KR20140046689A - Method for preparing rubusoside from stevioside using enzymes - Google Patents

Abstract

The present invention relates to a production method of rubusoside using the enzymatic conversion of stevioside, and a composition for producing the rubusoside denoted by chemical formula 2 which contains: one or more kinds of enzymes selected from α-galactosidase, β-galactosidase, lactase, pectinase, and naringinase; or microorganisms producing the said enzymes. [Reference numerals] (AA) Glucose

Description

Method for preparing rubusoside from stevioside using enzymatic conversion of stevioside {

The present invention relates to a process for the production of rubusoide using enzymatic conversion of stevioside.

Sugar has been the most commonly used sweetener, but due to the widespread adverse effects caused by over-consumption, the demand has been gradually decreasing in advanced countries at the end of the 1970s, and a variety of substitute sweeteners have been developed have.

The substitute sweetener currently used can be divided into a saccharide system and a non-saccharide system depending on the raw material. The saccharide system includes palatinose, fructooligosaccharide which reacts sugar with an enzyme, galactooligosaccharide enzymatically reacted with lactose, Oligosaccharides, and sugar alcohols such as sorbitol and maltitol, which hydrogenate starch sugar. Non-sugar based sweeteners include various synthetic sweeteners such as asphaltenes, saccharin, and acesulfame-K, and natural sweeteners extracted from plants such as tau Martin, stevioside, glycyrrhizin, dihydrochalcone, and monelin.

Most synthetic sweeteners have many advantages such as high stability against external conditions such as pH and heat, and possibility of mass production, but there is always controversy about toxicity and safety to human body when used as a sweetener. Due to these problems, there is a growing interest in exploring and using natural substitute sweeteners present in nature.

Natural sweeteners are natural sweetenable compounds contained in the roots, leaves and fruits of plants. There is little concern about the toxicity and safety of artificial synthetic sweeteners, and the sweetness is generally higher than that of sugar. And is showing an increasing tendency.

On the other hand, stevioside (Stevioside, ST) and the base side fusu (Rubusoside, R) Stevia is cultivated in South America It is a sweetener extracted from leaves of Rubus suavissimus S. Lee cultivated in rebaudiana and southern China. It is a glycoside of steviol. It is known that stevioside (13-O-β-sophorosyl-19-O-β-D-glucosyl-steviol) and lucusoid are 143 times and 114 times more sweet, respectively, than sugar at a concentration of 0.025%.

Rubus suavissimusLee is a rare natural sweetener that is included as a component of Chinese sweet potato. This tea plant grows in South China area. , It is influenced by climate. Lubuso side is also Stevia Small amounts can also be obtained from leaves of rebaudiana .

Stevia varieties grown in general are stevioside as a main ingredient among the above sweeteners, and are attracting attention as natural sweeteners that can prevent diabetes and obesity. In addition, it has excellent solubility and stability, and is economical due to the generation of complexion. Thus, marketability and potential are very high in line with recent food trends. Recently, it has been reported that it is used not only as a high-sweetener but also as an antidiabetic agent, antihypertensive agent, antitumor agent (antimutagenic), antiviral agent, intestinal flora improvement and improvement of kidney function. have.

In addition to being used as a sweetening ingredient in health beverages, most of the stevioside degradation products are naturally present and have pharmaceutical properties, which have anti-tumor and anti-inflammatory properties.

It has been reported that rubusoide has antiangiogenic and anti-allergic properties. It is an excellent natural solubilizer and it has been reported that various pharmaceutically important chemicals with very low water solubility such as paclitaxel, curcumin, capsaicin , cyclosporine, nystatin, and erythromycin have been reported to increase water solubility.

Such a rubusoda side has not been developed yet so far as the method of mass production of rubusoda side economically

As a fragmentary example, there are several studies on synthetic rubusoside, which is reported to be obtained as an intermediate in the chemical-enzymatic synthesis of rebaudioside A, ( Chryseobacterium ) culture broth was synthesized with stevioside but did not exclude isomers (ZY Jiang, YR Chen, H. Liu, Acta Microbiol. Sin. 51 (2011) 4349).

In addition, optimization studies have been reported for the production of enzymes that produce rubusoidal from stevioside. The enzyme used was beta-glucosidase, and Aspergillus aculeatus (Viscozyme L) was used to produce 193 mM of rubusoidal using 280 mM of stevioside, and the conversion was 68.9% ( Jin-A Ko, Young-Min Kim, etc. Mass Production of Rubusoside Using a Novel Stevioside-Specific β-Glucosidase from Aspergillus aculeatus J. Agric. Food Chem. 2012, 60, 62106216).

In addition, a study on the synthesis of rubusoidal from stevioside using beta-galactosidase derived from Aspergillus sp. Showed that 8% stevioside had 5,250 U of beta-galactose per gram The reaction was carried out at 60 ° C. for 72 hours using an enzyme cidase and the conversion of stevioside of 98.3% was confirmed [H. Wan, G. Tao, D. Kim, Y. Xia, Enzymatic preparation of a natural sweetener rubusoside from specific hydrolysis of stevioside with beta-galactosidase from Aspergillus sp. Journal of Molecular Catalysis B: Enzymatic 82 (2012) 12 17].

Accordingly, the inventors of the present invention discovered a rubusoidal-producing enzyme which is superior in the ability to convert rubusoidal from stevioside and has high thermal stability and excellent activity while studying a method for economically mass-producing rubusoidal, .

 Z.Y. Jiang, Y.R. Chen, H. Liu, Acta Microbiol. Sin. 51, 4349, 2011  Jin-A Ko., Food Chem. 60, 62106216, 2012,  H. Wan et al., Journal of Molecular Catalysis B: Enzymatic 82, 12, 17, 2012

An object of the present invention is to provide a lucoside-forming enzyme which is excellent in the ability to convert rubusoidal from stevioside and has high thermal stability and excellent activity.

It is still another object of the present invention to provide a composition for producing rubusoidal comprising the rubusoidal-forming enzyme.

It is still another object of the present invention to provide a method for producing rubusoidal using the rubusoidal-forming enzyme.

In order to achieve the above object, the present invention provides a lucoside-forming enzyme which is excellent in the ability to convert from stevioside to rubusoside, and has high heat stability and excellent activity.

In addition, the present invention provides a composition for producing rubusoidal comprising the rubusoidal-forming enzyme.

The present invention also provides a method for producing rubusoidal using the rubusoidal-forming enzyme.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for producing rubusoside having the following formula (2), which comprises an enzyme or a microorganism producing the enzyme.

The enzyme according to an embodiment of the present invention may be prepared by removing the terminal glucose of 2-O-? -D-Glucopyranosyl-α-D-glucose bound to 13C of Stevioside of the following formula It is possible to synthesize rubusoidal.

According to one embodiment of the present invention, the conversion of stevioside to rubusoidal was confirmed in the following 34 known carbohydrate degrading enzymes.

34 known carbohydrate degradation related enzymes [optimal pH range; Optimum temperature range ℃; Product name] is Trichoderma reesei cellulase (Novozyme) [pH 4.0-6.0; 45-55 DEG C; Celluclast 1.5 L], Aspergillus aculeatus pectinlyase [pH 4.0-5.0; 40-60 DEG C; Pectinex Ultra Clear], Aspergillus aculeatus polygalacturonase [pH 4.0-5.0; 40-60 DEG C; Pectinex Ultra SP-L], Aspergillus aculeatus polygalacturonase [pH 4.0-5.0; 40-60 DEG C; Pectinex 5XL], Aspergillus niger glucoamylase [pH 4.0-6.0; 45-55 DEG C; SAN-Extra L], Bacillus licheniformis Cyclomaltodextrin glucanotransferase [pH 5.0-5.5; 80-90 캜; Toruzyme 3.0 L (CGTase)], Aspergillus niger Pectinase [pH 3.0-4.5; 45-60 DEG C; Sumizyme PX], Aspergillus niger arabinase [pH 3.5-5.0; 50-60 DEG C; Sumizyme ARS], Trichoderma reesei Pectinase from Vision Biochem [pH 3.5-5.1; 55-66 DEG C; Crystalzyme PML MX], Aspergillus niger Pectinase, cellulase, mixed enzyme of hemicellulase and β-glucosidase [pH 2.5-5.0; 50-65 DEG C; Cytolase PCL5], Aspergillus niger pectinase [pH 3.2-4.7; 30-60 DEG C; SEBmash U13], Trichoderma reesei β-glucanase [pH 4.9-5.3; 50 C; Multifect B], Aspergillus niger Pectinase and hemicellulase mixed enzyme [pH 3.0-5.5; 35-45 캜; Rapidase TF], a mixed enzyme of Aspergillus niger cellulase with hemicellulase [pH 4.0-5.0; 45-50 DEG C; Cellulosin PE60], a mixed enzyme of Trichoderma reesei cellulase and β-glucosidase from Vision Biochem [pH 3.9-4.3; 55 C; Multifect CX15L], Trichoderma reesei cellulase [pH 5.0; 55 C; Multifect GC Extra], Aspergillus niger pectinase [pH 4.0-5.0; 45-50 DEG C; Rapidase Press L], Trichoderma reesei cellulase [pH 4.5-5.0; 60-65 DEG C; Rohament CL], Trichoderma reesei Hemicellulase (xylanase) [pH 4.5-5.1; 60-66 DEG C; Rohament GE], Trichoderma reesei Hemicellulase (xylanase) [pH 4.0-6.0; 45-60 DEG C; Sumizyme X], Trichoderma reesei Hemicellulase (xylanase) [pH 4.0-5.5; 50-65 DEG C; Optimash XL], Trichoderma reesei β-glucanase [pH 4.0-6.0; 40-70 DEG C; Filtrase Premium L], Aspergillus niger pectinase [pH 3.0-5.0; 50-60 DEG C; Sumizyme SPC], Trichoderma spp cellulase [pH 4.0-5.5; 50-60 DEG C; Lumicell YX1], Trichoderma spp Xylanase [pH 4.0-5.5; 50-60 DEG C; Lumixyl YX2], Trichoderma spp cellulase [pH 4.0-5.5; 50-60 DEG C; Lumicellulase Super], Aspergillus niger [beta] -galactosidase [pH 4.5-7.0; 45-55 DEG C; Sumilact L], Aspergillus niger a -galactosidase [pH 3.5-6.5; 40-50 DEG C; Validase AGS], Rhizopus oryzae cellulase [pH 4.0-6.0; 40-50 DEG C; Sumizyme MC], Penicillium spp naringinase [pH 4.0-6.0; 40-50 DEG C; Cellulase KN] and Kluyveromyces lactis [pH 7.0; 28 ° C] , Aspergillus oryzae [pH 4.3; 50 < 0 > C] , Bacillus circulans [pH 6.0; 60 ° C] derived β-galactosidase and Thermus Species [pH 7.0; 70 < 0 > C] derived lactase was used.

As a result, it has been found that pectinase, α-galactosidase, naringinase, β-galactosidase and heat-resistant lactase (hereinafter referred to as " Lactase) could produce rubusoidal from stevioside. More specifically Aspergillus Nigga (Aspergillus niger) let go of Lactobacillus α- derived claim (α-galactosidase), β- galactosidase (β-galactosidase), Pectinase claim (pectinase); Penny Room Solarium in (Penicillium naringinase derived from the spp. And Thermus It was confirmed that Lactase derived from Stevia spp. could switch from stevioside to rubusoidal.

Particularly, as a lucoside-forming enzyme having high heat stability and excellent activity, in the case of lactase, Thermus spp.) was very excellent in the ability to convert stevioside to rubusoidal, and the activity was maintained even at a high reaction temperature.

The composition for producing rubusoidal according to an embodiment of the present invention is an enzyme which is selected from the group consisting of α-galactosidase, β-galactosidase, Lactase, Pectinase, and naringinase can be used.

According to one embodiment of the present invention, pectinase (EC 3.2.1.15) is an enzyme that hydrolyzes a pectin substrate widely distributed in fruits and vegetables. It is composed of (1) α-1 of galacturonic acid, Polygalacturonase hydrolyzing 4-glycosidic linkages, (2) pectin lyase digesting α-1,4 linkages of methyl esterified galacturonic acid, ( 3) It has mixed activity of pectin esterase which hydrolyzes methy ester bond.

Naringinase (EC 3.2.1.40) according to one embodiment of the present invention has? -Galactosidase activity and rhamnosidase activity,? -Galactosidase (EC 3.2.1.22) Galactosidase (EC 3.2.1.23) or lactase is an enzyme which liberates galactose, which is bound to the end of the glycoprotein by an alpha -1,4-linkage, such as? -Galactoside (? galactoside linkage is hydrolyzed to liberate galactose.

A composition for producing lucusoside according to an embodiment of the present invention is an enzyme producing microorganism, and Aspergillus niger), Penny chamber in Solarium (Penicillium spp.), and Thermus spp.) can be used.

The enzyme according to one embodiment of the present invention is preferably produced based on recombinant DNA technology. (A) inserting a DNA sequence encoding said enzyme into a vector comprising at least one expression control sequence operably linked to said DNA sequence and regulating its expression, (b) recombinant expression (C) culturing the resulting transformant under conditions and conditions suitable for expression of the DNA sequence, and (d) isolating the enzyme from the culture. .

By "vector" is meant a DNA construct containing a DNA sequence operatively linked to a suitable regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert.

The " regulatory sequence " means nucleic acid sequences that are essential or advantageous for the expression of the enzyme. Such regulatory sequences include promoters, upstream activation sequences or enhancers, polyadenylation sequences, and transcription termination factors.

Examples of the host cell include well known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast, insect cells such as Spodoptera frugiperda, animal cells such as CHO and mouse cells, Tissue-cultured human cells and plant cells, and the like.

The transfection or transfection described above is described in Davis et al. Basic Methods in Molecular Biology, < RTI ID = 0.0 > 1986. < / RTI > Preferred examples include electroporation, transduction, calcium phosphate transfection, cationic lipid-mediated transfection, and the like.

Host cells can be cultured under conditions that allow for appropriate medium and expression of the enzyme protein and / or separation. The cultivation proceeds in a suitable nutrient medium containing carbon, nitrogen source and inorganic salt using known techniques. Suitable media are commercially available and may be prepared according to, for example, the components listed in the American Type Culture Collection catalog and the like, and their compositional ratios.

The enzyme from the culture can be isolated by methods known in the art. For example, the enzyme may be separated from the culture by methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. Further, the enzyme can be purified by a variety of methods known in the art including chromatography or electrophoresis.

Lactase according to one embodiment of the present invention may be used in the genus Thermus spp.). < / RTI >

According to one embodiment of the present invention, Thermus , which has the highest stevioside (substrate) conversion ability to rubusoidal, The conversion characteristics of the lactase - derived substrate were determined according to the substrate concentration.

As a result, Thermus spp.) showed that the production of rubusoidal was constantly increased for the same enzyme activity even at a high concentration of stevioside of 40% under the condition of confirming the reaction until 9 hours. In other words, 8.68 times more lubusoidal was used in the same concentration than in the case of Aspergillus sp., Which was previously reported by Wan et al. , And the production capacity was 38 times or more when 40% substrate was used.

The lactase (EMBL accession HB973086.1) according to an embodiment of the present invention comprises the amino acid sequence of SEQ ID NO: 1.

The lactase according to an embodiment of the present invention may be a lactase gene encoding the amino acid sequence of SEQ ID NO: 1 or a lactase gene comprising the nucleotide sequence of SEQ ID NO: 4.

The lactase gene according to an embodiment of the present invention comprises the nucleotide sequence as set forth in SEQ ID NO: 4. In view of the degeneracy of the genetic code, the nucleotide sequence of SEQ ID NO: 2 and the homology of 80% , Preferably 85% homology, more preferably 90% homology, and most preferably 95% homology are included in the lactase gene of the present invention.

In addition, the lactase (EMBL accession HB972972.1) according to one embodiment of the present invention is composed of the amino acid of SEQ ID NO: 2.

The lactase according to an embodiment of the present invention may be a lactase gene encoding the amino acid sequence of SEQ ID NO: 2 or a lactase gene comprising the nucleotide sequence of SEQ ID NO: 5.

The lactase gene according to an embodiment of the present invention comprises the nucleotide sequence as set forth in SEQ ID NO: 5. In view of the degeneracy of the genetic code, the nucleotide sequence of SEQ ID NO: 2 and the homology of 80% , Preferably 85% homology, more preferably 90% homology, and most preferably 95% homology are included in the lactase gene of the present invention.

In addition, the lactase (EMBLl accession HB972880.1) according to an embodiment of the present invention comprises the amino acid sequence of SEQ ID NO: 3.

The lactase according to one embodiment of the present invention may be a lactase gene encoding the amino acid sequence of SEQ ID NO: 3 or a lactase gene comprising the nucleotide sequence of SEQ ID NO:

The lactase gene according to an embodiment of the present invention comprises the nucleotide sequence as set forth in SEQ ID NO: 6. In view of the degeneracy of the genetic code, the nucleotide sequence of SEQ ID NO: 2 and the homology of 80% , Preferably 85% homology, more preferably 90% homology, and most preferably 95% homology are included in the lactase gene of the present invention.

The present invention

In the preparation of Rubusoside from Stevioside,

An enzyme or a microorganism that produces the enzyme is used as a microorganism to produce Rubusoside.

The enzyme according to an embodiment of the present invention may be selected from the group consisting of? -Galactosidase,? -Galactosidase, lactase, pectinase, (Naringinase) may be used.

The microorganism producing the enzyme according to an embodiment of the present invention is Aspergillus niger), Penny chamber in Solarium (Penicillium spp.), and Thermus spp.) can be used.

Lactase according to one embodiment of the present invention may be used in the genus Thermus spp.). < / RTI >

EMBL accession HB973086.1 according to one embodiment of the present invention is a lactase gene encoding the amino acid sequence of SEQ ID NO: 1 or a lactase gene comprising the nucleotide sequence of SEQ ID NO: 4 have.

The lactase gene according to an embodiment of the present invention comprises the nucleotide sequence as set forth in SEQ ID NO: 4. In view of the degeneracy of the genetic code, the nucleotide sequence of SEQ ID NO: 2 and the homology of 80% , Preferably 85% homology, more preferably 90% homology, and most preferably 95% homology are included in the lactase gene of the present invention.

In addition, the lactase (EMBL accession HB972972.1) according to an embodiment of the present invention may further comprise a lactase gene encoding the amino acid sequence of SEQ ID NO: 2 or a lactase gene comprising the nucleotide sequence of SEQ ID NO: Lt; / RTI >

The lactase gene according to an embodiment of the present invention comprises the nucleotide sequence as set forth in SEQ ID NO: 5. In view of the degeneracy of the genetic code, the nucleotide sequence of SEQ ID NO: 2 and the homology of 80% , Preferably 85% homology, more preferably 90% homology, and most preferably 95% homology are included in the lactase gene of the present invention.

The EMBLl accession HB972880.1 according to an embodiment of the present invention may further comprise a lactase gene encoding the amino acid sequence of SEQ ID NO: 3 or a lactase gene comprising the nucleotide sequence of SEQ ID NO: Lt; / RTI >

The lactase gene according to an embodiment of the present invention comprises the nucleotide sequence as set forth in SEQ ID NO: 6. In view of the degeneracy of the genetic code, the nucleotide sequence of SEQ ID NO: 2 and the homology of 80% , Preferably 85% homology, more preferably 90% homology, and most preferably 95% homology are included in the lactase gene of the present invention.

An enzyme according to an embodiment of the present invention may be immobilized on an immobilized bead.

The immobilized bead according to one embodiment of the present invention may use sodium alginate, triethanolamine alginate, or calcium alginate.

According to one embodiment of the present invention, the immobilized enzyme beads can be prepared by a conventional method, and more specifically, 0.1 to 10 g of 0.5 to 10% (w / v) sodium alginate beads of 50 to 500 U, Thermus spp.), and the like, but the present invention is not limited thereto.

According to one embodiment of the present invention, the free enzyme has an inconvenience to newly add the enzyme to the reactor every time it reacts, while the immobilized enzyme has an advantage that the enzyme can be recovered and used. In addition, in general, the immobilized enzyme is superior to the free enzyme in stability of the enzyme activity. In the present invention, an immobilized enzyme bead was prepared using an enzyme alginate bead, and the amount of lucusoid was determined using the enzyme bead.

As a result, it was confirmed that the longer the time when the free enzyme was used, the lower the formation of the produced rubusoid in the rubusoidal transfer reaction with the stevioside as the substrate. On the other hand, when the immobilized enzyme was used, And it was confirmed that it can be produced continuously more easily at a high yield.

The production method according to one embodiment of the present invention is characterized in that the stevioside and the enzyme are reacted at a temperature of 50 to 80 캜, preferably 60 to 70 캜, for 5 to 15 days, preferably 7 to 12 days (2-O-beta-D-Glucopyranosyl-alpha-D-glucose) bound to 13C of the stevioside was selectively decomposed to remove one terminal glucose to form rubusoid Can be synthesized.

The production method according to an embodiment of the present invention can produce 8 times as much lubusoidal as an enzyme by using 500-1000 U of enzyme per unit time than the conventional method of producing lubusoidal from the same substrate of stevioside In addition, when the amount of stevioside is more than 40%, it is possible to produce more than 38 times more lubusoidal than the conventional production amount.

The enzyme according to the present invention, the composition for producing rubusoside containing the enzyme, and the method for producing rubusoide using the enzyme can be used for anti-diabetic, antihypertensive, anti-tumor (anti-mutation) It has the advantage of mass production of rubusoide, which is an excellent natural solubilizer, in economical manner.

Further, the enzyme according to the present invention, the composition for producing rubusoside containing the enzyme, and the rubusoide produced from the method for producing rubusoide using the enzyme are further useful for the functional food and pharmaceutical industry There are advantages that can be utilized.

FIG. 1 is a graph showing the activity persistence and stability of an immobilized enzyme and a free enzyme according to an embodiment of the present invention,
2 shows the amino acid sequence (SEQ ID NO: 1) of EMBL accession HB973086.1 derived from Thermus spp. According to an embodiment of the present invention,
Figure 3 shows the amino acid sequence (SEQ ID NO: 2) of EMBL accession HB972972.1 derived from Thermus spp. According to one embodiment of the present invention,
4 shows the amino acid sequence (SEQ ID NO: 3) of EMBLl accession HB972880.1 derived from Thermus spp. According to an embodiment of the present invention,
5 shows the nucleotide sequence (SEQ ID NO: 4) of EMBL accession HB973086.1 derived from the genus Thermus spp. According to an embodiment of the present invention,
FIG. 6 shows the nucleotide sequence (SEQ ID NO: 5) of EMBL accession HB972972.1 derived from Thermus spp. According to an embodiment of the present invention,
7 shows a nucleotide sequence (SEQ ID NO: 6) of EMBLl accession HB972880.1 derived from Thermus spp. According to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail. Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Example 1

In order to identify enzymes that specifically produce rubusoid from stevioside (substrate) using carbohydrate-related enzymes produced from various microorganisms shown in the following Table 1, 0.5% stevioside was used as a substrate, The enzyme was added at 20% of the total reaction solution and 0.2 g of the powdery enzyme in 1 mL of the total reaction solution. The reaction was carried out at the optimum reaction temperature of each enzyme while maintaining the optimum pH as a buffer solution. The results were confirmed by TLC. The developing solvent was confirmed by developing two times using acetonitrile: water = 85: 15 (v / v).

The stevioside is represented by the following general formula (1).

As a result, as shown in Table 1, it was confirmed that the enzyme derived from a specific strain, that is, Aspergillus niger) let go of Lactobacillus α- derived claim (α-galactosidase), β- galactosidase (β-galactosidase), Pectinase claim (pectinase) and Penny chamber in Solarium (Penicillium (naringinase) from the spp., Thermus it was confirmed that luculose was specifically produced only in Lactase derived from Lactobacillus spp., and in particular, Lactase derived from Thermus spp. It was confirmed that it produces 3 to 17 times more rubusoidal.

The lactase derived from the genus Thermus spp. Was obtained from EMBL (European Molecular Biology Laboratory) under the accession number HB973086.1 (amino acid of SEQ ID NO: 1, nucleotide sequence of SEQ ID NO: 4), accession number HB972972.1 2, and the accession number HB972880.1 (amino acid of SEQ ID NO: 3, the nucleotide sequence of SEQ ID NO: 6).

[Example 2]

From the results of Example 1, it was confirmed that the highest thermostability of the thermosus ( Thermus) (EMBL accession number HB973086.1) was determined by the substrate concentration.

In order to investigate the conversion characteristics depending on the substrate concentration, the final stevioside concentration of the reaction solution was adjusted to 5% to 40%, the activity of stevioside per gram of stevioside was set to 3000 U, mM Tris-HCl at 70 < 0 > C for 9 hours.

As a result, as shown in Table 2, Thermus it was confirmed that the production of rubusoidal is continuously increased for the same enzymatic activity even when 40% of the high concentration of stevioside is used. These results were obtained by using 80 g / L stevioside as a substrate and 5,250 U / g stevioside using Aspergillus sp. Beta-galactosidase, Compared with 0.19 g / hour / 1,000 U, which is the amount of rubusoidalide produced after reaction at 60 ° C for 72 hours, it was 8.68 times higher in the same concentration of substrate when using lactase from Thermus spp. Lubusoidal can be used as an enzyme of 1000 U per unit time, and when 40% substrate is used, production capacity of 38 times or more can be confirmed.

[Example 3]

The free enzyme must be added to the reactor every time it reacts, whereas the immobilized enzyme has the advantage of being able to recover the enzyme. In addition, immobilized enzymes generally have better stability of enzyme activity than free enzymes. In this experiment, immobilized enzyme beads were prepared using alginate beads, and the amount of rubusoids was confirmed using the beads.

Immobilized enzyme beads were prepared by a conventional method, and 3% (w / v) of alginate was used. To 1 g of alginate beads was added 200 U of Thermus spp.) and 1% (final concentration) of stevioside was used to compare the stability and stability of the activity when using free enzyme and immobilized enzyme beads. The temperature used for the thermal stability test was 70 캜.

The Thermus The resultant lactase was found to have the highest ability to convert rubusoidal to stevioside ( Thermus (EMBL accession number HB973086.1) was used.

As can be seen from the results in Table 3 above, in sseomeoseu (Thermus All of the free enzymes and immobilized enzymes were found to be stable at 70 ℃.

However, the degree of formation of rubusoidal was confirmed by the fact that the longer the incubation time at 70 ° C was, the lower the formation of rubusoidal was observed in the case of the free enzyme, whereas in the case of using the immobilized bead enzyme, Side could be confirmed to generate more.

As a result, the same enzymes can be used many times in industry, which is an economical advantage of the enzyme reuse process, and it is confirmed that there is an advantage that a large amount of rubusoside can be produced stably and continuously with high efficiency.

[ Example  4]

The rubusoidal produced from the above Examples 1 to 3 was purified and confirmed to be rubusoidal by NMR analysis. The results are shown in Table 4.

As a result, it was confirmed that Rubusoside of Formula 2 was produced.

From the above results, it was found that Aspergillus niger- derived? -Galactosidase,? -Galactosidase, pectinase and penicillium Naringinase derived from Penicillium spp., Lactase derived from Thermus spp., Is bound to 13C of stevioside, and 2-O-β-D- Glucopyranosyl-aD-glucose) was removed to confirm the synthesis of rubusoidal.

In particular, Lactase derived from Thermus spp. Has excellent ability to convert stevioside to rubusoidal, and even when using a small amount of enzyme activity, It is possible to produce rubusoidal side, and it can be confirmed that there is little difference in activity or stability of the enzyme even when it is used for 12 days in the production of alginate-immobilized bead. Therefore, the reuse of enzyme and the production efficiency of rubusoidal And it can be confirmed that it can contribute greatly to commercial use.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Method for preparing rubusoside from stevioside using enzymes <160> 6 <170> Kopatentin 1.71 <210> 1 <211> 431 <212> PRT <213> Thermus spp. <400> 1 Met Asp Asp His Ala Glu Lys Phe Leu Trp Gly Val Ala Thr Ser Ala   1 5 10 15 Tyr Gln Ile Glu Gly Ala Thr Gln Glu Asp Gly Arg Gly Pro Ser Ile              20 25 30 Trp Asp Ala Phe Ala Arg Arg Pro Gly Ala Ile Arg Asp Gly Ser Thr          35 40 45 Gly Glu Pro Ala Cys Asp His Tyr Arg Arg Tyr Glu Glu Asp Ile Ala      50 55 60 Leu Met Gln Ser Leu Gly Val Arg Ala Tyr Arg Phe Ser Val Ala Trp  65 70 75 80 Pro Arg Ile Leu Pro Glu Gly Arg Gly Arg Ile Asn Pro Lys Gly Leu                  85 90 95 Ala Phe Tyr Asp Arg Leu Val Asp Arg Leu Leu Ala Ser Gly Ile Thr             100 105 110 Pro Phe Leu Thr Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Glu Glu         115 120 125 Arg Gly Gly Trp Arg Ser Glu Thr Ala Phe Ala Phe Ala Glu Tyr     130 135 140 Ala Glu Ala Val Ala Arg Ala Leu Ala Asp Arg Val Pro Phe Phe Ala 145 150 155 160 Thr Leu Asn Glu Pro Trp Cys Ser Ala Phe Leu Gly His Trp Thr Gly                 165 170 175 Glu His Ala Pro Gly Leu Arg Asn Leu Glu Ala Ala Leu Arg Ala Ala             180 185 190 His His Leu Leu Leu Gly His Gly Leu Ala Val Glu Ala Leu Arg Ala         195 200 205 Ala Gly Ala Arg Arg Val Gly Ile Val Leu Asn Phe Ala Pro Ala Tyr     210 215 220 Gly Glu Asp Pro Glu Ala Val Asp Val Ala Asp Arg Tyr His Asn Arg 225 230 235 240 Tyr Phe Leu Asp Pro Ile Leu Gly Lys Gly Tyr Pro Glu Ser Pro Phe                 245 250 255 Arg Asp Pro Pro Pro Val Ile Leu Ser Arg Asp Leu Glu Leu Val             260 265 270 Ala Arg Pro Leu Asp Phe Leu Gly Val Asn Tyr Tyr Ala Pro Val Arg         275 280 285 Val Ala Pro Gly Thr Gly Thr Leu Pro Val Arg Tyr Leu Pro Pro Glu     290 295 300 Gly Pro Ala Thr Ala Met Gly Trp Glu Val Tyr Pro Glu Gly Leu His 305 310 315 320 His Leu Leu Lys Arg Leu Gly Arg Glu Val Pro Trp Pro Leu Tyr Val                 325 330 335 Thr Glu Asn Gly Ala Ala Tyr Pro Asp Leu Trp Thr Gly Glu Ala Val             340 345 350 Val Glu Asp Pro Glu Arg Val Ala Tyr Leu Glu Ala His Val Glu Ala         355 360 365 Ala Leu Arg Ala Arg Glu Glu Gly Val Asp Leu Arg Gly Tyr Phe Val     370 375 380 Trp Ser Leu Met Asp Asn Phe Glu Trp Ala Phe Gly Tyr Thr Arg Arg 385 390 395 400 Phe Gly Leu Tyr Tyr Val Asp Phe Pro Ser Gln Arg Arg Ile Pro Lys                 405 410 415 Arg Ser Ala Leu Trp Tyr Arg Glu Arg Ile Ala Arg Ala Gln Thr             420 425 430 <210> 2 <211> 431 <212> PRT <213> Thermus spp. <400> 2 Met Thr Glu Asn Ala Glu Lys Phe Leu Trp Gly Val Ala Thr Ser Ala   1 5 10 15 Tyr Gln Ile Glu Gly Ala Thr Gln Glu Asp Gly Arg Gly Pro Ser Ile              20 25 30 Trp Asp Ala Phe Ala Gln Arg Pro Gly Ala Ile Arg Asp Gly Ser Thr          35 40 45 Gly Glu Pro Ala Cys Asp His Tyr Arg Arg Tyr Glu Glu Asp Ile Ala      50 55 60 Leu Met Gln Ser Leu Gly Val Arg Ala Tyr Arg Phe Ser Val Ala Trp  65 70 75 80 Pro Arg Ile Leu Pro Glu Gly Arg Gly Arg Ile Asn Pro Lys Gly Leu                  85 90 95 Ala Phe Tyr Asp Arg Leu Val Asp Arg Leu Leu Ala Ser Gly Ile Thr             100 105 110 Pro Phe Leu Thr Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Glu Glu         115 120 125 Arg Gly Gly Trp Arg Ser Glu Thr Ala Phe Ala Phe Ala Glu Tyr     130 135 140 Ala Glu Ala Val Ala Arg Ala Leu Ala Asp Arg Val Pro Phe Phe Ala 145 150 155 160 Thr Leu Asn Glu Pro Trp Cys Ser Ala Phe Leu Gly His Trp Thr Gly                 165 170 175 Glu His Ala Pro Gly Leu Arg Asn Leu Glu Ala Ala Leu Arg Ala Ala             180 185 190 His His Leu Leu Leu Gly His Gly Leu Ala Val Glu Ala Leu Arg Ala         195 200 205 Ala Gly Ala Arg Arg Val Gly Ile Val Leu Asn Phe Ala Pro Ala Tyr     210 215 220 Gly Glu Asp Pro Glu Ala Val Asp Val Ala Asp Arg Tyr His Asn Arg 225 230 235 240 Phe Phe Leu Asp Pro Ile Leu Gly Lys Gly Tyr Pro Glu Ser Pro Phe                 245 250 255 Arg Asp Pro Pro Pro Val Ile Leu Ser Arg Asp Leu Glu Leu Val             260 265 270 Ala Arg Pro Leu Asp Phe Leu Gly Val Asn Tyr Tyr Ala Pro Val Arg         275 280 285 Val Ala Pro Gly Thr Gly Thr Leu Pro Val Arg Tyr Leu Pro Pro Glu     290 295 300 Gly Pro Ala Thr Ala Met Gly Trp Glu Val Tyr Pro Glu Gly Leu Tyr 305 310 315 320 His Leu Leu Lys Arg Leu Gly Arg Glu Val Pro Trp Pro Leu Tyr Val                 325 330 335 Thr Glu Asn Gly Ala Ala Tyr Pro Asp Leu Trp Thr Gly Glu Ala Val             340 345 350 Val Glu Asp Pro Glu Arg Val Ala Tyr Leu Glu Ala His Val Glu Ala         355 360 365 Ala Leu Arg Ala Arg Glu Glu Gly Val Asp Leu Arg Gly Tyr Phe Val     370 375 380 Trp Ser Leu Met Asp Asn Phe Glu Trp Ala Phe Gly Tyr Thr Arg Arg 385 390 395 400 Phe Gly Leu Tyr Tyr Val Asp Phe Pro Ser Gln Arg Arg Ile Pro Lys                 405 410 415 Arg Ser Ala Leu Trp Tyr Arg Glu Arg Ile Ala Arg Ala Gln Thr             420 425 430 <210> 3 <211> 431 <212> PRT <213> Thermus spp. <400> 3 Met Thr Glu Asn Ala Glu Lys Phe Leu Trp Gly Val Ala Thr Ser Ala   1 5 10 15 Tyr Gln Ile Glu Gly Ala Thr Gln Glu Asp Gly Arg Gly Pro Ser Ile              20 25 30 Trp Asp Ala Phe Ala Gln Arg Pro Gly Ala Ile Arg Asp Gly Ser Thr          35 40 45 Gly Glu Pro Ala Cys Asp His Tyr Arg Arg Tyr Glu Glu Asp Ile Ala      50 55 60 Leu Met Gln Ser Leu Gly Val Arg Ala Tyr Arg Phe Ser Val Ala Trp  65 70 75 80 Pro Arg Ile Leu Pro Glu Gly Arg Gly Arg Ile Asn Pro Lys Gly Leu                  85 90 95 Ala Phe Tyr Asp Arg Leu Val Asp Arg Leu Leu Ala Ser Gly Ile Thr             100 105 110 Pro Phe Leu Thr Leu Tyr His Trp Asp Leu Pro Leu Ala Leu Glu Glu         115 120 125 Arg Gly Gly Trp Arg Ser Glu Thr Ala Phe Ala Phe Ala Glu Tyr     130 135 140 Ala Glu Ala Val Ala Arg Ala Leu Ala Asp Arg Val Pro Phe Phe Ala 145 150 155 160 Thr Leu Asn Glu Pro Trp Cys Ser Ala Phe Leu Gly His Trp Thr Gly                 165 170 175 Glu His Ala Pro Gly Leu Arg Asn Leu Glu Ala Ala Leu Arg Ala Ala             180 185 190 His His Leu Leu Leu Gly His Gly Leu Ala Val Glu Ala Leu Arg Ala         195 200 205 Ala Gly Ala Arg Arg Val Gly Ile Val Leu Asn Phe Ala Pro Ala Tyr     210 215 220 Gly Glu Asp Pro Glu Ala Val Asp Val Ala Asp Arg Tyr His Asn Arg 225 230 235 240 Phe Phe Leu Asp Pro Ile Leu Gly Lys Gly Tyr Pro Glu Ser Pro Phe                 245 250 255 Arg Asp Pro Pro Pro Val Ile Leu Ser Arg Asp Leu Glu Leu Val             260 265 270 Ala Arg Pro Leu Asp Phe Leu Gly Val Asn Tyr Tyr Ala Pro Val Arg         275 280 285 Val Ala Pro Gly Thr Gly Thr Leu Pro Val Arg Tyr Leu Pro Pro Glu     290 295 300 Gly Pro Ala Thr Ala Met Gly Trp Glu Val Tyr Pro Glu Gly Leu His 305 310 315 320 His Leu Leu Lys Arg Leu Gly Arg Glu Val Pro Trp Pro Leu Tyr Val                 325 330 335 Thr Glu Asn Gly Ala Ala Tyr Pro Asp Leu Trp Thr Gly Glu Ala Val             340 345 350 Val Glu Asp Pro Glu Arg Val Ala Tyr Leu Glu Ala His Val Glu Ala         355 360 365 Ala Leu Arg Ala Arg Glu Glu Gly Val Asp Leu Arg Gly Tyr Phe Val     370 375 380 Trp Ser Leu Met Asp Asn Phe Glu Trp Ala Phe Gly Tyr Thr Arg Arg 385 390 395 400 Phe Gly Leu Tyr Tyr Val Asp Phe Pro Ser Gln Arg Arg Ile Pro Lys                 405 410 415 Arg Ser Ala Leu Trp Tyr Arg Glu Arg Ile Ala Arg Ala Gln Thr             420 425 430 <210> 4 <211> 1296 <212> DNA <213> Thermus spp. <400> 4 atggatgatc atgctgaaaa atttttatgg ggtgtggcta cttccgcata ccagattgaa 60 ggtgctactc aggaggatgg tagaggtccg tctatctggg acgcattcgc tagaagacct 120 ggggctatta gagatggttc tacaggtgaa ccagcttgcg atcattatag aagatatgaa 180 gaagatattg ctttaatgca atcgcttggt gttagagctt acagattctc cgttgcttgg 240 ccaagaatat tgccagaagg tagaggtaga atcaacccta aaggtttagc tttttacgat 300 cgtttggttg acagattgtt ggctagtggt attacaccct ttttaaccct ataccactgg 360 gatcttcctt tggctttgga agagagaggt ggttggcgtt ctcgggaaac tgcttttgca 420 tttgctgagt acgccgaagc tgtagctaga gcattggctg atagagttcc tttctttgct 480 actttaaatg aaccatggtg tagcgctttt cttggtcatt ggacgggtga acacgctcca 540 ggattgagaa acctagaggc tgctctaaga gcagctcatc atttgttgtt gggacacgga 600 ttggcagtcg aggctttgag agctgcaggt gccagaagag ttggtattgt tttaaacttt 660 gctccagctt atggggaaga tccagaagct gtggatgttg ctgatcgtta ccacaataga 720 tattttcttg atccaatcct aggaaagggt tacccagaat caccatttag agaccctcca 780 cctgttccta ttttatcaag agatcttgaa ctagttgcta gacctttgga ctttttggga 840 gttaactact acgctccagt aagagttgct ccaggtactg gaacattgcc agtgagatat 900 ttgccccccg aaggaccagc aactgccatg ggttgggagg tttacccgga aggcttgcac 960 catttgttaa agagattggg tagagaagtt ccatggccat tgtatgttac tgaaaatggc 1020 gctgcttatc ctgatttgtg gactggtgag gcggtagttg aagatccaga aagagttgct 1080 tacttggaag cacatgtaga agctgcattg agagctagag aagaaggtgt tgacttgaga 1140 ggttatttcg tttggagttt gatggataat tttgaatggg catttggtta tactagaagg 1200 tttggattgt attacgttga ttttccttct caaagaagaa taccaaagag atctgcttta 1260 tggtatagag aaagaatcgc tcgtgctcaa acttaa 1296 <210> 5 <211> 1296 <212> DNA <213> Thermus spp. <400> 5 atgaccgaga acgccgaaaa attcctttgg ggagtggcca ccagcgccta ccagattgag 60 ggggccaccc aggaggacgg ccgggggcct tccatctggg acgccttcgc ccagcgcccc 120 ggggccatcc gggacgggag cacaggggag cccgcctgcg accactaccg ccgctacgag 180 gaggacatcg ccctgatgca atccctcggg gtgcgggcct accgcttctc cgtggcctgg 240 ccccggatcc tccccgaggg ccgggggcgg atcaacccca agggcctcgc cttctacgac 300 cgcctggtgg accggcttct cgcttccggg atcacgccct ttctcaccct ctaccactgg 360 gacctgcctt tggccctgga ggagcgggga ggctggcgga gccgggaaac cgccttcgcc 420 ttcgccgagt acgccgaggc ggtggcccgg gccctcgccg accgggtgcc cttcttcgcc 480 accctgaacg agccctggtg ctcggccttc ctcgggcact ggacggggga acacgccccc 540 ggcctcagga acctggaagc ggccctccgc gccgcccacc acctcctcct gggccacggc 600 ctcgccgtgg aggccttgag ggccgcgggg gcgaggcggg tggggatcgt cctcaacttc 660 gccccggcct acggcgagga ccccgaggcg gtggacgtgg ccgaccgcta ccacaaccgc 720 ttcttcctgg accccatcct gggcaagggg tatcccgaaa gccccttccg agaccccccg 780 cccgtcccca tcctctcccg cgacctggag ctcgtggcaa ggcccctgga cttcctgggg 840 gtgaactact acgcccccgt ccgcgtggcc ccggggacgg ggaccttgcc cgtgcgctac 900 cttcccccgg aagggccggc cacggccatg gggtgggagg tctaccccga ggggcttcac 960 ccctcttga agcgcctcgg ccgggaggtg ccctggcccc tttacgtcac ggaaaacggg 1020 gccgcctacc ccgacctctg gacgggagag gccgtggtgg aggaccccga gcgggtggcc 1080 tacctcgagg cccacgtgga ggccgccctc cgggcccggg aagaaggggt ggacctccgg 1140 ggctacttcg tctggagcct catggacaac tttgagtggg ccttcggcta cacccggcgc 1200 ttcggcctct actacgtgga cttccccagc cagaggcgca tccccaaaag gagcgccctc 1260 tggtaccggg agcggatcgc gcgggcccag acctaa 1296 <210> 6 <211> 1296 <212> DNA <213> Thermus spp. <400> 6 atgaccgaga acgccgaaaa attcctttgg ggagtggcca ccagcgccta ccagattgag 60 ggggccaccc aggaggacgg ccgggggcct tccatctggg acgccttcgc ccagcgcccc 120 ggggccatcc gggacgggag cacaggggag cccgcctgcg accactaccg ccgctacgag 180 gaggacatcg ccctgatgca atccctcggg gtgcgggcct accgcttctc cgtggcctgg 240 ccccggatcc tccccgaggg ccgggggcgg atcaacccca agggcctcgc cttctacgac 300 cgcctggtgg accggcttct cgcttccggg atcacgccct ttctcaccct ctaccactgg 360 gacctgcctt tggccctgga ggagcgggga ggctggcgga gccgggaaac cgccttcgcc 420 ttcgccgagt acgccgaggc ggtggcccgg gccctcgccg accgggtgcc cttcttcgcc 480 accctgaacg agccctggtg ctcggccttc ctcgggcact ggacggggga acacgccccc 540 ggcctcagga acctggaagc ggccctccgc gccgcccacc acctcctcct gggccacggc 600 ctcgccgtgg aggccttgag ggccgcgggg gcgaggcggg tggggatcgt cctcaacttc 660 gccccggcct acggcgagga ccccgaggcg gtggacgtgg ccgaccgcta ccacaaccgc 720 ttcttcctgg accccatcct gggcaagggg tatcccgaaa gccccttccg agaccccccg 780 cccgtcccca tcctctcccg cgacctggag ctcgtggcaa ggcccctgga cttcctgggg 840 gtgaactact acgcccccgt ccgcgtggcc ccggggacgg ggaccttgcc cgtgcgctac 900 cttcccccgg aagggccggc cacggccatg gggtgggagg tctaccccga ggggcttcac 960 ccctcttga agcgcctcgg ccgggaggtg ccctggcccc tttacgtcac ggaaaacggg 1020 gccgcctacc ccgacctctg gacgggagag gccgtggtgg aggaccccga gcgggtggcc 1080 tacctcgagg cccacgtgga ggccgccctc cgggcccggg aagaaggggt ggacctccgg 1140 ggctacttcg tctggagcct catggacaac tttgagtggg ccttcggcta cacccggcgc 1200 ttcggcctct actacgtgga cttccccagc cagaggcgca tccccaaaag gagcgccctc 1260 tggtaccggg agcggatcgc gcgggcccag acctaa 1296

Claims (12)

at least one selected from α-galactosidase, β-galactosidase, β-galactosidase, lactase, pectinase and naringinase. enzyme; Or microorganisms for producing the enzyme; Rubusoside (Rubusoside) production composition of the formula comprising a.

The method of claim 1,
The enzyme is characterized in that the synthesis of rubusoside by removing the terminal glucose of phosphoose (2-O-β-D-Glucopyranosyl-aD-glucose) bound to 13C of Stevioside. The method of claim 1,
Microorganism producing the enzyme is Aspergillus you are (Aspergillus niger), Penny chamber in Solarium (Penicillium spp.), and Thermus at least one selected from spp.). The method of claim 1,
The lactase can be obtained from the genus Thermus spp.). &lt; / RTI &gt; 5. The method of claim 4,
Wherein the lactase is an amino acid of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In preparing rubusoside from stevioside,
at least one selected from α-galactosidase, β-galactosidase, β-galactosidase, lactase, pectinase and naringinase. enzyme; Or a microorganism producing the enzyme; a method of producing ruboside, characterized in that using. The method according to claim 6,
The microorganism producing the enzyme is Aspergillus niga (Aspergillus niger),Penicillium( Penicillium spp.), and the summer genus (Thermus production method, characterized in that at least one selected from spp.). The method according to claim 6,
The lactase can be obtained from the genus Thermus spp.). &lt; / RTI &gt; The method of claim 8,
Wherein said lactase is an amino acid of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The method according to claim 6,
The enzyme is a production method, characterized in that using the immobilized in the immobilized beads. The method of claim 10,
Wherein the immobilized bead is sodium alginate, triethanolamine alginate, or calcium alginate. The method according to any one of claims 6 to 11,
The production method is the stevioside and the enzyme at a temperature of 50 to 80 ℃, reacted for 5 to 15 days Sophorose (2-O-β-D-Glucopyranosyl-aD-glucose) bound to 13C of stevioside A method for producing rubusoside by removing the terminal glucose of the.

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