KR20160107743A - METHOD FOR PREPARING STEVIOL FROM STEVIOGLYCOSIDE MIXTURE WITH HIGH YIELD USING β-GALACTOSIDASE - Google Patents

Abstract

The present invention relates to a process for preparing high yield steviol from a steviol glycoside mixture using? -Galactosidase. According to the present invention, since steviol is produced directly from a steviol glycoside mixture rather than a purified product such as stevioside, not only the production process of steviol can be simplified, but the production yield of steviol can be increased. Therefore, the process for producing steviol according to the present invention enables mass production of steviol on a commercial scale.

Description

METHOD FOR PREPARING STEVIOL FROM STEVIOGLYCOSIDE MIXTURE WITH HIGH YIELD USING < RTI ID = 0.0 > β-GALACTOSIDASE < / RTI > from a steviolglycoside mixture using?

The present invention relates to a process for preparing high yield steviol from a steviol glycoside mixture using? -Galactosidase.

In general, stevioside is a generic name of a compound in the Stevia rebaudiana plant that is about 200 times as sweet as sugar, more precisely steviol glycoside. Steviol glycosides contain stevioside, rebaudioside A, rebaudioside C, dulcoside and the like, and steviol is a compound that contains the sugar It is an aglycone.

Steviol can be used to enhance cognitive function (International Patent Application Publication No. 2009/071277), can be used to enhance hair growth (International Patent Application Publication No. 2011/009863), antimicrobial activity and plant growth And the like.

Various methods for producing steviol from stevioside have been attempted. Acid hydrolysis of stevioside is difficult to use due to the problem of rearrangement of steviol prepared under acidic conditions to isosteviol (Kohda et al 1976 Phytochemistry 15: 981-983), using sodium peridodate (Ogawa et al 1980; Tetrahedron 36: 2641-2648) require an excessively expensive sodium peridotate (greater than about 10 molar equivalents) and a highly diluted system to achieve useful yields It is not economical.

Recently, a patent for a method for producing steviol from stevioside using commercially available cytocholate PCL5 (International Patent Application Publication No. 2011/08903110, Korean Patent Publication No. 2012/0127443) is available. However, this enzyme is an enzyme prepared for the degradation of pectin, and it is estimated that a very small amount of enzymes other than pectinase, which is a main component, is involved in steviol formation. Therefore, The disadvantage is that a large amount of the enzyme product must be used for a long period of time, as indicated by 50% or more, preferably 90% or more by weight of the side. Korean Patent Registration No. 2012/0138757 discloses a method for producing steviol in which a glycoside moiety is separated from stevioside using Aspergillus oryzae to obtain a non-steric steviol, but a steviol glycoside mixture And steviol is produced after the stevioside is purified from the stevioside. Thus, there is a problem that the production yield of the steviol is less than expected. Therefore, there is a continuing need to simplify the manufacturing process of steviol and to mass-produce steviol efficiently by producing steviol at a high yield.

The present inventors have made intensive efforts to develop a method for economically mass-producing steviol directly from the steviol glycoside mixture. As a result, they have found that the steviol glycoside mixture has excellent conversion to steviol, By discovering a steviol-forming enzyme, the present invention has been completed.

The present invention provides a method for producing steviol using? -Galactosidase. The process for producing steviol according to the present invention comprises

A) preparing a steviol glycoside mixture;

B) reacting the steviolglycoside mixture with? -Galactosidase; And

C) recovering the steviol.

In the method for producing steviol according to the present invention, the? -Galactosidase may be derived from Sulfolobus solfataricus .

In the method for producing steviol according to the present invention, the? -Galactosidase may be prepared by a) culturing a recombinant microorganism to express? -Galactosidase; And b) recovering the expressed? -Galactosidase. The recombinant microorganism may be E. coli . The β-galactosidase can be recovered by salting out, centrifugation, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography or affinity chromatography.

In the process for preparing steviol according to the present invention, the step b) may be carried out at a temperature of from 70 캜 to 95 캜.

In the method for producing steviol according to the present invention, the steviol glycoside mixture may be used in an amount of 40 mg / ml to 90 mg / ml.

In the method for producing steviol according to the present invention, the? -Galactosidase may be used at an activity of 50 Unit / ml to 200 Unit / ml.

In the method for producing steviol according to the present invention, the amount of the steviol in the step (C) and the amount of the steviol glycoside mixture in the step (B), the activity of the? -Galactosidase, Can be established.

[Relation 1]

Y = + 26.65 - 0.97 x A + 4.34 x B - 0.38 x C + 0.62 x AB + 0.015 x AC + 1.41 x BC - 2.36 x A ² - 4.82 x B ² - 9.81 x C ²

Y, steviol production (mg / ml); A, steviol glycoside mixture (mg / ml); B, the activity of? -Galactosidase (U / ml); C, reaction temperature (캜).

In the process for producing steviol according to the present invention,

A) preparing a steviol glycoside mixture;

B) reacting the steviol glycoside mixture with 60 mg / ml of? -Galactosidase activity of 108 U / ml at 80 ° C; And

C) recovering a steviol of 25 mg / ml or more.

According to the present invention, since steviol is directly produced from a steviol glycoside mixture rather than a purified product such as stevioside, the production process of steviol can be simplified and the production yield of steviol can be increased. Therefore, the process for producing steviol according to the present invention enables mass production of steviol on a commercial scale.

FIG. 1 is a graph comparing the activity of expression enzymes according to the induction conditions of Example 1. FIG.
Fig. 2 shows the result of SDS-PAGE analysis of the enzyme according to Example 2. Fig.
(lane 1: protein marker; lane 2: stationary SSbgal)
Figure 3 shows the steviol production after steviol glycoside mixture and enzyme reaction.
Ste: Stevio seed (5%);
lane G: glucose (100 mM);
lane E: enzyme;
lane R: rubusoside,
lane S: steviol;
lane R: Enzyme activity of β-galactosidase 10% (v / v) reacted with 0.5-5% steviol glycoside at 8 ° C for 8 hours;
Lane M: steviol glycoside mixture;
lane 1, 6, 11, 16: 1% (v / v) β-galactosidase in the presence of 5% (w / v) steviol glycoside at 80 ° C for 0.5 h, 1 h, 2 h and 8 h;
lane 2, 7, 12, 17: 2.5% (v / v) β-galactosidase reacted with 5% (w / v) steviol glycoside at 80 ° C. for 0.5 h, 1 h, 2 h and 8 h;
lane 3, 8, 13, 18: 5% (v / v) β-galactosidase reacted with 5% (w / v) steviol glycoside at 80 ° C. for 0.5 h, 1 h, 2 h and 8 h;
lane 4, 9, 14, and 7.5% (v / v) β-galactosidase reacted with 5% (w / v) steviol glycoside at 80 ℃ for 0.5 h, 1 h, 2 h and 8 h;
10% (v / v) β-galactosidase reacted with 5% (w / v) steviol glycoside at 0.5 h, 1 h, 2 h,
Figure 3 shows the steviol glycoside mixture according to Example 2 and the steviol production result data after the enzyme reaction.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Various scientific events, including the terms contained herein, are well known and available in the art. Although any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing of the present application, some methods and materials have been described. Should not be construed as limiting the invention to the particular methods, protocols and reagents, as they may be used in various ways in accordance with the context in which those skilled in the art use them.

As used herein, the singular forms include plural objects unless the context clearly dictates otherwise. As used herein, unless otherwise stated, "or" means "and / or ". Moreover, it is to be understood that other forms, for example, "having," "being", and "consisting of" as well as the term "comprising"

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated. The subject matter provided herein should not be construed as limiting the following embodiments in various aspects or as a reference throughout the specification.

The present invention is to propose an enzymatic method of highly expressing a heat resistant? -Galactosidase gene in an E. coli system and producing steviol from a steviol glycoside mixture in high yield by using the? -Galactosidase.

According to one embodiment,

A) preparing a steviol glycoside mixture;

B) reacting the steviolglycoside mixture with? -Galactosidase; And

C) Step of recovering steviol

≪ RTI ID = 0.0 > steviol < / RTI >

As used herein, the term " stevia "refers to the perennial herbaceous perennial herbaceous perennial herbaceous perennials and perennial herbaceous perennials. The Stevia genus includes, for example, Stevia rebaudiana, Stevia eupatoria, Stevia ovata, Stevia plummerae, Stevia salicifolia, Stevia serrata. ≪ / RTI >

As used herein, the term " steviol glycoside "refers to a compound that is present in the leaves of Stevia and as a sweet substance. The steviol glycosides in the stevia leaves are, for example, stevioside, rebaudioside, rebaudioside A, rebaudioside C, dulcoside A, as well as rebaudioside, Side B, D, E, F, rubuososide, steviol monoside or steviol bioside.

The term " steviol "as used herein refers to an aglycone, excluding the sugar moiety, from steviol glycosides. The chemical structure of the steviol glycoside is as follows:

[Chemical Formula 1]

For details of the R group, see Table 1 below.

[Table 1]

As used herein, the term " beta-galactosidase "refers to a non-reducing terminal beta-D-galactosidase (beta -D-galactosidase) in beta-D- galactopyranoside, -D-galactose), or an enzyme that catalyzes the transfer reaction of galactose.

The β-galactosidase is characterized by being an enzyme having excellent heat resistance, which is isolated from Sulfolobus solfataricus . To obtain? -Galactosidase from the sulpalobase sulfatricus can be obtained by directly isolating or purifying? -Galactosidase from a strain or by expressing? -Galactosidase gene from a strain and expressing it in a recombinant expression vector and purifying it . The procedure for obtaining [beta] -galactosidase from Sulfolobus sulfatariksus is by conventional methods (Sambrook, J. and Russell, DW Molecular Cloning 3rd Ed., Cold Spring Harbor Laboratory, 2001).

According to one exemplary embodiment, the beta -galactosidase is

a) culturing the recombinant microorganism to express? -galactosidase; And

b) recovering the expressed? -galactosidase.

As used herein, the term "recombinant microorganism" means a recombinant microorganism in which a gene encoding a? -Galactosidase according to the present invention is inserted on a chromosome or transformed with the recombinant vector. The transformed recombinant microorganism is a host cell which has high efficiency of DNA introduction and high expression efficiency of the introduced DNA, and is all microorganisms including prokaryotic and eukaryotic cells. Bacteria, yeast, fungi and the like are available . In the examples of the present invention, E. coli was used, but it is not limited thereto, and any kind of microorganism can be used as long as the β-galactosidase can be sufficiently expressed.

As used herein, the term "transformation" refers to the introduction of DNA into a host to allow DNA to replicate either as a chromosomal factor or by chromosome integration completion, introducing foreign DNA into cells, . ≪ / RTI > For example, the transformation methods include electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection or lithium acetate-DMSO method no.

The recovery of the? -Galactosidase can be carried out by conventional biochemical separation techniques such as treatment with a protein precipitant (salting-out method), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration) , Adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like, but the present invention is not limited thereto.

According to this embodiment, the reaction of the steviol glycoside mixture with? -Galactosidase can be carried out at a temperature of from 70 ° C to 95 ° C, preferably at a temperature of 80 ° C. Within this temperature range, it was confirmed that the steviol glycoside mixture exhibits high reaction specificity to the? -Galactosidase enzyme. Also, the reaction of the steviol glycoside mixture according to the above embodiment with? -Galactosidase can be carried out at a pH of from 5.5 to pH 6.5, preferably at a pH of 6.0.

According to this embodiment, the steviol glycoside mixture may be used in an amount of 40 mg / ml to 90 mg / ml, preferably about 90 mg / ml as a substrate.

According to this embodiment,? -Galactosidase can be used at an activity of 50 Unit / ml to 200 Unit / ml, preferably about 100 Unit / ml.

According to the present invention, since steviol is directly produced from a steviol glycoside mixture rather than a purified product such as stevioside, the production process of steviol can be simplified and the production yield of steviol can be increased. Therefore, the process for producing steviol according to the present invention enables mass production of steviol on a commercial scale.

Hereinafter, various embodiments are provided to facilitate understanding of the present invention. The following examples are provided to facilitate understanding of the invention and are not intended to limit the scope of the invention.

<Examples>

Example 1: Expression of? -Galactosidase

Sulfolobus solfataricus β-galactoside (ssbgal) (amino acid residues 1-489, GenBank accession no. M34696) was cloned in the synthetic genes, and (GenScript, Piscataway, NJ, USA ), pET28a (Novagen, Darmstadt, Germany) polyhistidine The ssbgal enzyme, which has both a N-terminal and a C-terminal tag, was expressed using E. coli Rosetta BL21 (De3) pPlyS. A small amount of expression was induced by inoculating the seedlings into 100 mL of LB medium containing kanamycin (50 μg.ml ^-1 ) and inducing with 0.1 mM IPTG or 0.5 mM to 10 mM lactose at 37 ° C. Here, when OD ₆₀₀ was 0.5, the cultured seeds were added and incubation for induction was carried out for 16 hours. After incubation, the cells were suspended in 20 mM Bis-Tris buffer (pH 6.0), followed by sonication. After centrifugation (12,000 xg, 30 min), the cell lysate was reacted with 200 mM lactose for 30 min at 80 ° C. Large scale expression was achieved by incubating 0.5% (w / v) glycerol with carbon source at 37 ° C, 350 rpm, and an aeration rate of 2 vvm using a 7-L fermentor (Biotron, Gyeonggi-do, Korea) Respectively. When the OD ₆₀₀ reached 0.5, the induction for expression at the final 10 mM lactose concentration was initiated and proceeded to 24 h. After the expression, the cells were suspended in 20 mM Bis-Tris buffer (pH 6.0), and 10 mL of cell lysate was sonicated by sonication using a 10 mL Ni-Sepharose resin (GE Healthcare, Buckinghamshire, UK) Purification was carried out. At this time, elution was carried out with a buffer solution containing 20 mM Tris (pH 7.5), 0.3 M imidazole and 0.2 M NaCl. The fractions having enzyme activity were collected and dialyzed with 20 mM Bis-Tris buffer (pH 6.0). Next, the galactosidase activity was confirmed by ο-nitrophenyl β-D-galactopyranoside as a substrate. The production of ο-nitrophenol was determined at 410 nm, and the activity of galactosidase was determined based on this. The reaction was stopped with Na ₂ CO ₃ (1 M) at 40 ° C for 10 min at 80 ° C in 40 mM Bis-Tris buffer (pH 6.0), enzyme solution and ο-nitrophenyl β-D-galactopyranoside The galactosidase activity of one unit (U) was defined as the amount of enzyme producing 1 μM ο-nitrophenol per minute. The results are shown in Fig. The activity of the enzyme SSbgal enzyme produced by the fermenter was 26.5 U / mg. The final OD ₆₀₀ was 23 and was obtained at 18 h after induction. The purified SSbgal was obtained in a single Ni-Sepharose column chromatographic step and was obtained with a high yield of 92.26% (Table 1). Pure proteins were identified using SDS-PAGE analyses. The molecular weight was about 60 kDa. This is shown in FIG.

Step Purification process Total volume (ml) Enzym activity (U / ml) Total activity (U) Total Protein (mg) Specific activity (U / mg) Purification fold Yields (%) One Crude enzyme 11 58.35 641.85 26.45 24.27 One 100 2 Ni-NTA chromatography 6 98.7 592.2 6.86 86.33 3.56 92.26

Example 2: Steviol glycoside mixture and enzyme reaction and enzyme reaction product analysis

Recombinant SSbgal was eluted with acetonitrile: water [85:15 (v / v)] as a developing solvent in the presence of 10.0% (w / v) steviol glycoside mixture at 80 ° C and pH 6.0. -coated silica gel 60 F ₂₅₄ plates (Merck, Darmstadt, Germany). TLC plates were dipped in a methanol solution containing 5% (w / v) sulfuric acid and 0.5% (w / v) N- (1-Naphthy) ethylenediamine dihydrochloride and developed by baking at 120 ° C for 10 min. After the enzyme reaction, the supernatant was discarded at 12,000 xg for 10 min. After washing the pellet with water several times, the produced steviol was analyzed by TLC method using phosphomolybdic acid staining solubiton. [http://www.chemistry.mcmaster.ca/adronov/resources/Stains_for_Developing_TLC_Plates.pdf]. 15 mg of purified steviol was dissolved in DMSO-d6 and its structure was confirmed by NMR spectrum [Unity Inova 500 spectrometer (Varian, Palo Alto, CA, USA)]. This is shown in FIG.

As can be seen from FIG. 3, stevioside, rubucoside, steviol mono-glycoside, steviol monoglycosyl ester, steviolbiose, rebaudioside AF and dulcoside were identified as steviol glycoside mixtures .

Example 3 Optimization of Steviol Production Conditions

The steviol production conditions were determined according to steviol glycoside concentration (A), enzyme amount (B) and reaction temperature (C). Table 3 shows codes of three conditions for CCD (Central composite design).

Condition Symbol Code level -One 0 +1 Steviol glycosides (mg / ml) A 40 60 80 Enzyme (U / ml) B 72 108 144 Temperature (℃) C 70 80 90

Design expert 9.0.1.3 program was used. All conditions were evaluated based on steviol production. The results of steviol synthesis according to the conditions are shown in Table 3 below.

Run number A: SG mixture (mg / ml) B: enzyme (U / ml) C: temperature (占 폚) Steviol (mg / ml) Experimental Predicted One 60.00 108.00 80.00 26.60 26.66 2 93.64 108.00 80.00 22.85 17.61 3 40.00 144.00 90.00 17.02 16.52 4 80.00 72.00 90.00 1.10 1.43 5 60.00 108.00 80.00 26.58 26.66 6 40.00 72.00 90.00 4.22 4.54 7 40.00 72.00 70.00 11.27 7.76 8 60.00 108.00 80.00 27.60 26.66 9 60.00 108.00 80.00 26.02 26.66 10 80.00 144.00 90.00 10.59 14.39 11 40.00 144.00 70.00 12.63 13.08 12 60.00 168.54 80.00 21.83 21.66 13 80.00 144.00 70.00 11.93 12.69 14 60.00 108.00 63.18 0.00 -0.69 15 60.00 108.00 80.00 26.10 26.66 16 60.00 108.00 80.00 26.60 26.66 17 80.00 72.00 70.00 2.30 6.39 18 26.36 108.00 80.00 19.28 20.55 19 60.00 47.46 80.00 6.38 6.29 20 60.00 108.00 96.82 0 0

* Reaction pH - 6.0.

Based on the results of Table 3, the relation of steviol production according to the three reaction conditions was derived as follows.

[Relation 1]

Y = + 26.65 - 0.97 x A + 4.34 x B - 0.38 x C + 0.62 x AB + 0.015 x AC + 1.41 x BC - 2.36 x A ² - 4.82 x B ² - 9.81 x C ²

Y, steviol production (mg / ml); A, steviol glycoside mixture (mg / ml); B, enzyme activity (U / ml); C, reaction temperature (캜).

The maximum steviol production estimated from the above Relation 1 is 26.66 mg / ml, which is a reaction of 108 Unit SSbgal / ml and 60 mg / ml steviol glycoside mixture at a temperature of 80 ° C. This was confirmed to be very similar to the actual experimental value of 26.58 mg / ml.

Variables Optimum levels Stevia glycoside mixture (mg / ml) 60 Enzyme activity (U / ml) 108 Reaction temperature (캜) 80 reaction Estimate Experimental value Steviol (mg / ml) 26.66 26.58

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

A) preparing a steviol glycoside mixture;
B) reacting the steviolglycoside mixture with? -Galactosidase; And
C) Step of recovering steviol
&Lt; / RTI &gt;
The method according to claim 1,
Wherein the? -Galactosidase is derived from Sulfolobus solfataricus .
The method according to claim 1,
The? -Galactosidase
a) culturing the recombinant microorganism to express? -galactosidase; And
and b) recovering the expressed? -galactosidase.
The method of claim 3,
Wherein the recombinant microorganism is E. coli .
The method of claim 3,
The β-galactosidase is recovered through salting out, centrifugation, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography or affinity chromatography, &Lt; / RTI &gt;
The method according to claim 1,
Wherein step b) is carried out at a temperature of from 70 캜 to 95 캜.
The method according to claim 1,
Wherein said steviol glycoside mixture is used in an amount of from 40 mg / ml to 90 mg / ml.
The method according to claim 1,
Wherein the? -Galactosidase is used at an activity of 50 Unit / ml to 200 Unit / ml.
The method according to claim 1,
Wherein the following relationship is established between the amount of steviol in step (C) and the amount of steviol glycoside mixture in step (B), the activity of? -Galactosidase, and the reaction temperature.
[Relation 1]
Y = + 26.65 - 0.97 x A + 4.34 x B - 0.38 x C + 0.62 x AB + 0.015 x AC + 1.41 x BC - 2.36 x A ² - 4.82 x B ² - 9.81 x C ²
Y, steviol production (mg / ml); A, steviol glycoside mixture (mg / ml); B, the activity of? -Galactosidase (U / ml); C, reaction temperature (캜).
A) preparing a steviol glycoside mixture;
B) reacting the steviol glycoside mixture with 60 mg / ml of? -Galactosidase activity of 108 U / ml at 80 ° C; And
C) recovering steviol of 25 mg / ml or more
&Lt; / RTI &gt;

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