KR20150000258A - Method for Preparing Natural High Intensity Sweetener Rebaudiside A by Using Enzymatic Conversion - Google Patents

Abstract

The present invention relates to a manufacturing method of rebaudioside A from stevioside hugely included in stevia extracts. Specifically, the manufacturing method of the present invention is allowed to manufacture rebaudioside A from stevioside by making a reaction solution react wherein the reaction solution includes (a) maltodextrin, UMP, ATP, stevioside, phosphoric acid compound, maltodextrin phosphorylase, glucose-1-phophate thymidylytrasferase, inorganic pyrophosphatase, acetate kinase, uridylate kinase, and UDP-glucosyltransferase. According to the present invention, the cost of production of stevia sweetener can be reduced since rebaudioside A is economically manufactured from stevioside without using high-priced UDP-glucose.

Description

[0001] The present invention relates to a method for preparing a natural high-sweetener using an enzyme conversion method,

The present invention relates to a method for preparing rebaudioside A from stevioside, which is contained in a large amount of stevia extract, and more particularly, to a method for producing rebaudioside A from a stevia extract which comprises (a) maltodextrin, UMP, ATP, stevioside, A reaction solution containing a cellulase, glucose-1-phosphate thymidyl transferase, inorganic pyrophosphatase, acetate kinase, free delayed kinase and UDP-glycosyltransferase is reacted to generate rebaudioside A ≪ RTI ID = 0.0 > A < / RTI > from stevioside.

Sugar (sucrose) is the most commonly known and widely used sweetener, and is used in large quantities in soft drinks such as instant coffee, coffee drinks, juices and carbonated drinks. However, according to social trends for well-being, there is an increasing tendency to use sugar-rich sweeteners that can reduce sugar or cause sugar-causing diseases such as obesity and diabetes, and artificial sweeteners such as saccharin, The use of sweeteners is becoming increasingly regulated due to the toxicity and allergenic hazards and stability problems, and the need for new functional sugar derivatives is increasing.

Stevia Sweetener is a high-sweetening substance extracted from Stevia, a perennial plant of Asteraceae originating in South American Paraguay. Worldwide consumption of stevia sweeteners is about 30% in Korea, 27% in Japan, 30% in China, and 13% in other countries. In Korea, consumption of food as an ingredient is almost the same as that of enzyme-treated stevia (the official name for food additives), which is a product that improves the sweetness by adding glucose using enzymes. Among the stevia sweeteners, the enzyme-treated stevia has the following advantages: (1) The sweetness is close to sugar, no bitter taste, and the sweetness is as high as 100 to 250 times that of sugar, but is virtually calorie. ② It is stable to heat and acid, and there is little change in sweetness during processing and preservation. ③ It is difficult to become a nutrient source of microorganisms, and it is practically non-oozing in the oral cavity. ④ It does not cause a mail rod reaction, and it is hard to be browned by food processing. ⑤ Relieves the taste of salted fish. ⑥ Relieve sickness. ⑦ Freezing point is small. ⑧ Do not increase infiltration pressure. ⑨ There is synergy with other sweeteners, which is effective in reducing sweetness cost. ⑩ It is refreshing taste and it relaxes the rough taste of carbohydrate sweetener. However, despite the above advantages and the refining process and enzyme reaction in the stevia leaf extract, the enzyme-treated stevia has a slow rise in sweetness compared to other sugar sweeteners (sugar, fructose, etc.) There is a characteristic that the sweetness remains relatively long, and the so-called sweetness finish is bad. In addition, the stevioside, which is contained in a large amount in the above-mentioned stevia sweetener, is accompanied by a bitter taste or a bitter taste distinctive to the aftertaste apart from the sweetness, and thus has disadvantages that it gives a sense of discomfort when used in coffee beverages, soft drinks and alcoholic beverages (Korean Patent No. 0,888,694 ).

The sweetness component of stevia is close to sugar and high in sweetness is rebaudioside A. Rebaudioside A has recently been used as a sweetener in Coca Cola and Pepsi as it is licensed for use as food by the FDA. The content of rebaudioside A in stevia extract is about 30%, and stevioside is about 50% (Korean Patent Laid-Open Publication No. 2001-0111560). As a result, the demand for products containing high-grade rebaudioside A having an excellent sweetness is increasing. However, when only high-rebaudioside A is separated, stevioside, which is a by-product, As the demand for Boudioside A increases, the problem of treating stevioside, which has less good quality than Rebaudioside A, is becoming a challenge for commercialization.

 To solve these problems, many companies have developed sweeteners using stevioside. In this study, β-1,4-galactosyltransferase was acted in an aqueous solution containing stevia extract as a main component and β-1,4-galactosyl sugar compound to produce a galactosyl group on the stevioside (Japanese Patent Laid-Open No. 58-94367), a method of synthesizing a stevioside oligosaccharide by the action of dextran sucrase in an aqueous solution containing stevioside and sugar (Korean Patent No. 10-1199821) , A method of converting stevioside containing stevioside, which is a by-product obtained from the production process of rebaudioside A, to rebaudioside A by an enzyme conversion method (-1,3-glucanase) (Korean Patent Laid- -0115699), a method for improving the quality of stevioside by converting stevioside into stevioside by enzymatic reaction in a reaction system in which water and a hydrophobic organic solvent are mixed (Korean Patent Registration No. 1994-528), a method of improving solubility of stevioside and rebaudioside A in a mixture of stevioside and rebaudioside A, (Japanese Patent Application Laid-Open No. 56-121453, Korean Patent Laid-Open Publication No. 10-2004-0102322), a method of irradiating a stevia plant with a gamma ray to produce a large amount of rebaudioside A, (Japanese Patent Application Laid-Open No. 2002-34502) and the like are disclosed. In order to separate ST-G1 and ST-G2, which are excellent in sweetness, cyclodextrin glucanotransferase (hereinafter referred to as "CGTase") was added to an aqueous solution containing stevia extract and α-glucosyl sugar compound (Korean Unexamined Patent Publication No. 1992-252), a method of producing a stevia sweetener, which comprises treating the stevia extract with CGTase and < RTI ID = 0.0 > ss-amylase < / RTI & ST-G1, ST-G2, and ST-G2, which are high-sweetness components with respect to the total steviol glycoside contents in the case of the preparation method of stevia sweetener, , The total content of RA-G1 and RA-G2 is as low as 40 to 60%, resulting in unsatisfactory sweetness, There is an enzyme treated stevia still have a bitter taste or unpleasant, it is difficult to use in coffee or drinks.

Accordingly, the present inventors have made intensive efforts to develop an effective treatment method for reducing stevioside in the stevia extract, and as a result, have developed a process for economically converting stevioside contained in the stevia extract into enzymatic treatment to rebaudioside A , Thereby completing the present invention.

It is an object of the present invention to provide a process for preparing stevioside from rebaudioside A.

In order to achieve the above object, the present invention provides a method for preparing rebaudioside A from stevioside and UDP-glucose in the presence of UDP-glycosyltransferase,

Maltodextrin phosphorylase, glucose-1-phophate thymidyltransferase, inorganic pyrophosphatase, acetate kinase, and glass delayed canine (Rebaudioside A) which is characterized in that UDP-glucose is regenerated by adding a substrate composed of uridylate kinase and a substrate composed of acetylphosphate, maltodextrin, UMP and ATP. . ≪ / RTI >

According to the invention, rebaudioside A can be produced economically from stevioside without using expensive UDP glucose, and the cost of producing stevia sweetener can be lowered.

Figure 1 shows the route of synthesis of Rebaudioside A from Stevioside.
Fig. 2 shows the result of purification of UDP-glucosyltransferase derived from Stevia by SDS-PAGE.
FIG. 3 shows Rebaudioside A produced by an enzyme reaction by HPLC.
Figure 4 shows the effect of temperature (A) on UDP-glucosyltransferase activity and the effect on pH (B).
Figure 5 is a graph coli K-12 substr. Expression of MG1655-derived genes was confirmed by SDS-PAGE (1: total protein without induction, 2: total protein with induction, 3: souble protein,
FIG. 6 shows the HPLC analysis results of the reference material UDP-glucose (A) and the reaction solution (B).
7 shows the amount of UDP-glucose produced (A) and the relative activity of rebaudioside A production (B) according to the amount of enzyme.
FIG. 8 shows the concentrations of stevioside and rebaudiosideA depending on the reaction time of the reaction solution using the enzyme of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a method for preparing rebaudioside A from stevioside and UDP-glucose in the presence of UDP-glycosyltransferase,

Maltodextrin phosphorylase, glucose-1-phophate thymidyltransferase, inorganic pyrophosphatase, acetate kinase, and glass delayed canine (Rebaudioside A) which is characterized in that UDP-glucose is regenerated by adding a substrate composed of uridylate kinase and a substrate composed of acetylphosphate, maltodextrin, UMP and ATP. .

The conversion of stevioside contained in the stevia extract to rebaudioside A having high sweetness through enzyme treatment is shown in Fig.

However, in the conversion reaction through the conventional enzymatic treatment, expensive UDP-glucose was used as a saccharide donor, and the economical power for industrial use was lowered.

To overcome these disadvantages, the present invention introduces a system for synthesizing UDP-glucose using low-cost maltodextrin, UTP, and phosphate. Since various enzymes such as maltodextrin phosphorylase, glucose-1-phophate thymidyltransferase, inorganic pyrophosphatase, acetate kinase, and uridylate kinase are involved in the UDP-glucose synthesis system (FIG. 1), the present invention clones these enzymes to construct a transformant After expression, the enzymes were purified and applied to the synthesis of UDP-glucose.

In one aspect of the invention, an enzyme that converts stevioside to rebaudioside A is Stevia rebaudiana (UDP-glycosyltransferase) derived from UDP-glycosyltransferase was prepared and a recombinant plasmid containing a gene encoding UDP-glycosyltransferase was prepared and cloned into E. coli BL21 to obtain a large amount of enzyme And then purified and used.

Thus, the UDP-glycosyltransferase of the present invention can be used as a carrier for Stevia rebaudiana . ≪ / RTI >

In the present invention, the UDP-glycosyltransferase may be characterized in that the UDP-glycosyltransferase is encoded by the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the maltodextrin phosphorylase, glucose-1-phophate thymidyltransferase, inorganic pyrophosphatase, acetate kinase kinase) and uridylate kinase ( Escherichia coli K-12 substr.). Using the gene derived from MG1655, a recombinant plasmid containing each of the above genes was prepared and cloned into Escherichia coli BL21 to express a large amount of the enzyme, followed by purification.

Thus, in the present invention, maltodextrin phosphorylase, glucose-1-phophate thymidyltransferase, inorganic pyrophosphatase, acetate kinase, kinase) and uridylate kinase Escherichia coli K-12 substr. MG1655. ≪ / RTI >

In another embodiment of the present invention, the reaction conditions include 5% maltodextrin, 2 mM UMP, 2 mM ATP, 1 mM MgCl ₂ , 100 mM sodium phosphate buffer (pH 7.5), 0.1 mg / ml Stevioside, 100 μl of galU, 100 mU of ppa, 100 mU of UGT76G1, 50 mU of ackA and 100 μg of pyrH were added to make 1 ml total, and reacted at 30 ° C for 8 hours. Rebaudioside A was produced at a yield of 90% (Fig. 8).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One: Stevia rebaudiana Derived UDP - glycosyltransferase  Construction and expression of transformants

Stevia rebaudiana A pET41a / UGT76G1 recombinant plasmid was constructed by cloning the UDP-glycosyltransferase (accession number; AY345974) (UGT76G1) from the Nco I / Bam HI site of the pET41a vector (novagen, USA). In order to measure the enzyme activity, a recombinant plasmid was introduced into E. coli BL21 to construct a transformant.

The transformant was cultured in an LB liquid medium supplemented with 50 μg / ml kanamycin at 37 ° C. until the OD ₆₀₀ value reached 0.5, 0.1 mM IPTG was added, and the mixture was cultured at 25 ° C. for 12 hours with shaking at 180 rpm Overexpression of the enzyme was induced. The cultures were centrifuged to recover precipitated cells and washed twice with 0.85% NaCl. The recovered cells were sonicated with 50 mM Tris-HCl (pH 7.5) supplemented with 1 mM PMSF and 1x protease inhibitor cocktail, and centrifuged to recover the supernatant. For purification using Hisp-tag, the supernatant was passed through a Ni-NTA agarose carrier and chromatographed. When the enzyme was eluted, the enzyme active fraction was eluted with an eluent containing 250 mM imidazole, 300 mM NaCl and 20 mM Tris-Cl (pH 8.0), and the purification was confirmed by SDS-PAGE (FIG. The purified enzyme was concentrated and desalted with Amicon Ultra-15 (Millipore, 5K NMWL device).

To measure the activity of UGT76G1, stevioside was used as a substrate. 1 mM MgCl ₂ , 1 mg / ml Stevioside, and 100 μg of the enzyme supernatant were added to a final volume of 1 ml, and the mixture was reacted at 30 ° C for 1 hour. The reaction was analyzed using HPLC equipped with ZORBAX NH ₂ (4.6 x 250 5 μm, Agilent) column. As a mobile phase, 30% water and 70% acetonitrile were flowed at a flow rate of 1 ml / min and analyzed at a column temperature of 30 ° C., and absorbance was measured at 210 nm. Rebaudioside A was generated from stevioside through enzyme reaction (Fig. 3).

 The unit of enzyme (U) was defined as the amount of enzyme required to produce 1 μmol of rebaudioside A per unit time (1 min).

Activity and recovery rate of UGT76G1 according to the purification step Total vol. (ml) Total protein (mg) Total activity (U) Specific activity (U / mg) Purification
(Fold) Yield (%) Crude extract 3 4.48 3.17 0.71 1.0 100 Ni-NTA affinity chromatography 0.5 0.61 0.54 0.89 1.26 17.03

Example  2: Stevia rebaudiana  Derived UDP - glycosyltransferase  Investigate characteristics

Reaction temperature and reaction pH were investigated to determine the optimal reaction conditions of UGT76G1 enzyme purified from Streptomyces sp.

Relative activity was measured at 20 ° C to 80 ° C at 10 ° C intervals using the enzyme digested after overexpression, and it was found that 37 ° C was the optimal reaction temperature (FIG. 3A). 50 mM citrate-NaOH buffer (pH 3.0-6.0), 50 mM Tris-HCl buffer (pH 5.0-8.5), 50 mM sodium phosphate buffer (pH 5.0-8.0) and 50 mM A buffer solution of Glycine-NaOH buffer (pH 8.0-11.0) was used. As a result of measuring the relative activity after reacting in each buffer solution, the activity was maximum at 50 mM sodium phosphate buffer pH 7.5 (FIG. 4B).

Example  3: UDP - glucose regeneration system Involved in Escherichia coli K-12 subst . MG1655  Investigation of the characteristics of strain-derived enzymes

The enzymes required for the UDP-glucose production system for economical processing are Escherichia coli K-12 substr. Amplified from MG1655 using PCR, and cloned into an expression vector. Thus, pET302 / ppA with activity of pET30a / malP, glucose-1-phophate thymidylytrasferase (accession NO. EG11319) activity and maltodextrin phosphorylase (accession NO. EG10560) activity and pET302 / galU and inorganic pyrophosphatase (accession NO. EG10755) , pET302 / ackA, and uridylate kinase (accession NO. EG11539), respectively, with the activity of acetate kinase (accession NO. EG10027).

The recombinant plasmids were introduced into Escherichia coli [BL21 (DE3)] to construct a transformant. The cells were cultured in LB liquid medium supplemented with antibiotics at 37 ° C for 1 hour and 30 minutes. When the absorbance at 600 nm reached 0.5, IPTG was added to induce overexpression of the enzyme. Lt; / RTI > The cultures were centrifuged to recover precipitated cells and washed twice with 0.85% NaCl. To the recovered cells, 50 mM Tris-HCl (pH 7.5) supplemented with 1 mM PMSF and 1x protease inhibitor cocktail was added to the recovered cells, disrupted by ultrasonication, and centrifuged to recover the supernatant. The presence or absence of expression of the recovered enzyme supernatant was confirmed by SDS-PAGE (Fig. 5) and used for the enzyme reaction experiment.

The purified Escherichia coli K-12 substr. The following standard reaction experiments were performed to examine the activities of the MG1655-derived enzymes (maltodextrin phosphorylase, glucose-1-phophate thymidylytrasferase, inorganic pyrophosphatase, acetate kinase and uridylate kinase).

(I) malP: The activity of malP can be expressed as the amount of UDP-glucose produced by reacting maltodextrin as a substrate. The reaction conditions were as follows: 100 mM phosphate (pH 7.5), 5% maltodextrin, 5 mM MgCl ₂ , 5 mM UTP, 0.25 U galU and 1 U ppa, And reacted for 30 minutes. The reaction product, UDP-glucose, was analyzed by HPLC equipped with NH ₂ column (5 μm, 4.6 × 250 mm, aglient). The mobile phase was measured at 260 nm while flowing 67 mM Na ₂ HPO ₄ -KH ₂ PO ₄ (pH 5.3) at a flow rate of 1.5 ml / min. Enzyme units were defined as the amount of enzyme required to change 1 μmol substrate per minute (Table 2).

Escherichia coli K-12 substr. Activity and recovery rate of MG1655-derived malP according to purification steps Total vol. (ml) Total protein (mg) Total activity (U) Specific activity (U / mg) Purification
(Fold) Yield (%) Crude extract One 8.04 2.17 0.27 1.0 100 Ni-NTA affinity chromatography 0.1 0.64 0.29 0.46 1.70 13.56

(Ii) galU: The standard reaction solution was prepared by adding 50 mM phosphate (pH 7.5), 5 mM MgCl ₂ , 5 mM glucose-1-phosphate, 2 mM UTP and 1 mg of galU protein to 1 ml The reaction was carried out at 30 DEG C for 1 minute. The product, UDP-glucose, was analyzed by HPLC with NH ₂ column (Table 3).

Escherichia coli K-12 substr. Activity and recovery rate of MG1655-derived galU according to purification step Total vol. (ml) Total protein (mg) Total activity (U) Specific activity (U / mg) Purification
(Fold) Yield (%) Crude extract One 10.19 68.27 6.70 1.0 100 Ni-NTA affinity chromatography 0.1 2.80 27.38 9.77 1.46 40.10

(Iii) Standard reaction solution of ppa: ppa was prepared by adding 50 mM Tris-HCl (pH 7.5), 5 mM MgCl ₂ , 10 mM Na ₄ P ₂ O ₇ and 1 mg of ppa protein Followed by reaction at 30 ° C for 1 minute. After the reaction was completed, 20 μl of the reaction solution was added to 500 μl of a phosphorus-determining reagent solution (17% H ₂ SO ₄ : 2.5% Na ₂ MoO ₄ - (NH ₄ ) ₂ SO ₄ : 10% L-ascorbic acid: : 1: 1: 2), and 480 μl of H ₂ O was added thereto, followed by reaction at 45 ° C for 10 minutes. The product was identified by its absorbance at 660 nm (Table 4).

Escherichia coli K-12 substr. Activity and Recovery Rate of Purified Stage of pp16 from MG1655 Total vol. (ml) Total protein (mg) Total activity (U) Specific activity (U / mg) Purification
(Fold) Yield (%) Crude extract One 14.02 56.08 4.00 1.0 100 Ni-NTA affinity chromatography 0.1 4.91 28.53 5.81 1.45 50.87

(Iv) ackA: AckA protein was added to 50 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 10 mM ATP, 800 mM potassium acetate, and 700 mM neutralized hydroxylamine to make 1 ml of final ackA protein. Lt; / RTI > After completion of the reaction, 1 ml of 10% trichloroacetic acid and 1 ml of 1.25% FeCl ₃ dissolved in 1 N HCl are added and reacted at 30 ° C for 5 minutes. Hydroxamate acetate production was measured at an absorbance of 540 nm (Table 5).

Escherichia coli K-12 substr. Activity and Recovery Rate of Purified Stage of ackA Derived from MG1655 Total vol. (ml) Total protein (mg) Total activity (U) Specific activity (U / mg) Purification
(Fold) Yield (%) Crude extract One 12.35 1808.66 146.45 1.0 100 Ni-NTA affinity chromatography 0.1 3.89 869.61 223.55 1.53

Example  4: UDP - glucose Biosynthesis of

Escherichia < / RTI > prepared in Example 3 coli K-12 substr. The production of UDP-glucose was confirmed by reacting MG1655-derived enzymes with Maltodextrin and UTP. 10 μg malP enzyme solution, 10 μg galU, 15 μg ppa and 50 μg pyrH were added to 5% maltodextrin, 2 mM UMP, 2 mM ATP, 5 mM acetyl phosphate, 1 mM MgCl ₂ and 100 mM sodium phosphate buffer And allowed to react at 30 ° C for 6 hours. The reaction product was analyzed by using a NH ₂ column (5 μm, 4.6 × 250 mm, aglient) at 67 ° C. Na ₂ HPO ₄ -KH ₂ PO ₄ (pH 5.3) at a flow rate of 1.5 ml / min at 30 ° C. do. As a result, UDP-glucose was obtained at a yield of 99.9% (FIG. 6).

Example  5: UDP - glucose regeneration system Using Stevioside The party  reaction

For the efficient operation of the UDP-glucose regeneration system, the optimal reaction unit (U) of each enzyme was obtained. GalU, ppa, ackA, and pyrH were added to 5 mM maltodextrin, 2 mM UMP, 1 mM MgCl ₂ , 0.1 mg / ml stevioside, 5 mM acetyl phosphate and 100 mM sodium phosphate buffer , And the amount of UGT76G1 enzyme was changed to 1 ml in total, followed by reaction at 30 for 3 hours. The reaction was analyzed using HPLC equipped with ZORBAX NH ₂ (4.6 x 250 5 um, Agilent) column. In the mobile phase, 30% water and 70% acetonitrile were flown at a flow rate of 1 / min and analyzed under the condition of a column temperature of 30, and the absorbance was measured at 210 nm.

As a result, in the case of malP, galU and ppa, malP 80 mU, gal U 100 mU and ppa 50 mU were selected as the most efficient enzymatic reaction unit (U) for producing UDP-glucose. In case of ackA and pyrH, rebaudiosideA was produced When relative activity was measured, ackA 50 mU, pyrH 100 was selected as the most efficient enzyme reaction unit (U) (Fig. 7).

We investigated whether the UDP-glucose regeneration system produced rebaudioside A when stevioside was introduced into the reaction. The reaction conditions were maltodextrin, 2 mM UMP, 2 mM ATP, 1 mM MgCl ₂ , 100 mM sodium phosphate buffer (pH 7.5), 0.1 mg / ml stevioside, 2 mM acetyl phosphate, malP 80 mU, galU 100 mU, ppa 50 mU, UGT76G1 100 mU, ackA 50 mU, and pyrH 100 ㎍ were added to make 1 ml total, and then reacted at 30 ° C. Analysis of the reactants was performed using HPLC equipped with ZORBAX NH ₂ (4.6 x 250 5 um, Agilent) column. In the mobile phase, 30% water and 70% acetonitrile were flowed at a flow rate of 1 ml / min and analyzed under the conditions of a column temperature of 30 ° C. and absorbance was measured at 210 nm. As a result, it was confirmed that rebaudioside A was produced at a yield of 90% or more in 8 hours (FIG. 8).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for Preparing Natural High Intensity Sweetener Rebaudiside          A by Using Enzymatic Conversion <130> p13-b139 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 458 <212> PRT <213> Artificial Sequence <220> <223> Stevia rebaudiana UDP glycosyltransferase <400> 1 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile   1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu              20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr          35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg      50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro  65 70 75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His                  85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser             100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr         115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu     130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu                 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser             180 185 190 Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile         195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu     210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser                 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro             260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp         275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln     290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val                 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala             340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu         355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn     370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val                 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln             420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu         435 440 445 Glu Ser Leu Val Ser Tyr Ser Ser Leu     450 455

Claims (4)

A method for preparing rebaudioside A from stevioside and UDP-glucose in the presence of UDP-glycosyltransferase,
Maltodextrin phosphorylase, glucose-1-phophate thymidyltransferase, inorganic pyrophosphatase, acetate kinase, and glass delayed canine (Rebaudioside A) which is characterized in that UDP-glucose is regenerated by adding a substrate composed of uridylate kinase and a substrate composed of acetylphosphate, maltodextrin, UMP and ATP. How to.
The method of claim 1, wherein the UDP-glycosyltransferase is Stevia rebaudiana &Lt; / RTI &gt;
3. The method according to claim 2, wherein the UDP-glycosyltransferase E is encoded by the nucleotide sequence of SEQ ID NO: 1.
The method according to claim 1, wherein the maltodextrin phosphorylase, glucose-1-phophate thymidyltransferase, inorganic pyrophosphatase, acetate kinase, ) And uridylate kinase are Escherichia coli K-12 substr. RTI ID = 0.0 &gt; MG1655. &Lt; / RTI &gt;

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