JPS62293A - Production of l-rhamnose - Google Patents

Abstract

PURPOSE:To obtain L-rhamnose in high yield from the residue solution, by treating an L-rhamnose-containing flavonoid glycoside with an enzyme to liberate L-rhamnose, removing the flavonoid glycoside from a reaction solution and collecting L-rhamnose form the residue solution. CONSTITUTION:A rhamnose-containing flavonoid glycoside such as hesperidin, naringin, etc., is treated with an enzyme to cleave rhamnoside bond part, produced by Aspergillus niger, Penicillium purpurogenum, etc., to liberate rhamnose. Then settled and precipitated flavonoid compound is filtered and removed, a very small amount of the dissolved flavonoid compound is removed from the reaction solution by adsorption chromatography, or by treatment with an organic solvent and high-purity L-rhamnose is collected.

Description

Detailed Description of the Invention: An enzyme that selectively cleaves the rhamnosidic bond is applied to the doglycoside, [only monorhamnose is released from the flavonoid glycoside, and then the flavonoid with the rhamnosidic bond cleaved is extracted from the reaction solution. The present invention relates to a process for producing highly purified rhamnose from the remaining solution by precipitating and removing the compound.

[Mono-rhamnose is a monosaccharide that is difficult to chemically synthesize, but it is a compound that has been developed for various uses in recent years and its demand is increasing.] Rhamnose naturally exists in the cell walls of microorganisms and flavonoid glycosides. In particular, hesveridin is present in large amounts in unshiu mandarin oranges, a specialty of Japan, naringin is present in grapefruit and zapon, and neohesveridin is present in naramican and heshu. ing. These flavonoid glycosides have not been generally utilized due to their lack of utility, but the present inventors have noted that they contain rhamnose, which is of no use.
As an excellent raw material source for L-rhamnose, we have developed a manufacturing method for obtaining highly purified L-rhamnose in high yield from the flavonoid glycoside using a novel process, and have completed the present invention.

The method of the present invention involves treating flavonoid glycosides containing rhamnose, such as hesveridin, naringin, voncillin, linarin, and neohesveridin, with an enzyme that selectively cleaves the rhamnoside binding moiety, such as hesveridinase and naringinase produced by bacterial cells. 1l
After removing most of the flavonoid compounds that precipitate out by filtration, trace amounts of the dissolved flavonoid compounds are removed from the reaction solution by adsorption chromatography or treatment with an organic solvent, resulting in high purity [
- Characterized by rhamnose scales.

According to the method of the present invention, L-rhamnose can be obtained in high yield from a flavonoid glycoside having shi-rhamnose.

In order to selectively release only L-rhamnose from a flavonoid glycoside substrate containing L-rhamnose, an enzyme that cleaves the rhamnoside bond of the substrate is used. The enzyme is
Not only pure enzymes but also enzymes in various stages of purification can be used; for example, a culture solution of microbial cells that produce the same stimulant may be used.

The enzyme used is Aspergillus (Aspergillus).
For example, Aspergillus niger J
The enzyme produced by AN 2531 (commercially available as naringinase), Penicillium n.
For example, Penicillium purpurogenum
num) IFO7756 (commercially available as hesveridinase), an enzyme produced by Hansenula spp.
Enzymes produced by Penicillium spp.
An enzyme belonging to the genus Aspergillus or one having a similar reaction is used.

After the rhamnoside bond is cleaved by the enzyme or bacterial cell 18 nutrient solution, the reaction solution is cooled, for example, on ice, and most of the cleaved flavonoid compounds of shi-rhamnose are precipitated and removed from the reaction solution by filtration. I can do it.

The flavonoid compound of ylm is dissolved in the reaction solution, but in order to remove it, the flavonoid compound is completely extracted and removed using a polar organic solvent that is poorly soluble in water in an acidic state, or the flavonoid compound is dissolved in the reaction solution. A method is used in which the reaction solution is passed through an adsorbent column, such as a styrene oVB adsorbent column. Next, the aqueous solution is passed through an anion exchange resin column to de-heat and concentrate, and then by adding methanol, highly purified [-rhamnose can be isolated as crystals in good yield.

As for reaction conditions, the reaction is carried out at the optimum pH of the enzyme used, and although it varies depending on the type of enzyme, it is usually
3 to 7 are suitable. Removal of flavonoid compounds can be achieved by extraction with polar organic solvents such as iso-butanol, cyclohexanone, iso-amyl alcohol or n-butanol under acidic conditions, preferably below pH 3, or with 5P- 207, HP-20, S
This is done by adsorption and removal using a styrene PVB adsorbent (approximately 3%) such as -8f31, S-862 or XAD-4.

EXAMPLES Next, the present invention will be explained in more detail using Examples, but the present invention is not limited to these.

Example 1 25 g of hesperidin was dissolved in 2 g of 0.058 sodium hydroxide solution, the OH was adjusted to 3.5 with hydrochloric acid, 100 g of commercially available hesperidinase was added thereto, and the mixture was heated at 50°C.
The reaction mixture was shaken for 4 days at the same time. Cleavage of the rhamnosidic bond was approximately 100% of theoretical. After the reaction was completed, the mixture was cooled on ice to precipitate and remove most of hesveretin-7-glucoside. Next, after adjusting the pH to 1 by adding la acid, trace amounts of hesperetin-7-glucoside present in the aqueous solution were extracted and removed twice with cyclohexanone 500-. The aqueous phase is anion exchange resin IR-120 fl
The R salt was concentrated, then methanol was added and cooled to obtain 5.5 g of L-rhamnose monohydrate as white crystals (yield near 5%, HP: 93.5°C, [α]o+9.1). .

Example 2 After dissolving 25 g of Tairinjin in 2g of 0.05N sodium hydroxide solution, the DH was adjusted to 4.5 with hydrochloric acid, and 80q of commercially available Narindinase was added to this,
The reaction was carried out by incubation at ℃ for 4 hours.

Realize the reaction solution! The same procedure as for V141 was followed to obtain 6.0 g of L-rhamnose monohydrate as white crystals (yield near 6%, HP: 93.0°C, [α]D+9.
0).

Example 3 After 25 g of hesveridin was hydrolyzed according to Example 1, most of the precipitated hesveretin-7-glucoside was cooled and removed, and I)H was converted to 3.5 g by adding hydrochloric acid. Adjusted to. On the other hand, a column with an inner diameter of 2 cm and a length of 30 CIR was packed with styrene DVB adsorbent 5p-207.
Ion-exchanged water and salt with a pH of 3! ! After thorough washing with an acidic solution, the above prepared solution was passed through the column to adsorb and remove the remaining hesveretin-1-glucoside. Subsequently, it was desalted and concentrated using an anion exchange resin l1l-120 column, and methanol was added and cooled to obtain L-rhamnose monohydrate 5,0g as white crystals (yield:
67%, HP: 93.0°C, [α]. +9,0).

Example 4 A medium containing 0.5 part of peptone, 0.3 part of yeast extract, and 0.1 part of glucose and adjusted to pH 5.0 was inoculated with SP strain of Hanseniura and incubated at 40°C for 48 hours. 100 d of the culture solution was added to 100 ml of an aqueous solution containing 0.2% hesveridin, and then added to an acetate buffer.
I adjusted IH to 4. This was photographed at 40° C. for 2 hours to react. This reaction selectively cleaved about 50% of L-rhamnose bound to hesveridin. The progress of the reaction was confirmed by thin layer chromatography (developing solution: ethyl acetate/isopropyl alcohol/water system).

Examples 5 to 8 were carried out in the same manner as Examples 1 to 4, and the results are shown in Table 1.

[Leaving space below] Table 1 Patent Applicant Kanebuchi Chemical Industry Co., Ltd. Representative Patent Attorney Sota Asahina and 1 other person 1 (::l:
, (+'l゛・;, :! dried, I, -1:

Claims (1)

[Scope of Claims] 1. After releasing only L-rhamnose from the flavonoid glycoside by acting with an enzyme that selectively cleaves the rhamnoside bond of the flavonoid glycoside containing L-rhamnose, the rhamnoside bond is removed from the reaction solution. 1. A method for producing L-rhamnose, which comprises precipitating and removing the cleaved flavonoid compound and obtaining highly purified L-rhamnose from the remaining solution. 2. A patent claim for obtaining even higher purity L-rhamnose by extracting and removing trace amounts of flavonoid compounds remaining in the aqueous solution containing the high-purity L-rhamnose in an acidic state with a polar organic solvent that is immiscible with water. The manufacturing method described in Scope 1. 3. The method according to claim 1, wherein even higher purity L-rhamnose is obtained by adsorbing and removing trace amounts of flavonoid compounds remaining in the aqueous solution containing high-purity L-rhamnose using an adsorption chromatography in an acidic state. Manufacturing method.