KR20070073724A - Method for preparing derivatives of sugar transfer compounds using sugar transfer enzyme and derivatives prepared therefrom - Google Patents

Abstract

The present invention relates to a method for preparing a derivative of a sugar transfer compound using a sugar transfer enzyme and a derivative prepared therefrom. The final active concentration of a sugar transferase in a solution containing 0.1-1 M sugar is 0.1-3.0 U / was added such that the following ml, catechin it in the reactor of 15-50 ℃, resveratrol (resveratrol), Accu blank (aucubin), para-nitrophenol (p -nitrophenol), p-nitrophenyl - O -α-D- gluconic nose Llano P- nitrophenyl- O- α-D-glucopyranoside, tyrosol, alpha-acarbose, nodakenin, ferulic acid, quercetin and alcohol Reacting by adding at least one sugar receptor selected from the group consisting of a final active concentration of 0.01-2% to transfer glucose or fructosyl groups of sugar to the receptor to generate derivatives of the sugar transition compound; and Party There is provided a method for preparing a derivative of a sugar transfer compound, and a derivative prepared therefrom, comprising the step of isolating and purifying the derivative of the transition compound. The sugar transition derivative produced by the present invention has an effect of increasing solubility in water or preventing decomposition or oxidation by light and heat, and thus may be usefully used in food, cosmetics, and pharmaceutical industries.

Description

Method for preparing derivatives of glycotransfer compounds using sugar transfer enzymes and derivatives prepared therefrom {Method for preparing derivatives of glyco-compounds by using glycosyltransferases and the derivatives

1 is a TLC result of analyzing the distribution of the reaction product when the solution of each catechin and the glucan sucrase were added to the sucrose solution according to the present invention.

S, sucrose; F, fructose;

Lane 1, 0.5M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 2, catechin

Lane 3, 0.5M sucrose + catechin + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 4, epicatechin

Lane 5, 0.5M sucrose + epicatechin + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 6, epigallocatechin

Lane 7, 0.5M sucrose + epigallocatechin + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 8, epicatechin gallate

Lane 9, 0.5M sucrose + epicatechin gallate + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 10, catechin gallate

Lane 11, 0.5M sucrose + catechin gallate + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 12, epigallocatechin gallate;

Lane 13, 0.5M sucrose + epigallocatechin gallate + L. mesenteroides NRRL 1299 glucan sucrase

Figure 2 is a TLC result of analyzing the distribution of the reaction product when reacted by adding the resveratrol solution and glucan sucrase to the sucrose solution according to the present invention.

S, sucrose; G, glucose;

Lane 1, resveratrol

Lane 2, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 3, 0.2M sucrose + resveratrol + L. mesenteroides NRRL 1299 glucan sucrase

Figure 3 is a TLC result confirming the receptor product synthesized after reacting sugar and dextran sucrase using acubin.

Lane 1, Accubin

Lane 2, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 3, 0.2M sucrose + acubin + L. mesenteroides NRRL 1299 glucan sucrase

Figure 4 is a TLC result of analyzing the distribution of the reaction product when reacted by adding ethanol and glucan sukrase to the sucrose solution according to the present invention.

S, sucrose; G, glucose; F, fructose;

Lane 1, 0.5M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 2, 0.5M sucrose + ethanol + L. mesenteroides NRRL 1299 glucan sucrase

* Figure 5 is a TLC result of analyzing the distribution of the reaction product when the reaction by adding paranitrophenol, paranitrophenyl- O- α-D-glucopyranoside and glucan sucrase to the sucrose solution according to the present invention to be.

F, fructose; S, sucrose;

Lane 1, paranitrophenol;

Lane 2, paranitrophenyl- O- α-D-glucopyranoside;

Lane 3, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 4, 0.2M sucrose + paranitrophenols + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 5, 0.2M sucrose + paranitrophenyl- O- α-D-glucopyranoside + L. mesenteroides NRRL 1299 glucan sucrase

6 is a TLC result of analyzing the distribution of the reaction product when the reaction was added to the sucrose solution in the addition of tyrosol and glucan sucrase.

Lane 1, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 2, 0.2M sucrose + tyrosol + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 3, tyrosol

7 is a TLC result of analyzing the distribution of the reaction product when the reaction was added to the sucrose solution and the acarbose and levane sukrases according to the present invention.

Lane 1, acarbose;

Lane 2, 0.2M sucrose + buffer + L. mesenteroides NRRL 512 levansukraase;

Lane 3, 0.2M sucrose + acarbose + L. mesenteroides NRRL 512 levansoukrases

8 is a TLC result of analyzing the distribution of the reaction product when reacted by the addition of fructosyl acarbose and glucan sucrase to the sucrose solution according to the present invention.

Lane 1, fructosyl acarbose;

Lane 2, 0.2M sucrose + fructosyl acarbose + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 3, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase

9 is a TLC result of analyzing the distribution of the reaction product when the reaction with the addition of alcohols and glucan sucrase to the sucrose solution according to the present invention.

Lane 1, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 2, 0.2M sucrose + methanol + L. mesenteroides NRRL 1299 glucansu kraase;

Lane 3, 0.2M sucrose + 1-propanol + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 4, 0.2M sucrose + 1-butanol + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 5, 0.2M sucrose + 2-propanol + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 6, 0.2M sucrose + 3-methyl-1-butanol + L. mesenteroides NRRL 1299 glucan sucrase

10 is a TLC result of analyzing the distribution of the reaction product when the reaction was added to the sucrose solution with nodakine and glucan sukrase.

Lane 1, nodakenin;

Lane 2, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 3, 0.2M sucrose + nodakenin + L. mesenteroides NRRL 1299 glucan sucrase

11 is a TLC result of analyzing the distribution of the reaction product when the reaction was added to the sucrose solution and alcohols and levane sucrase.

Lane 1, 0.2M sucrose + buffer + L. mesenteroides NRRL 512 levansukraase;

Lane 2, 0.2 M sucrose + methanol + L. mesenteroides NRRL 512 levansukraase;

Lane 3, 0.2M sucrose + ethanol + L. mesenteroides NRRL 512 levansukraase;

Lane 4, 0.2M sucrose + 1-propanol + L. mesenteroides NRRL 512 levansukraase;

Lane 5, 0.2M sucrose + 1-butanol + L. mesenteroides NRRL 512 levansukraase;

Lane 6, 0.2M sucrose + 3-methyl-1-butanol + L. mesenteroides NRRL 512 levansukraase

12 is a TLC result of analyzing the distribution of the reaction product when the reaction was added to the ferulic acid (ferulic acid) and levane sucrase in the sucrose solution according to the present invention.

Lane 1, ferulic acid

Lane 2, 0.2M sucrose + buffer + L. mesenteroides NRRL 512 levansukraase;

Lane 3, 0.2M sucrose + ferulic acid + L. mesenteroides NRRL 512 levansukraase;

13 is a TLC result of analyzing the distribution of the reaction product when the reaction was added to quercetin and glucan sucrase in a sucrose solution according to the present invention.

Lane 1, quercetin

Lane 2, 0.2M sucrose + buffer + L. mesenteroides NRRL 1299 glucan sucrase;

Lane 3, 0.2M sucrose + quercetin + L. mesenteroides NRRL 1299 glucan sucrase

The present invention relates to a method for producing a variety of sugar-transfer substances using enzymes and sugars, and more specifically, using green tea catechins, resveratrol, acubin, and ethanol using sugar-transferases. , p-nitrophenol (p -nitrophenol), the para-nitrophenyl-O -α-D- gluconic nose Llano side (p -nitrophenyl- O -α-D- glucopyranoside), tee brush (tyrosol), alpha-acarbose (α- acarbose, methanol, 1-propanol, 1-butanol, 1-butanol, isopropanol, 3-methyl-1-butanol, 3-methyl-1-butanol, and nodakine The present invention relates to a method for preparing derivatives of sugar-transfer compounds such as (nodakenin), ferulic acid, and quercetin, and derivatives prepared therefrom.

The main constituent of the various physiological activities of green tea is catechins contained in green tea leaves. The catechins contained in the green tea leaves are catechin, epicatechin, epigallocatechin, epicatechin gallate, catechin gallate, and epigallocatechin gallate. gallate). There is interest in these green tea catechins increased, which Biol strong antioxidant properties catechin compound (Sakanaka etc. Antibacterial substances in Japanese green tea extract against Streptococcus mutans, a bacterium carcinogenic. Agric.. Chem., 53, 2307, 1989 ), antibacterial activity (Yeo etc., antimicrobial effect of tea extracts from green tea, oolong tea and black tea. Korean J. Soc . Food Nutr . , 24 (2), 293-298, 1995), as well as various activities such as antiulcer, anticancer and anti-allergic activity have been reported.

However, these catechin compounds do not dissolve well in water, and are easily decomposed and oxidized by light or heat to cause browning. Therefore, there are many limitations in using them as food additives, cosmetics, and medicines. On the other hand, biotechnological methods, that is, modified products synthesized by transferring these catechins through the enzymatic reaction, have not only increased light stability, but also have good water-soluble properties. It has been reported to have tasteless properties (Satoshi etc. The syntheses of Catechin-glucosides by Transglycosylation with Leuconostoc mesenteroides sucrose phosphorylase, Biosci . Biotech . Biochem ., 57 (12), 2010-2015, 1993). This report is about synthesizing catechin-glucoside using sucrose phosphorylase, which is different from the method for making a sugar-transfer compound using the glucan sucrase of the present invention.

As a domestic patent for green tea catechins, various application products and manufacturing methods using catechin-containing green tea are registered as patent technologies. These patented technologies simply separate, purify and add green tea from using catechins. In addition, the patented technology for the preparation of sugar-transfer catechin-inducing substance by the enzyme reaction is epigallocatechin, epicatechin gallate to the substrate starch through the enzymatic reaction of Bacillus subtilis bacteria a modified product of the epi go synthesized by the transition from dextrin per catechins - O -α- D- gluconic nose side Llano, catechin -O-α-D- gluconic nose production method (transition manufacture of water per side in the pyrano epi go Method, and the technology for the Patent Publication No. 2003-0032930), but in the present invention, all kinds of green tea catechins using low-cost sugar as a substrate using the kimchi fermentation bacteria Leuconostoc mesenteroides It succeeded in transferring glucose to these substances and also improved its functionality.

Resveratrol compound, which is found in red wine, is one of the phenolic compounds. It is a compound having antioxidant effect and various functionalities. However, this substance is easily oxidized by enzymes such as tyrosinase or polyphenol oxidase, reducing its functional activity. When the glycosyl illustration (glycosylation) that each transition through a chemical method is reported here represents the effect of preventing oxidation from these enzymes (Gilly etc., Glycosylation of resveratrol protects it from enzymic oxidation. Biochem. J. 374, 157-163 (2003). However, as in the present invention, there is no report on improving sugar functionality by transferring sugar to resveratrol using a sugar transfer enzyme, particularly glucan sucrase, which is a biological method instead of a chemical method.

As another example, acubin has the effects of wound healing and antimicrobial effects (such as the effects of Iridoid compounds on wound healing, Journal of the Korean Institute of Oral Medicine, 24 (2), 137-143,1999) and hepatic and animal cancer cells. RNA biosynthesis inhibitory effect (Chang etc., Liver protective activities of aucubin derived from traditional oriental medicine.Commun.Mol.Pathol.Pharmacol. 102, 189-204, 1998) There is no report on the method of synthesizing and the properties of the product.

As such, compounds that can be usefully used in the food, cosmetics, and pharmaceutical industries, such as catechin, resveratrol, and acubin, are easily deteriorated due to low solubility in water, or easy decomposition or oxidation by light and heat. There is a problem that has not been studied, and there is a continuing need for research on a method for improving the functionality or increasing the stability of such a compound.

It is an object of the present invention to use catechins, resveratrol, acubin, paranitrophenol, paranitrophenyl- O- α-D-glucopyranoside, tyrosol, alpha-acarbose, using glycan sucralase or levan sucralase, It provides a method for enzymatically synthesizing derivatives of sugar-transfer compounds such as nodakenin, ferulic acid, quercetin and alcohols.

Another object of the present invention is to provide a derivative of a sugar transfer compound prepared from the above method.

According to the first aspect of the present invention, a sugar transferase is added to a solution containing 0.1-1 M sugar so that the final active concentration of the reactor is 0.1-3.0 U / ml, and then the catechin in the reactor at 15-50 ° C. , resveratrol (resveratrol), Accu blank (aucubin), para-nitrophenol (p -nitrophenol), p-nitrophenyl - O -α-D- gluconic nose Llano side (p -nitrophenyl- O -α-D- glucopyranoside), tyrosine The final active concentration is at least one sugar receptor selected from the group consisting of sol, tyrosol, alpha-acarbose, nodakenin, ferulic acid, quercetin and alcohol. Adding 0.01-2% by weight to react to transfer glucose or fructosyl groups of sugar to the receptor to form derivatives of sugar transition compounds; And there is provided a method for producing a derivative of the sugar-transfer compound comprising the step of separating and purifying the derivative of the sugar-transfer compound.

According to a second aspect of the present invention, there are provided sugar inducing substances of the substrate compounds prepared by the above method.

Hereinafter, the present invention will be described in detail.

The present inventors have found green tea catechins, resveratrol, acubin, ethanol, paranitrophenol, paranitrophenyl- O- α-D-glucopyranoside, tyrosol, alpha-acarbose, methanol, 1-propanol, butanol, isopropanol, A method of synthesizing a sugar-transducing substance of compounds used as a substrate by transferring sugar of glucose to the receptor using 3-methyl-1-butanol, nodakenin, and quercetin as receptors, in particular, has been found. In the case of alpha-acarbose and ferulic acid, levansucrase is used to make a compound (beta-di-fructofuranosyl- (2 → 4 ^IV ) -alpha-acarbose) to which fructose is transferred. Glucose-transferred sugar-transfer compounds were synthesized, and in the case of alcohols such as methanol, ethanol, 1-propanol, butanol, and 3-methyl-1-butanol, Using the La kinase was synthesized a number of compounds of this dangjeon fructose transition. The general functional characteristics of these products are expected to increase their solubility in water while maintaining their original functional characteristics and relatively stable to heat or light, increasing their applicability to food additives, cosmetics and pharmaceutical materials. .

Glucan sucrase is an enzyme that synthesizes glycosylated compounds by transferring them to a compound containing glucose units of sugar when a compound other than sugar is added to the enzyme reaction solution. The compound other than sugar added at this time is called a receptor and this reaction is called a receptor reaction. Depending on the type of enzyme, the binding structure of the transferred glucose to the receptor and the reaction efficiency of the compound as a receptor are different, and the type of receptor product to be biosynthesized also depends on the concentration ratio between the enzyme and the receptor.

In the present invention, the catechin, resveratrol, acubin, paranitrophenol, paranitrophenyl- O- α-D-glucopyranoside, tyrosol, alpha by enzymatic method using glucan sucrase produced from a specific microorganism strain -Sugar synthesis compounds such as acarbose, nodakenin, ferulic acid, quercetin and alcohols were biosynthesized. Particularly, in the case of alpha-acarbose, a compound having a fructose-transferred compound (beta-di-fructofuranosyl- (2 → 4 ^IV ) -alpha-acarbose) was prepared using levansukraase and used as a receptor to produce glucose. The sugar-transfer compound was synthesized, and alcohols such as methanol, ethanol, 1-propanol, butanol, and 3-methyl-1-butanol and ferrolic acid were also variously converted to fructose using levansukraase. Sugar transfer compounds were synthesized.

Glucan sucrase used in the present invention may be obtained from a strain of Leuconostoc mesenteroides . Separation of the glucan sucrase from the microorganism is, for example, inoculated with a strain containing a leukonostock mesenteroid species strain in a medium containing yeast extract, peptone, K ₂ HPO ₄ and sucrose, and then isolates only the cells from the culture medium. This is achieved by purifying glucan sucrase. Isolation and purification of the glucans sucrase can be done by general methods known in the art.

Preferred strains of leuconosstock mesentoid species for obtaining glucan sucrase are Leuconostoc mesenteroides ) NRRL B-1299, NRRL B-742, NRRL B-1355, NRRL B-512, NRRL B-1149, NRRL B-1142 or mutants thereof. Especially Leuconostoc mesenteroides ) NRRL B-1299 is most preferred.

Levansukraase used in the present invention is Leuconostoc mesenteroides) strains. Separation of Levansukraase from the microorganism is, for example, inoculated with a strain containing a leukonostock mesenteroid species strain in a medium containing yeast extract, peptone, K ₂ HPO ₄ and sucrose, and then isolates only the cells from the culture medium and This is achieved by purifying Levansukraase. Isolation and purification of the glucans sucrase can be done by general methods known in the art.

Leuconostoc is a preferred strain of Leuconostok mesenteroid species for obtaining Levansukraase. mesenteroides ) NRRL B-512 (ATCC 10830), NRRL B-1299, NRRL B-742, NRRL B-1355, NRRL B-1149, NRRL B-1142, or mutants thereof. Especially Leuconostoc mesenteroides ) NRRL B-512 (ATCC 10830) is most preferred.

The glucan sucrase was added to a solution containing 0.1-1 M of sugar, which was catechin, resveratrol, acubin, paranitrophenol, paranitrophenyl- O- α-D-glucopyranoside, tyrosol, Reaction with sugar receptors, such as alpha-acarbose, nodakenin, and alcohol, breaks down the sugar and transfers sugar glucose to the sugar receptor, thereby synthesizing derivatives of various sugar transfer compounds. In this case, the glucan sucrase is added so that the reactor final activity concentration is 0.1-3.0 U / ml, and in particular 0.5-1.0 U / ml, and the sugar acceptor has a reactor final activity concentration of 0.03- It is preferable to add so that it may become 3 weight%. In this case, the reaction is generally performed for 1-18 hours at all sugars, preferably at 15-50 ° C, more preferably at 28-37 ° C, until all the sugars are decomposed.

Derivatives of the resulting sugar transfer compounds can be isolated and purified by conventional methods. For example, the gel may be first purified by gel permeation chromatography (Gel Permeation Chromatography), Biogel P-2 column chromatography or Sephadex LH-20 column chromatography, and then purified using HPLC (High performance Liquid Chromatography). Derivatives were isolated. The column used was a μ-Bondapak C ₁₈ column or a Hypersil APS-2 NH ₂ column.

When catechin as the sugar receptor is used in the reaction, at least one catechin derivative is selected from the group consisting of the following formulas 1 to 5 and 6-1 to 6-6.

[Formula 1]

Catechin-alpha-di-isomaltooligosaccharide

(Catechin- O- α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 2]

Epicatechin-alpha-di-isomaltooligosaccharide

(Epicatechin- O- α-D-isomaltooligosaccharides)

(Wherein n is 0-5)

[Formula 3]

Epigallocatechin-alpha-di-isomaltooligosaccharide

Epigallocatechin-O-α-D-isomaltooligosaccharides

(Wherein n is 0-5)

[Formula 4]

Epicatechin Gallate-alpha-di-isomaltooligosaccharide

(Epicatechingallate-O-α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 5]

Catechingallate-alpha-di-isomaltooligosaccharide

(Catechingallate-O-α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 6-1]

Epigallocatechingallate-7-alpha-di-glucopyranoside

(Epigallocatechingallate-7-O-α-D-glucopyranoside)

[Formula 6-2]

Epigallocatechingallate-7,4 "-alpha-di-glucopyranoside

(Epigallocatechingallate-7.4 "-O-α-D-glucopyranoside)

[Formula 6-3]

Epigallocatechingallate-7,4'-alpha-di-glucopyranoside

(Epigallocatechingallate-7,4'-O-α-D-glucopyranoside)

[Formula 6-4]

Epigallocatechingallate-4 "-alpha-di-glucopyranoside

(Epigallocatechingallate-4 "-O-α-D-glucopyranoside)

[Formula 6-5]

Epigallocatechin gallate-4'-alpha-di-glucopyranoside

(Epigallocatechingallate-4'- O- α-D-glucopyranoside)

[Formula 6-6]

Epigallocatechingallate-4 ', 4 "-alpha-di-glucopyranoside

(Epigallocatechingallate-4 ', 4 " -O- α-D-glucopyranoside)

That is, when catechin is used for the reaction, catechin- O- α-D-isomaltooligosaccharide (Formula 1), which is a form in which several glucose is bound to catechin as a sugar transfer compound, is produced, and epicatechin is used for the reaction. Epicatechin- O- α-D-isomaltooligosaccharide (Formula 2) is produced, and in the case of epigallocatechin, epigallocatechin- O- α-D-isomalto oligosaccharide (Formula 3) is produced. In the case of epicatechin gallate is epicatechin gallate - O -α-D- iso-malto-oligosaccharides (IV) is generated, for catechin gallate catechins include gallate - O -α-D- iso-malto-oligosaccharides (Formula 5) is produced, and in the case of epigallocatechin gallate, epigallocatechin gallate- O- α-D-isomalto oligosaccharides of formulas 6-1 to 6-6 are produced. In this case, it is preferable to use glucan sucrase as a sugar transfer enzyme.

When resveratrol is used for the reaction as a sugar receptor, a resveratrol derivative of the following formula (7) is produced as a sugar transition compound. In this case, it is preferable to use glucan sucrase as a sugar transfer enzyme.

[Formula 7]

Resveratrol-alpha-di-isomaltooligosaccharide

(Resveratrol- O- α-D-isomaltooligosaccharides)

(Wherein n is 0-5)

When acubin is used as a sugar acceptor for the reaction, an acubin derivative represented by the following formula (8) is produced. In this case, it is preferable to use glucan sucrase as a sugar transfer enzyme.

[Formula 8]

(Wherein n is 0-5)

When paranitrophenol or paranitrophenyl- O- α-D-glucopyranoside is used for the reaction as the sugar acceptor, a paranitrophenol derivative of the following formula (9) is produced. In this case, it is preferable to use glucan sucrase as a sugar transfer enzyme.

[Formula 9]

Paranitophenyl-alpha-di-isomaltooligosaccharide

( p -Nitrophenyl- O -α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

When tyrosol is used in the reaction as the sugar acceptor, a tyrosol derivative represented by the following Chemical Formula 10 is produced. In this case, it is preferable to use glucan sucrase as a sugar transfer enzyme.

[Formula 10]

Tyrosol-alpha-di-isomaltooligosaccharide

(Tyrosol- O- α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

When acarbose is used as the sugar acceptor in the reaction, an alpha-acarbose derivative of the formula (11) is produced, and when this derivative is used in the reaction, an alpha-acarbose derivative of the formula (12) is produced. At this time, the sugar transferase is preferably Levansukraase.

[Formula 11]

[Formula 12]

When nodakenin is used for the reaction as a sugar receptor, a nodakenin derivative represented by the following formula (13) is produced. In this case, it is preferable that glucan sucrase is used as the sugar transfer enzyme.

[Formula 13]

(Wherein n is 0-5)

In addition, when alcohol is used for the reaction as a sugar acceptor, at least one alcohol derivative selected from the group consisting of the following Chemical Formulas 14 to 24 is produced.

[Formula 14]

Ethyl-alpha-di-isomaltooligosaccharide

(Ethyl- O- α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 15]

Methyl-alpha-di-isomaltooligosaccharide

(Methyl- O -α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 16]

1-propyl-alpha-di-isomaltooligosaccharide

(1-Propyl- O- α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 17]

1-butyl-alpha-di-isomaltooligosaccharide

(1-Butyl- O- α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 18]

Isopropyl-alpha-di-isomaltooligosaccharide

(2-Propyl- O- α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 19]

3-methyl-1-butyl-alpha-di-isomaltooligosaccharide

(3-Methyl-1-butyl- O -α-D-isomaltooligosaccharide)

(Wherein n is 0-5)

[Formula 20]

(Wherein n is 0-5)

[Formula 21]

(Wherein n is 0-5)

[Formula 22]

(Wherein n is 0-5)

[Formula 23]

(Wherein n is 0-5)

[Formula 24]

(Wherein n is 0-5)

In addition, when quercetin is used in the reaction as a sugar acceptor, at least one quercetin derivative selected from the group consisting of the following formulas 25-1 and 25-2 is produced.

[Formula 25-1]

Quercetin-4'-alpha-di-glucopyranoside

(Quercetin-4'- O -α-D-glucopyranoside)

[Formula 25-2]

Quercetin-3'-alpha-di-glucopyranoside

(Quercetin-3'- O -α-D-glucopyranoside)

In addition, when phenolic acid is used in the reaction as a sugar acceptor, a phenolic acid derivative represented by the following Chemical Formula 26 is produced.

[Formula 26]

Penulic-Beta-D-Fructooligosaccharides

(Ferulic-β-D-fructooligosaccharide)

(Wherein n is 0-5)

As alcohol, for example, when ethanol is used in the reaction, ethyl- O- α-D-isomaltooligosaccharide (Formula 14) (using glucan sucrase as the sugar transferase) or ethyl-O-β-D -Fructooligosaccharide (Formula 20) (when using levansukraase as a sugar transfer enzyme) is produced. When methanol is used in the reaction, methyl- O- α-D-isomaltooligosaccharide (Formula 15) (when using glucan sucrase as the sugar transferase) or methyl- O- β-D-fractoligosaccharide (Formula 15) 21) (when using levansukraase as a sugar transfer enzyme) is produced. In addition, when 1-propanol is used for the reaction, 1-propyl- O -α-D-isomaltooligosaccharide (Formula 16) (when using glucan sucrase as the sugar transferase) or 1-propyl- O- β -D-Fructooligosaccharide (Formula 22) (when using levansukraase as a sugar transfer enzyme) is produced. In addition, when 1-butanol is used for the reaction, 1-butyl- O- α-D-isomaltooligosaccharide (Formula 17) (when using glucan sucrase as the sugar transferase) or 1-butyl- O- β -D-Fructooligosaccharide (Formula 23) (when using Levansukraase as a sugar transfer enzyme) is produced. When isopropanol is used for the reaction, isopropyl- O- α-D-isomaltooligosaccharide (Formula 18) (when using glucan sucrase as the sugar transfer enzyme) is produced. When 3-methyl-1-butanol is used for the reaction, 3-methyl-1-butyl- O- α-D-isomaltooligosaccharide (Formula 19) (when glucan sucrase is used as a sugar transferase) Or 3-methyl-1-butyl- O- β-D-fractoligosaccharide (Formula 24) (when using levansukraase as a sugar transfer enzyme). In addition, when quercetin is used in the reaction, quercetin-4'-alpha-di-glucopyranoside (Formula 25-1) and quercetin-3'-alpha-di-glucopyranoside (Formula 25-2, sugar Glucan sucrase as a transfer enzyme). In addition, when ferulic acid is used for the reaction, ferulic-beta-di-fructooligosaccharide (Formula 26) is produced (when using levan sucrase as a sugar transfer enzyme).

In particular, when n is 1-5 in the derivatives of the sugar-transfer compounds thus prepared, derivatives of the formulas 6-1, 6-2, 6-4, and n in the formula 7 is 0-5 When n is 2-5 in formula 8, n is 2-5 in formula 9, n is 1-5 in formula 10, n is 0-5 in formula 11 and 12, in formula 13 n is 2-5, n is 3-5 in Formula 14, n is 3-5 in Formula 16, n is 2-5 in Formula 18, n is 2-5 in Formula 19 When n is 2-5 in formula 20, n is 2-5 in formula 21, n is 2-5 in formula 22, n is 2-5 in formula 23, n in formula 24 When silver is 1-5, the derivatives and the derivatives of formulas 25-1, 25-2 and 26 are all considered to be novel.

Derivatives of such sugar-transfer compounds are believed to be useful for food, cosmetics and medicines because they have an effect of increasing solubility in water or preventing decomposition or oxidation by light and heat. For example, in the case of catechins, conventional catechins do not dissolve well in water and are easily decomposed and oxidized by light or heat to cause browning, whereas the catechin derivatives of the present invention have higher solubility and higher resistance to light or heat. It has stability and is suitable for use in food additives, cosmetics and pharmaceuticals. The derivative of the sugar transfer compound of the present invention is not limited to its content when used in food, cosmetics and pharmaceuticals, but is preferably 0.001-10% by weight based on the total weight.

* Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 (Preparation of Enzyme Solution)

L. mesenteroides NRRL B-1299 was inoculated in LB medium containing 5 g of yeast extract, 5 g of peptone, 5 g of K ₂ HPO ₄ and 20 g of sucrose in 1 L of water, followed by incubation at 28 ° C. The glucan sucrase was purified from this enzyme solution and used. Purification of enzyme was performed after equilibrating DEAE-Sepharose (2.5 × 35cm) with 20mM sodium phosphate buffer (pH6.8), applying crude enzyme solution, and removing unbound protein with 3 times the buffer of column. , The adsorbed enzyme was separated by NaCl (0-1M) solution prepared in the same buffer solution. The active protein fractions were collected, dialyzed with the same buffer solution, and then applied to the column three times to separate and purify the glucan sucrose activity.

L. mesenteroides NRRL B-512 was added to LWG liquid medium (0.3% (w / v) yeast extract, peptone, 0.3% (w / v) K ₂ HPO ₄ , and mineral solution (2% MgSO ₄ · 7H ₂ O, 0.1 % NaCl, 0.1% FeSO ₄ · 7H ₂ O, 0.1% MnSO ₄ · H ₂ O, 0.13% CaCl ₂ · 2H ₂ O, 2% (w / v) glucose) for 12 to 16 hours After culturing until the cells were separated only from the culture medium to prepare a crude enzyme solution was used as the leban sucrase enzyme solution.

Example 2

1) sugar transferase reaction

In this example, 0.5M sugar solution was prepared in order to produce a sugar transition product, and the glucan sucrase enzyme obtained in Example 1 was used.

The catechins were reacted at 28 ° C. after mixing glucan sucrase, 0.5M sucrose and 0.5% catechins in a 1: 2: 2 (v / v / v) reactor. The reaction was carried out at 28 ° C. until all of the sugar in the reactor was consumed (about 3.5 hours).

Resveratrol reaction conditions were mixed with glucan sucrase, 0.2M sucrose and 1% resveratrol (dissolved in 50% acetone) in a 1: 1: 1 (v / v / v) in the reactor and reacted at 28 ℃. The reaction was run until all the sugar in the reactor was consumed (18 hours).

Reaction conditions of Accucuin were glucan sucrase, 0.2M sucrose and 0.5% Accucuin in a 1: 1: 1 (v / v / v) mixture in the reactor, followed by reaction at 28 ° C. The reaction was run until all the sugar in the reactor was consumed (2 hours).

The reaction conditions of ethanol were glucan sucrase, 0.5M sucrose and 80% ethanol solution in a 1: 1: 1 (v / v / v) in the reactor was mixed and reacted at 28 ℃. The reaction was run until all the sugar in the reactor was consumed (2 hours).

The reaction conditions of paranitrophenol and paranitrophenyl- O- α-D-glucopyranoside were determined using glucan sucrase, 0.2M sucrose and 1% paranitrophenol or 1% paranitrophenyl- O- α-D-glu. Copyranoside was mixed in a reactor of 1: 1: 1 (v / v / v), respectively, and then performed at 28 ° C. until all of the sugar was consumed (3 hours).

The final activity of the enzyme is 0.5 U / ml for catechins, and other accucuins, resveratrol, ethanol, paranitrophenol, paranitrophenyl- O- α-D-glucopyranoside, tyrosol, methanol, 1 For propanol, butanol, isopropanol, 3-methyl-1-butanol, and nodakenin, it was 0.83 U / ml. One unit of enzyme is expressed in μmol of fructose free from sugar per ml of enzyme per minute.

In the case of alpha-acarbose, beta-di-fructofuranosyl- (2 → 4 ^IV ) -alpha-acabose was first synthesized using levansukraase under the above conditions, and then glucansucura. The sugar transfer compound was synthesized by the above method using an azease (0.5 U / ml), and in the case of alcohols such as methanol, ethanol, 1-propanol, 1-butanol, and 3-methyl-1-butanol, Using an aze, various glycotransfer compounds to which fructose was transferred in the above-described manner were synthesized. Confirmation of production of sugar transfer product and complete reaction of sugar was performed by taking 1 μl of the reaction solution and dropping it onto a Merck K6F TLC plate, followed by nitromethane: normalpropyl alcohol: water = 2: 5: 1.5 (v / v / v) or It was developed using a developing solvent prepared with acetonitrile: water = 85: 15 (v / v), and the components of the separated carbohydrate were 0.5% (w / v) α-naphthol on a high pressure thin layer chromatograph (HPTLC) plate. It was analyzed using a color reagent containing 5% (v / v) sulfuric acid.

2) Check the composition of the reaction product

The composition of the produced product was also confirmed using HPLC. In order to purify the reaction, a Bio Gel P-2 column was prepared, the sample was administered, eluted with water, and the degree of sugar separation was confirmed by TLC. The isolated sugar was concentrated in vacuo and then confirmed using TLC. The composition and distribution of the synthetic product were confirmed using TLC and MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer), and the results of the receptor reaction are shown in FIGS. 1 to 11.

Leuconostoc Catechin- O- α-D-glucopyranoside (P1 in Fig. 1), catechin- O- α-D-isomaltoside from the reaction of glucan sucrase and catechin obtained from mesenteroides NRRL B-1299 (Fig. 1). A catechin isomaltooligosaccharide receptor reaction product such as (P2 in FIG. 1) was synthesized, and epicatechin- O- α-D-glucopyranoside (P3 in FIG. 1) and epicatechin- O from the reaction of epicatechin (FIG. 1). Epicatechin isomaltooligosaccharide receptor reaction products, such as -α-D-isomaltoside (P4 in FIG. 1), were synthesized, and epigallocatechin- O- α-D- was synthesized from the reaction of epigallocatechin (FIG. 1). Epigallocatechin isomaltooligosaccharide receptor reaction products such as glucopyranoside (P5 in FIG. 1) and epigallocatechin- O- α-D-isomaltoside (P6 in FIG. 1) were synthesized. Also, from the reaction of epicatechin gallate, two epicatechin gallate- O- α-D-glucopyranosides (P7 and P8 in FIG. 1) and one epicatechin gallate- O- α-D-isomaltoside ( Epicatechin gallate oligosaccharide receptor reaction products such as P9) of FIG. 1 were synthesized, and two catechin gallate- O- α-D-glucopyranosides (P10 of FIG. 1) were synthesized from the reaction of catechin gallate (FIG. 1). , P11) and catechin gallate isomaltoligosaccharide receptor reaction products, such as one catechin gallate- O- α-D-isomaltoside (P12 in FIG. 1), were synthesized, and the reaction of epigallocatechin gallate (FIG. 1) from epigallocatechin gallate- O- α-D-glucopyranoside (P13 in Fig. 1) and epicatechin gallate- O- α-D-isomaltoside (P14 in Fig. 1). Gallocatechin gallate isomaltoligosaccharide receptor reaction products were synthesized.

In addition, Leuconostoc mesenteroides NRRL-glucan sucrase two resveratrol from Ajay and reaction (2) of resveratrol obtained from the B-1299 - O -α-D- gluconic nose Llano side (in FIG. 2, P15, P16) and one resveratrol - O -α It was confirmed that resveratrol isomaltooligosaccharide receptor reaction product was synthesized including -D-isomaltoside (P17 in FIG. 2).

In addition, Leuconostoc Acubin isomaltooligosaccharide receptor reaction products, such as acubigenin- O- α-D-isomaltoside (P18 in FIG. 3), from the reaction of glucan sucrase obtained from mesenteroides NRRL B-1299 with acubin (FIG. 3). It was confirmed that this was synthesized.

In addition, Leuconostoc Elution of ethyl- O- α-D-glucopyranoside (P19 in FIG. 4) with ethyl- O- α-D-isomalto from reaction of glucans sucrase obtained from mesenteroides NRRL B-1299 with ethanol (FIG. 4) It was confirmed that the reaction product of ethanol isomaltooligosaccharide receptor such as side (P20 of Figure 4).

In addition, Leuconostoc mesenteroides NRRL B-1299 glucan sucrase Kinase reaction of the para-nitrophenol from para (Fig. 5) obtained from nitrophenyl - O -α-D- gluconic nose Llano side (P21 in Figure 5), p-nitrophenyl - O -α -D-isomaltoside (P22 of FIG. 5), paranitrophenyl- O- α-D-isomaltotrioside (P23 of FIG. 5), etc., Leuconostoc Paranitrophenyl - O- α-D-isomaltoside (FIG. 5) from the reaction of glucan sucrase obtained from mesenteroides NRRL B-1299 with paranitrophenyl- O- α-D-glucopyranoside (FIG. 5) P22) and paranitrophenyl- O- α-D-isomaltotrioside (P23 of FIG. 5) and other paranitorophenol isomaltooligosaccharide receptor reaction products were identified.

In addition, Leuconostoc Receptor reaction products of tyrosol- O- α-D-isomaltooligosaccharides (P24, P25, P26 in FIG. 6) were identified from the reaction of glucan sucrase and tyrosol obtained from mesenteroides NRRL B-1299 (FIG. 6). .

In addition, Leuconostoc Receptor reaction product of beta-di-fractopuranosyl- (2 → ^4IV ) -alpha-acarbose (P27 in FIG. 7) from the reaction of avanose with levansukraase obtained from mesenteroides NRRL B-512 (FIG. 7). Confirmed them. In addition, Leuconostoc Alpha-di-glucopyranosyl- (1 →) from the reaction of glucansucrase obtained from mesenteroides NRRL B-1299 with beta-di-fractopuranosyl- (2 → 4 ^IV ) -alpha-acarbose (FIG. 8). The receptor reaction product of 1) -beta-di-fractopuranosyl- (2 → 4 ^IV ) -alpha- acarbose (P28 in FIG. 8) was identified.

In addition, Leuconostoc Receptor reaction products of methyl- O- α-D-isomalto-oligosaccharides (P29 in FIG. 9) were identified from the reaction of glucans sucrase obtained from mesenteroides NRRL B-1299 with methanol (FIG. 9). In addition, Leuconostoc Receptor reaction products of 1-propyl- O- α-D-isomaltooligosaccharides (P30, P31, P32 in Fig. 9) from the reaction of glucans sucrase obtained from mesenteroides NRRL B-1299 with 1-propane (Fig. 9). It was confirmed. In addition, the receptor of 1-butyl- O- α-D-isomalto-oligosaccharides (P33, P34, P35 in Fig. 9) from the reaction of glucans sucrase obtained from Leuconostoc mesenteroides NRRL B-1299 with 1-butanol (Fig. 9). The reaction product was identified. In addition, Leuconostoc Receptor reaction products of isopropyl- O- α-D-isomaltooligosaccharides (P36 in FIG. 9) were identified from the reaction of glucan sucrase and isopropane obtained from mesenteroides NRRL B-1299 (FIG. 9). Furthermore, isopropyl- O- α-D-isomalto-oligosaccharides (P37, P38, P39 of Fig. 9) were obtained from the reaction of glucan sucrase obtained from Leuconostoc mesenteroides NRRL B-1299 with 3-methyl-1-butanol (Fig. 9). Receptor reaction product of

In addition, Leuconostoc Receptor reaction products of nodakenin- O- α-D-isomaltooligosaccharides (P40 in FIG. 10) were confirmed from the reaction of glucans sucrase obtained from mesenteroides NRRL B-1299 with nodakenin (FIG. 10).

In addition, Leuconostoc Receptor reaction products of methyl-O-β-D-fracttooligosaccharide (P41 in FIG. 11) were identified from the reaction of levansukraase and methanol obtained from mesenteroides NRRL B-512 (FIG. 11). In addition, Leuconostoc Receptor reaction products of ethyl-O-β-D-fracttooligosaccharides (P42, P43 in FIG. 11) were identified from the reaction of levane sucrase and ethanol (FIG. 11) obtained from mesenteroides NRRL B-512. In addition, the receptor reaction products of 1-propyl-O-β-D-fractoligosaccharides (P44 and P45 in Fig. 11) from the reaction of 1-propanol with levansukraase obtained from Leuconostoc mesenteroides NRRL B-512 (Fig. 11). Confirmed them. In addition, Leuconostoc Receptor reaction products of 1-butyl-O-β-D-fracttooligosaccharide (P46, P47 in FIG. 11) were identified from the reaction of 1-butanol with levansukraase obtained from mesenteroides NRRL B-512 (FIG. 11). . In addition, 3-methyl-1-butyl-O-β-D-fractoligosaccharide (Fig. 11) was obtained from the reaction of 3-methyl-1-butanol with levansoucrase obtained from Leuconostoc mesenteroides NRRL B-512 (Fig. 11). Receptor reaction products of P48, P49) were identified.

In addition, Leuconostoc Quercetin-4'-alpha-di-glucopyranoside (P50 in FIG. 12) and quercetin-3'-alpha-di-glulune from the reaction of glucansucase and quercetin obtained from mesenteroides NRRL B-1299 (FIG. 12). The receptor reaction product of copyranoside (P51 in FIG. 12) was identified. In addition, Leuconostoc Receptor reaction products of ferulic-beta-di-fractooligosaccharides (P52, P53 in FIG. 13) were identified from the reaction of lebansucrase and ferulic acid obtained from mesenteroides NRRL B-512 (FIG. 13).

Green tea catechins, resveratrol, acubin, paranitrophenol, paranitrophenyl- O- α-D-glucopyranoside, tyrosol, alpha-acarbose using glucan sucrase and levan sucrase according to the present invention And enzymatic synthesis of sugar transition compounds such as nodakenin, ethanol, methanol, 1-propanol, butanol, isopropanol, 3-methyl-1-butanol, quercetin, and ferulic acid, and inducing these sugar transitions through sugar transition Substances have an effect of increasing solubility in water or preventing decomposition or oxidation by light and heat, and thus may be useful for food, cosmetics, and pharmaceutical industries.

Claims (6)

The alpha-acarbose derivative represented by the following formula (11) or (12). [Formula 11]

[Formula 12]

Nodakenin derivative represented by the following formula (13). [Formula 13]

(Wherein n is 2-5) An alcohol derivative represented by any one of the following Formulas 14, 16, 18 and 19. [Formula 14]

Ethyl-alpha-di-isomaltooligosaccharide (Ethyl- O- α-D-isomaltooligosaccharide) (Wherein n is 3-5) [Formula 16]

1-propyl-alpha-di-isomaltooligosaccharide (1-Propyl- O- α-D-isomaltooligosaccharide) (Wherein n is 3-5) [Formula 18]

Isopropyl-alpha-di-isomaltooligosaccharide (2-Propyl- O- α-D-isomaltooligosaccharide) (Wherein n is 2-5) [Formula 19]

3-methyl-1-butyl-alpha-di-isomaltooligosaccharide (3-Methyl-1-butyl- O -α-D-isomaltooligosaccharide) (Wherein n is 2-5) An alcohol derivative represented by any one of the following Formula 20 to Formula 24. [Formula 20]

(Wherein n is 2-5) [Formula 21]

(Wherein n is 2-5) [Formula 22]

(Wherein n is 2-5) [Formula 23]

(Wherein n is 2-5) [Formula 24]

(Wherein n is 1-5) A quercetin derivative represented by any one of the following Formulas 25-1 and 25-2. [Formula 25-1]

Quercetin-4'-alpha-di-glucopyranoside (Quercetin-4'-O-α-D-glucopyranoside) [Formula 25-2]

Quercetin-3'-alpha-di-glucopyranoside (Quercetin-3'-O-α-D-glucopyranoside) Penulic acid derivative represented by the following formula (26). [Formula 26]

Penulic-Beta-D-Fructooligosaccharides (Ferulic-β-D-fructooligosaccharide)

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