccharides are compared in Table 1, the number of dextran production stars increases as the degree of polymerization increases from maltotriose to maltohexaose. This is because D is said to act on the glucose unit from the non-reducing end of the maltooligosaccharide and leave maltose
This agrees well with the mode of action of Dase. However, for example, if this enzyme was allowed to act completely using maltotetraose as a substrate, it was thought that dextran would be produced with a yield of 50% and maltose would remain, but as shown in Table 1, the yield was low. Met. This is because this enzyme causes side reactions such as by-producing various oligosaccharides such as panose and glucose.

[0013] Also, the reason why the yield of soluble starch is lower than that of amylose is that this enzyme is a non-reducing terminal α-1,4 in starch.
transfer the glucose residue, but the branched part i.e. α-1
, 6-bonded glucose residues as side chains, and this is thought to be because many unreacted portions remain. Therefore, if the degree of polymerization of the starch decomposition product used as a raw material is simply high, the unreacted portion will increase, and the yield will decrease. A better raw material for the production of dextran is one that is a linear chain with α-1,4 bonds, does not contain other bonds or branches in its structure, and has a high degree of polymerization. In order to efficiently obtain such a raw material, it is most appropriate to selectively cleave only the branches in starch, and what is obtained in this way is amylose with a degree of polymerization of about 17. Therefore, if debranching enzymes such as isoamylase or pullulanase are allowed to act on starch together with DDase, dextran can be produced from starch in high yield (65%).

Table 1 also shows the results of producing dextran by applying DDase to hydrogenated products of starch decomposition products such as low sugar syrup and maltooligosaccharides. Similarly, these hydrogenates have been found to be useful substrates in the production of dextran. Maltotriitol and maltotetriitol are hydrogenated products of maltotriose and maltotetraose, respectively, but it is clear that the hydrogenated products have a considerably higher yield of sugar in the production of dextran. The reason why such a large difference was observed even though the degree of polymerization of low-saccharification starch syrup and low-saccharification reduced starch syrup was the same is due to the substrate specificity of DDase. The present inventor has demonstrated that the mechanism of action of DDase is to produce dextran from maltooligosaccharides (α-1
, 4 to α-1,6 transition), but also catalyze side reactions such as α-1,4 to α-1,4 transition or α-1,6 to α-1,6 transition. It was revealed that. We also confirmed the fact that two molecules of maltose are converted into panose and glucose. These DDa
Due to the various transfer activities possessed by se, when dextran is produced from a substrate such as an ordinary malto-oligosaccharide, a considerable amount of various oligosaccharides are produced as by-products, resulting in a decrease in the yield of sugar. On the other hand, with hydrogenated substances, unlike maltose, maltitol does not interact with DDase, so there are almost no by-product oligosaccharides and the amount thereof is extremely small, making it possible to produce dextran with a high sugar yield. It is considered that

[0015]

[Table 1]

[0016]

[Example] (Example 1) Dissolve 1g of low sugar reduced starch syrup (commercial product) in 20ml of water, and add 0.1U/
Add enzyme to make 30 milliliters of DDase.
The reaction was carried out at ℃ for 70 hours. After placing this in boiling water for 15 minutes to denature and deactivate DDase, 1 ml of commercially available starch liquefaction amylase (500 U/ml) was added and reacted at 37° C. for 2 hours. This was again placed in boiling water for 15 minutes to inactivate amylase, and then 50 ml of ethanol was added and placed at 4°C to precipitate the produced dextran. The amount of dextran obtained by drying this precipitate was 0.64 g, and the yield based on sugar was 64%.

(Example 2) 0.1 g of short-chain amylose was dissolved in 5 ml of water, an enzyme was added thereto at a concentration of 0.2 U/ml DDase, and the mixture was incubated at 30°C for 2 hours.
The reaction was allowed to proceed for 0 hours. Place this in boiling water for 15 minutes
After denaturing and inactivating Dase, commercially available starch liquefying α-amylase (500 U/ml) was added to it.
The reaction was carried out at 7°C for 30 minutes. This was again placed in boiling water for 15 minutes to inactivate amylase, and then 9 ml of ethanol was added and placed at 4°C to precipitate dextran. The amount of dextran obtained by drying this precipitate was 0.07 g, and the yield was 70%.

(Example 3) 1g of corn starch
After suspending it in ml of water and boiling it to gelatinize it, add 5
Five milliliters of an aqueous solution containing 0 U of isoamylase and 20 U of DDase were mixed and reacted overnight at 30°C. This was placed in boiling water for 15 minutes to denature and deactivate both enzymes, and then added to the commercially available starch liquefaction α-amylase (50
0 U/ml) was added thereto, and the mixture was reacted at 37°C for 2 hours. This was again placed in boiling water for 15 minutes to inactivate amylase, and then 20 ml of ethanol was added and placed at 4°C to precipitate dextran. The amount of dextran obtained by drying this precipitate was 0.65 g.
The yield was 65%.

[0019]

[Effects] Amylose can be obtained in large amounts by a simple method such as allowing isoamylase to act on starch. Moreover, since starch itself is cheaper than sucrose, which is the raw material for producing dextran using DSase, it is thought that the method of producing dextran from amylose will be able to supply dextran at a lower cost than conventional methods. Reduced starch syrup can be an excellent raw material for producing dextran compared to starch hydrolysates such as starch syrup that have been known so far. In addition, since low sugar starch syrup is produced inexpensively using starch as a raw material, dextran produced using it can also be supplied at a lower cost than conventional methods.

Claims (3)

[Claims] 1. A method for producing dextran, which comprises allowing dextrin dextranase to act on a reduced amylose or starch degradation product. 2. The method for producing dextran according to claim 1, which uses dextrin dextranase obtained by extracting and purifying dextrin dextranase-producing bacteria with an organic solvent. 3. The method for producing dextran according to claim 2, wherein a strain belonging to Gluconobacter oxidans is used as the dextrin dextranase producing bacterium.