JP2527345B2 - Method for producing oligosaccharide or glycoside containing a-galactosyl group using a-galactosylfructoside as a starting material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention uses α-galactosylfructoside as a substrate for α-galactosidase to efficiently and inexpensively produce α.
-A method for producing a useful oligosaccharide or glycoside containing a galactosyl group.

[Prior Art] In recent years, oligosaccharides having an α-galactose group at the non-reducing end, such as raffinose and starchose, have been recognized as effective as growth factors for Bifidobacteria, which are useful enteric bacteria, and these oligosaccharides are used as sweeteners. It is being considered for use. Sources of raffinose and starchiose include raffinose in soybean oligosaccharides or beets. However, soybean oligosaccharides, which are considered as a raw material for the above raffinose and starkose, are small in quantity, so it is difficult to supply them in large quantities, and the price is also high. Raffinose in beet also has the drawback that the supply period is limited to October to March, and that the price is currently high.

On the other hand, as a method for synthesizing an oligosaccharide or a glycoside, a method utilizing a glycosyltransferase activity of a glycosyltransferase or a hydrolase has been developed. For example, there is production of fructooligosaccharides by β-fructofuranosidase and β-galactooligosaccharides by β-galactosidase. Similarly, a method for producing useful compounds such as oligosaccharides and glycosides having an α-galactosyl group by utilizing the glycosyl transfer activity of α-galactosidase can be considered. In this case, an α-galactosyl group is supplied. Inexpensive raw materials are needed. However, at present, there are few oligosaccharides and polysaccharides containing α-linked galactose in the natural world, and there are only raffinose family oligosaccharides such as raffinose, starchose, verbascose and planteose or their derivatives, and they are supplied in large quantities. Is difficult.

[Problems to be Solved by the Present Invention] Therefore, an object of the present invention is to provide an inexpensive raw material having an α-galactosyl group, and further to utilize the transfer reaction of α-galactosidase by using this substrate. It is intended to provide a method for synthesizing oricosugars and glycosides having an α-galactosyl group.

[Means for Solving Problems] As a result of various studies conducted by the present inventors, they found that α-galactosylfructoside can be used as a donor of a galactosyl group in the transglycosylation action of α-galactosidase, and the present invention has been completed. It was That is, the present invention allows levansucrase to act on a compound containing a galactose and a fructose residue (for example, sucrose, raffinose, starchose, etc.) to synthesize α-galactosylfructoside, and use this α-galactosylfructoside as a substrate. It is characterized in that the α-galactosyl group is transferred by the transferring action of α-galactosidase to synthesize a useful oligosaccharide, glycoside or polysaccharide containing the α-galactosyl group.

The present invention will be described in detail below.

Commercially available galactose can be used as the galactose which can be used, as well as natural oligosaccharides containing α-galactosyl group or β-galactosyl group, glycosides or polysaccharides (β-galactanase, β-galactosidase, α).
-Galactosidase etc.) or galactose prepared by hydrolysis using an acid can also be used. Levansucrase is a glycosyltransferase, but when this enzyme acts on sucrose and galactose, the synthesis reaction of levan in which fructose is bound by β-2.6 bond, the synthesis reaction of α-galactosylfructoside by the transfer to galactose And catalyzes three reactions, a hydrolysis reaction. The ratio of the three reactions is influenced by the concentration ratio of the donor sucrose and the acceptor galactose and the reaction temperature. In order to synthesize α-galactosylfructoside efficiently, it is better to increase the molar ratio of the acceptor and the donor, preferably 1: 1 to 4: 1. Also, the higher the substrate concentration, the better, and 20 to 50% is desirable. As for the reaction temperature, the lower the reaction temperature, the more easily the rearrangement reaction occurs, and the temperature is preferably 10 to 30 ° C. The reaction pH and reaction time may be such that Levansucrase acts to produce α-galactosylfructoside,
A pH of 3.0 to 10 and a reaction time of 1 to 48 hours are applied.

α-galactosidase is originally a hydrolase,
Increasing air quality also catalyzes the transglycosylation action. Therefore, utilizing the glycosyl transfer activity of α-galactosidase, using α-galactosylfructoside as a donor,
Raffinose, Starkiose, Galactobiose, α
-When synthesizing galactosylglycerin, etc., the higher the substrate concentration, the better. Usually, it is preferable to use a concentration of 10 to 50%. The action conditions of α-actosidase vary depending on the enzyme used, but the pH is in the range of 3.0 to 10.0, preferably 5.0 to 8.0, and the reaction temperature is in the range of 20 to 80 ° C, preferably 50 to 70 ° C. The reaction time varies depending on the amount of enzyme used, but is preferably 1 to 48 hours until the substrate α-galactosylfructoside is decomposed by about 90%. However, the present invention is not limited to the above conditions or reaction modes.

Here, levansucrase (EC 2,4,1,10) that can be used to synthesize α-galactosylfructoside is a solution of a compound containing galactose and a fructose residue such as sucrose or raffinose. Any enzyme capable of acting to transfer a fructose residue of a compound containing a fructose residue such as sucrose or raffinose to the C1 position of galactose and producing a non-reducing α-galactosylfructoside may be used. The origin and type are not limited. For example, Bacillus subtilis, Aerobacter levanicum, Acetobacter suboxydans, Gluconobacter oxydans, Streptococcus mutan
s), Streptococcus salivarius (Streptococc
levansucrase produced by many microorganisms such as us salivalius) can be used for the purpose of the present invention.

Further, α-galactosidase may be any enzyme that decomposes oligosaccharides or glycosides containing an α-galactosyl group such as melibiose, raffinose, starchose, or phenyl-α-galactoside, and catalyzes a glycosyl transfer action, The origin and type of enzyme are not limited. For example, Pycnoporus cinnabarinus, Streptococcus bovis,
Diplococcus pneumoni
ae), Mortiellela vinace
a) and other microorganisms and Vicia sativa
Α-galactosidase produced by plants such as As a method for producing levansucrase or α-actosidase from these microorganisms, liquid culture or solid culture is usually used. In the case of liquid culture, the culture supernatant can be used as it is, and in the case of solid culture, the extract can be used as it is as an enzyme preparation. In addition, depending on the case, it is possible to use the bacterial cells as they are as an enzyme agent. In addition, an enzyme purified by a known method can be used if necessary. It is also possible to immobilize these enzymes or bacterial cells that produce the enzymes in a column or immobilize them on a membrane and use them in a continuous or batch reaction repeatedly.

[Effect of the Invention] Currently, when enzymatically synthesizing useful oligosaccharides or glycosides containing an α-galactosyl group, α is present in large amounts at low cost.
The source of the galactosyl group is unknown in nature. The α-galactosylfructoside of the present invention can be synthesized in a large amount by levansucrase from a compound having galactose and a fructosyl group. Levansucrase is a transferase and can be efficiently synthesized at low cost. By using this α-galactosylfructoside as a substrate, useful oligosaccharides or glycosides containing an α-galactosyl group can be enzymatically inexpensively and massively synthesized by the transfer action of α-galactosidase.

 Next, details of the present invention will be described with reference to examples.

[Examples] Enzyme activity Levansucrase activity assay method 0.5% of 20% (w / v) sucrose solution and 100 mM phosphate buffer (pH 7.0) Incubate for 10 minutes at 30 ℃. The reaction solution was heated for 10 minutes to inactivate the enzyme, and the amount of glucose produced was measured by the glucose oxidase method. One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmole of glucose per minute under this condition.

Assay method for α-galactosidase activity 10 mM para-nitrophenyl-α-galactoside 0.2 ml and 1
0.05 ml of α-galactosidase solution is added to 0.2 ml of 00 mM acetate buffer (pH 5.5) and reacted at 40 ° C for 10 minutes. After the reaction, 0.
The reaction was stopped by adding 0.5 ml of 2M Na ₂ CO ₃ and the amount of liberated para-nitrophenol was measured by measuring the absorbance at 400 nm with a spectrophotometer. One unit of enzyme activity was defined as the amount of enzyme that produced 1 μmole of para-nitrophenol per minute under these conditions.

Preparation of enzyme preparation Levansucrase 5% soybean meal extract (20 times volume of 0.2% NaOH solution at 100 ° C
Extract for 1 hour),% sucrose, 0.6% diammonium phosphate, 0.02% potassium iodide, 0.02% magnesium sulfate,
200 ml of medium (pH 7.0) containing 0.3% calcium acetate was dispensed into a 1 l Erlenmeyer flask and sterilized by autoclaving at 120 ° C for 15 minutes, and then Bacillus subtilis var.saccharolyti
The cus was inoculated and statically cultured at 37 ° C for 40 to 44 hours. The cells obtained by centrifugation were suspended in 20 mM phosphate buffer (pH 7.0), treated with a homogenizer for 1 minute, and then centrifuged to obtain a supernatant. This was used as a crude enzyme solution in the following experiments.

α-galactosidase sucrose 2%, peptone 3%, yeast extract 0.5
_{%, (NH 4) 2 SO} 4 1%, KH 2 PO 4 0.5%, MgSO 4 · 7H 2 O0.05%
Pycronoporus cinn after sterilizing medium 51 (pH 5.0) consisting of
The abarinus IFO6139 strain was inoculated and shake-cultured at 30 ° C for 10 days. Ammonium sulfate was added to the supernatant obtained by centrifugation to 0.75 saturation, and the mixture was allowed to stand overnight. Collect the precipitate and pH of 200 ml
5.0 was dissolved in an acetate buffer and dialyzed against the same buffer to obtain a crude α-galactosidase enzyme preparation.

Example 1 Preparation of α-galactosylfructoside 1 ml of levansucrase solution (20 units / ml) was added to 100 ml of pH 7.0 phosphate buffer containing 10 g of sucrose and 20 g of galactose and reacted at 30 ° C. for 48 hours. It was The reaction solution was subjected to activated carbon column chromatography to adsorb oligosaccharides and then eluted with a concentration gradient of 0% to 20% ethyl alcohol. Concentrated and dried eluate to transfer product
I got 9g. The transfer product was identified as α-galactosylfructoside because it had no reducing property and was completely hydrolyzed to galactose and fructose by saccharase.

Preparation of oligosaccharide by α-galactosidase α-galactosidase solution (8 units / ml) prepared as above in 5 ml of pH 5.0 acetate buffer containing α-galactosylfructoside (1 g) and sucrose (1 g) 0.5 ml was added and reacted at 70 ° C. for 24 hours. After the reaction solution was boiled and heated for 10 minutes to inactivate the enzyme, 1 μl of the reaction solution was spotted on a paper, and a composition of n-butanol: pyridine: water = 6: 4: 3 was developed in a solvent system four times and then the phloroglucin method When ketose coloring was performed as described above, a spot was detected at a position corresponding to raffinose. The reaction solution was subjected to activated carbon column chromatography to adsorb oligosaccharides and then eluted with a concentration gradient of ethyl alcohol. The trimeric fraction was collected and concentrated to obtain a freeze-dried standard product (1.1 g).

This preparation (a) produces equimolar amounts of galactose and sucrose when hydrolyzed with α-actosidase, and (b) produces equimolar melibiose and fructose when hydrolyzed with β-fructosidase. ,
(C) Hydrolysis of α-galactosidase and β-fructosidase produces 1 mol of galactose, 1 glucose, and 1 mol of fructose, and (d) mobility in paper chromatography is consistent with raffinose. From the above, it was identified as raffinose.

Example 2 (Preparation of oligosaccharide having α-galactosyl group) Levansucrase was allowed to act on 5 ml of pH 7.0 phosphate buffer containing 1 g of sucrose and 2 g of galactose in the same manner as in Example 1 and then 0.2 ml of the α-galactosidase solution used in 1- was added, and the mixture was further reacted at 70 ° C. for 24 hours. 1 μl of the reaction solution was spotted on paper, and the reaction product was examined by ketose coloration by paper chromatography. As a result, spots considered to be raffinose were confirmed. As in the case of Example 1-, the trimer segment was isolated by activated carbon column chromatography, and the structure was confirmed by the enzymatic method. As a result, it was found to be raffinose.

Example 3 (Preparation of glycoside having α-galactosyl group) 0.3 ml of the α-galactosidase solution used in Example 1 was added to 3 ml of pH 5.0 acetate buffer containing α-galactosylfructoside (500 mg) and 1 g of glycerin. ml was added and reacted at 70 ° C. for 48 hours. The reaction solution was heated for 10 minutes to heat inactivate the enzyme. 0.3 ml of 1N NaOH solution was added to the reaction solution and heated for 60 minutes to decompose reducing sugars, treated with an ion exchange resin, and then the reaction product was examined by paper chromatography.As a result, spots were confirmed at positions corresponding to glucose. Was done. The section corresponding to this glucose was extracted from the paper and examined for properties. (A) This preparation has no reducing power (b) α
-Hydrolysis with galactosidase produces equimolar amounts of glassose and glycerin. Based on the above, this sample was identified as galactosylglycerin.

Claims (1)

(57) [Claims] 1. A method for producing an oligosaccharide or a glycoside having an α-galactosyl group, which comprises utilizing α-galactosylfructoside as a donor and utilizing the transglycosylation action of α-galactosidase.