KR20230120591A - A method of UGT enzyme of thermostability or high efficiency for producing rebaudioside - Google Patents

Abstract

The present invention relates to a UGT enzyme (UDP-glycosyltransferase) variant with improved stability or conversion activity and a method for producing a UGT enzyme using the same. The UGT enzyme variant of the present invention stably can produce rebaudioside or significantly improve the production efficiency of rebaudioside A, to be able to effectively produce rebaudioside with excellent characteristics, and to be expected to have a wide range of industrial applications including food, feed, and medicine utilizing rebaudioside.

Description

Method for producing rebaudioside heat resistance or high efficiency UGT enzyme {A method of UGT enzyme of thermostability or high efficiency for producing rebaudioside}

The present invention relates to a UGT enzyme (UDP-glycosyltransferase) mutant having improved stability or conversion activity and a method for preparing the same.

As the World Health Organization (WHO) recommends lowering the daily sugar intake according to concerns about disease (obesity) caused by sugar intake, policies to reduce the intake of various sugars are actively discussed under the leadership of governments in developed countries. Accordingly, there is an increasing need for various alternative sweetener materials to replace sugar and high fructose in the market, and alternative sweetener materials are continuously being developed and commercialized.

Alternative sweeteners are continuously changing to synthetic high sweeteners (Saccharin, Aspartame, Sucralose, etc.), synthetic sugar alcohols (Maltitol, Xylitol), and high sweeteners (Rebaudioside A, Liquorice). However, customer needs for natural sweeteners continue to grow due to concerns over the safety of synthetic sweeteners. It is currently unable to do so.

In this regard, a natural high sweetener that has recently received considerable attention is a stevia sweetener extracted from the leaves of stevia. Stevia sweetener has high potential as an alternative sweetener as it is reported to have no calories, is positive for blood glucose and insulin levels, and has no side effects on the human body.

Meanwhile, stevia is an Asteraceae perennial plant native to Paraguay, South America, and its scientific name is Stevia rebaudiana Bertoni. Since stevia leaves contain components that are 200 to 300 times sweeter than sugar, these sweet components are extracted and used as natural sweeteners. The sweetening components of stevia extract include various steviol glycosides such as stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside M, rebaudioside I, and rebaudioside E. is included.

Stevioside has a strong sweet taste and almost no calories, so there are various methods for producing stevioside (KR 10-1995-0005996), but there is a disadvantage that the aftertaste is not clear and a bitter taste can be felt. Rebaudioside A is a diterpenoid-based compound having a molecular formula of C44H70O23 and a molecular weight of 967.01. Rebaudioside A or (4α)-13-[(2-O) It is named as -β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-oxy]kaur-6-en-8-oic acid β-D-glucopyranosyl ester. It can be isolated from the leaves of Stevia (Stevia rebaudiana Bertoni.) and is known to be soluble in solvents such as pyridine, methanol, and ethanol.

Uridine diphosphate (UDP)-glycosyltransferase (UGT) is an enzyme that transfers sugar from UDP-sugar to various metabolites such as hormones and secondary metabolites. UGT enzymes with high heat resistance have stability to high heat during processing reactions, resistance to chemical denaturants, high storage stability, increased reaction rate and catalytic activity, low viscosity of reaction mixtures, improved mass transfer during reactions, reduced risk of contamination in microbial enzyme production And it may have a resistance effect on organic solvents, but research on this is insufficient.

Under this background, the present inventors have completed enzyme variants with excellent heat resistance, reduced synthesis of Rebaudioside I and increased production efficiency of Rebaudioside A.

One object of the present invention is to provide a UGT enzyme (UDP-glycosyltransferase) mutant comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

Another object of the present invention is a polynucleotide encoding the UGT enzyme variant; an expression vector comprising the polynucleotide; and to provide a transformant into which the expression vector is introduced.

Another object of the present invention is to provide a composition for producing rebaudioside, comprising the UGT enzyme variant as an active ingredient.

Another object of the present invention is (a) culturing the transformant; and (b) isolating the UGT enzyme variant from the culture of the transformant.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention provides a UGT enzyme (UDP-glycosyltransferase) mutant comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

The term of the present invention, "UGT (UDP (Uridine diphosphate)-glycosyltransferase)" refers to transfer of a monosaccharide moiety from a glycosyl donor to a glycosyl acceptor molecule means an enzyme that has the activity of

In the present invention, the UGT enzyme has a glycosyltransfer activity (β-1,3 glycosyltransfer) or rebaudioside that converts stevioside to rebaudioside A as a substrate and rebaudioside A to rebaudioside I as a substrate. It may have glycotransfer activity to convert D to Rebaudioside M.

In one embodiment of the present invention, the UGT enzyme variant defined by the amino acid sequence of SEQ ID NO: 2 was named '76_7', and the UGT enzyme variant defined by the amino acid sequence of SEQ ID NO: 4 was named '76_4'.

As such, the UGT enzyme variant is not only the amino acid sequence described in SEQ ID NO: 2 or 4, but also 70% or more, specifically 80% or more, more specifically 90% or more, more specifically 95% or more, More specifically, amino acid sequences exhibiting a similarity of 98% or more, most specifically, 99% or more, including without limitation, any protein having substantially glycosyltransfer activity, and also having substantially the same or corresponding biological activity as the UGT enzyme variant. If it is an amino acid sequence having, it is obvious that a protein variant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added in the amino acid sequence described in SEQ ID NO: 2 or 4 is also included within the scope of the present invention.

As used herein, the term "similarity" refers to the percentage of identity between two polynucleotide or polypeptide moieties. Homology between sequences from one moiety to another can be determined by known art techniques. For example, homology can be determined by aligning the sequence information and directly aligning the sequence information between two polynucleotide molecules or two polypeptide molecules using a readily available computer program. The computer program may be BLAST (NCBI), CLC Main Workbench (CLC bio), MegAlignTM (DNASTAR Inc), etc., but any program capable of determining homology may be used without limitation. In addition, homology between polynucleotides can be determined by hybridizing polynucleotides under the condition of forming stable duplexes between homologous regions, then digesting the polynucleotides with a single-strand-specific nuclease to determine the size of the digested fragments, but is not limited thereto. no.

In the present invention, the enzyme variant comprising the amino acid sequence of SEQ ID NO: 2 may have heat resistance at 40 - 65°C, specifically 45 - 55°C, but is not limited thereto.

In the present invention, "heat resistance" refers to the property of exhibiting original activity without losing activity of an enzyme even in a high temperature environment, and the heat resistance of an enzyme has various advantages in the process of producing a target product. Specifically, resistance to organic solvents, improved thermal stability during reaction, resistance to chemical inhibitors, high storage stability, increased reaction rate and enzyme activity, low viscosity of reactants, improved mass transfer during reaction, reduced risk of contamination during microbial enzyme production, etc. can have an advantage.

In the present invention, the variant comprising the amino acid sequence of SEQ ID NO: 2 may have a higher conversion rate from Rebaudioside D to Rebaudioside M than that of the wild type, but is not limited thereto. .

In one embodiment of the present invention, the 76_7 enzyme model confirmed that the conversion rate from stevioside to rebaudioside A was about 50 times higher than that of the control group (FIG. 7, FIG. 9), and rebaudioside D to rebaudioside. It was also confirmed that the conversion rate to Side M was superior to that of the control group (FIG. 10).

In the present invention, the enzyme variant comprising the amino acid sequence of SEQ ID NO: 4 may have a conversion rate from stevioside to rebaudioside A, and specifically may have a higher conversion rate than the wild type, but is not limited thereto.

The term "Stevioside" of the present invention is a natural sweetener contained in the leaves of Stevia Rebaudiana Bertoni, and the main sweetening component is in the form of steviol glycosides. It is used as a low-calorie sweetener because its sweetness reaches 200 times that of sugar, and is known to have excellent stability compared to sugar. It has a feature that does not increase blood sugar, so it can be a sugar substitute for people with diabetes or obesity, but it has a bitter taste and an unpleasant aftertaste (aftertaste).

The term of the present invention, "Rebaudioside (Reb)" has the strongest sweet taste among steviol glycosides, the highest sweetness among the sweetness components of stevia, and excellent sweetness, and has a refreshing taste and less bitter taste. However, it is not economical because the amount of rebaudioside contained in the raw material is small.

Rebaudioside A can be produced by transferring sugar to stevioside, and rebaudioside I can be produced by transferring sugar to rebaudioside A. In addition, when a sugar is transferred to Rebaudioside D, Rebaudioside M can be produced. In the present invention, an enzyme variant comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 may have a glycosyltransferase activity.

The activity of the UGT enzyme mutant comprising the amino acid sequence of SEQ ID NO: 4 has a conversion rate (AUC of RebA / AUC of ST before reaction) from stevioside, which is a substrate, to rebaudioside A is about 50% or more, specifically 55% or more, more Specifically, it may be 60% or more. More specifically, the conversion rate may be in the range of 50% to 100% and in the range of 55% to 80%.

In one embodiment of the present invention, when the 76_4 enzyme variant was treated, it was confirmed that the conversion rate from stevioci to rebaudioside A was about 60% or more (FIG. 12). It was found that the conversion rate from stevioside to I and the conversion rate from rebaudioside A to rebaudioside I were significantly reduced, indicating that rebaudioside A could be produced from stevioside with high efficiency (FIGS. 13 and 14). ).

Another aspect of the present invention is a polynucleotide encoding the UGT enzyme variant; an expression vector comprising the polynucleotide; and a transformant into which the expression vector is introduced.

The "UGT" is as described above.

The polynucleotide encoding the UGT enzyme variant may consist of any one nucleotide sequence selected from the nucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 5, but not only the nucleotide sequence described in SEQ ID NO: 3 or SEQ ID NO: 5, but also the sequence and 80 % or more, specifically 90% or more, more specifically 95% or more, more specifically 97% or more homology, as long as it is a nucleotide sequence capable of encoding a protein having substantially glycosyltransferase activity may be included without

An expression vector containing the polynucleotide of the present invention is an expression vector capable of expressing a target protein in a suitable host cell, and refers to a nucleic acid construct comprising essential regulatory elements operably linked to express a nucleic acid insert. Target proteins can be obtained by transforming or transfecting the prepared recombinant vector into a host cell.

Expression vectors containing the polynucleotides provided in the present invention are not particularly limited thereto, but E. coli-derived plasmids (pMAL, pYG601BR322, pBR325, pUC118 and PUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5), Yeast-derived plasmids (YEp13, YEp24 and YCp50) and Ti-plasmids usable for Agrobacterium mediated transformation. Specific examples of phage DNA include λ-phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus, double-stranded plant viruses (eg CaMV), single-stranded viruses Alternatively, viral vectors derived from gemini viruses may also be used.

In addition, as the vector of the present invention, a fusion plasmid (eg, pJG4-5) to which a nucleic acid expression activating protein (eg, B42) is linked may be used. In addition, in order to facilitate the purification of the target protein recovered in the present invention, the plasmid vector may additionally include other sequences, if necessary. The fusion plasmid may include MBP, GST, GFP, His-tag, Myc-tag, etc. as a tag, but is not limited by the above examples.

In addition, in the case of a fusion protein expressed by a vector containing the fusion sequence, it can be purified by affinity chromatography. For example, when MBP (maltose-binding protein) is used, an amylose resin column can be used, and when hexahistidine is used, a desired target protein can be easily recovered using a Ni-NTA His-binding resin column. .

In order to insert the polynucleotide of the present invention into a vector, a method of cutting the purified DNA with an appropriate restriction enzyme and inserting it into an appropriate restriction site or cloning site of the vector DNA can be used.

In the present invention, the term "transformant" refers to a cell or microorganism that is mutated to express a target protein after a polynucleotide encoding a target protein is introduced into a host cell using an expression vector. Specifically, the transformant may be a recombinant E. coli expressing the UGT enzyme.

Any transformation method can be used for the transformation method of the present invention, and can be easily performed according to a conventional method in the art. In general, transformation methods include the CaCl2 precipitation method, the Hanahan method with increased efficiency by using a reducing material called DMSO (dimethyl sulfoxide) in the CaCl2 method, the electroporation method, the calcium phosphate precipitation method, the protoplast fusion method, and the silicon carbide fiber. agitation method using agitation method, agrobacteria-mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine, and desiccation/inhibition-mediated transformation method. Methods for transforming a vector containing a polynucleotide encoding a UGT enzyme variant of the present invention are not limited to the above examples, and transformation or transfection methods commonly used in the art may be used without limitation.

Another aspect of the present invention provides a composition for producing rebaudioside comprising the UGT enzyme variant.

The "UGT" and "rebaudioside" are as described above.

The rebaudioside may be rebaudioside A, rebaudioside I, or rebaudioside M, but is not limited thereto.

In addition, the composition for producing rebaudioside of the present invention may further include any suitable excipient commonly used in the composition for producing rebaudioside. Such an excipient may be, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent, but is not limited thereto.

The composition may further include stevioside, rebaudioside A or rebaudioside D as a substrate, but is not limited thereto.

Another aspect of the present invention is (a) culturing the transformant; and (b) isolating the UGT enzyme variant from the culture of the transformant.

The "UGT" and "transformant" are as described above.

In the present invention, the term "culture" is a material obtained as a result of culturing a strain, and may include all substances secreted from a medium, a cultured strain, and a cultured microorganism. For example, inorganic salt components, amino acids, vitamins, nucleic acids and / or other components that can be generally contained in a culture medium may be included in addition to nutrient sources necessary for culturing cells, such as carbon sources and nitrogen sources.

Another aspect of the present invention is a step comprising reacting a UGT enzyme variant or a transformant expressing the same with stevioside, rebaudioside A or rebaudioside D; including, rebaudioside production methods are provided.

Recovering rebaudioside from the reaction product of the UGT enzyme and stevioside, rebaudioside A or rebaudioside D; may be further included.

The "UGT", "stevioside", and "rebaudioside" are as described above.

In the present invention, the reaction may be caused by a UGT enzyme mutant or a transformant expressing the same.

The glycosyltransfer reaction of the UGT enzyme variant is carried out at pH 6 to 9, specifically at pH 6.5 to 8.5, more specifically at pH 7 to pH 8 for 10 hours to 25 hours, specifically 13 hours to 22 hours, more specifically 15 hours to 25 hours. It may be carried out by reacting for 20 hours.

In the present invention, since the UGT enzyme variant can stably produce rebaudioside even at high temperature or significantly improve the efficiency of rebaudioside A production, it can effectively produce rebaudioside with excellent properties, It can be expected to be used in a wide range of industries such as food, feed, and medicine that utilize side.

1 shows a UGT (UDP-glycosyltransferases) enzyme model selection process.
Figure 2 is the result of confirming the final purity of the selected UGT enzyme variant.
Figure 3 is the result of confirming the amount of reactant change, product change amount, Rebaudioside A production amount, and Rebaudioside I production amount of WT and the selected 9 enzyme models.
Figure 4 is the result of confirming the conversion ratio from stevioside to rebaudioside A of the WT and the selected 9 enzyme models, and the glycosyltransferase activity (B) of the enzyme model compared to the WT.
Figure 5 is the result of confirming the conversion ratio from stevioside to rebaudioside A, from rebaudioside A to rebaudioside I, and the glycosyltransferase activity of the enzyme model compared to WT in the WT and nine selected enzyme models. am.
6 shows the results of comparison of modified midpoint (Tm) (A) and comparison of Tm change compared to WT UGT (B).
7 is a result of confirming the conversion rate from stevioside to rebaudioside A at each temperature.
8 is a result of confirming the conversion rate (total glycosyltransferase activity) from stevioside to rebaudioside A and from rebaudioside A to rebaudioside I at different temperatures.
9 is a result of confirming the conversion rate from stevioside to rebaudioside A.
10 is a result of confirming the conversion rate from Rebaudioside D to Rebaudioside M.
11 is a picture showing a self-produced Python script in the present invention.
12 is a result of confirming the conversion rate from stevioside to rebaudioside A.
13 is a result of confirming the conversion rate from Rebaudioside A to Rebaudioside I.
14 is a result of confirming the conversion ratio from Rebaudioside A to Rebaudioside I in a single reaction.
15 is a result of confirming the conversion ratio from Rebaudioside A to Rebaudioside I in a single reaction.
16 is a result showing a reaction kinetics graph and Kcat/Km values (Assay kit).

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Experimental Example 1. UGT (UDP-glycosyltransferases) enzyme model design

After finding 30 homologous structures with UGT76G1 homology using Phyre2, the mutant sequences were listed, multiple sequence comparisons and sequence matching mutations were identified, and then 41 constraint residues (restriction residues) were selected. After that, residues were selected by coding a residue information file and a limit value file for modeling with Rosetta. Then, by repeating the process of re-packing and minimizing the UGT76G1 structure using Rosetta script coding and randomly selecting the redesigned residues, recombination mutation combinations were calculated and filtered. After selecting 10 models with the highest scores among them, mutation sites were identified in the redesigned structure (FIG. 1, Table 1). 76_10 is insoluble, and after selecting 9 types that can be purified, purification was performed.

Experimental Example 2. Recombinant expression vector construction and UGT enzyme purification

The pMal_Tev_LL vector made by modifying the pMal_c2 vector was used as a protein expression vector, and the pMal_Tev_LL vector had 10x His tag, MBP tag, Long linker, Tev protease recognition and cleavage site from the N-terminus behind the promoter, and multiple restriction sites (Multiple cloning). site) is a vector. Specifically, pMal_Tev_LL vector was digested with EcoRI/SalI restriction enzyme to make a linear vector, electrophoresed with 1% agarose gel, and then purified with a gel extraction kit. Then, to construct DNA to be inserted into the backbone vector (pMal_Tev_LL), the natural UGT enzyme and the designed protein sequences from 76_1 to 76_10 were optimized for E. coli codons. It was designed to include the EcoRI/SalI restriction enzyme sequence present in the backbone vector and 15 additional sequences at the 5' and 3' ends of the optimized sequence, and then synthesized by DNA oligomer synthesis service. Then, the prepared backbone vector and insert DNA were recombined with an Infusion-based subcloning kit, and then subcloned through transformation, colony isolation, and sequencing in E. coli, and MalEF primers were used for sequencing. The subcloned vector expresses 10x His tag - MBP tag - Long linker - Tev cleavage site (ENYLQG) - (EcoRI) - insert DNA Construct - (SalI).

The prepared protein expression vector was transformed into E.Coli, cultured in 3L LB, 37°C until the O.D. Thereafter, E.coli was centrifuged, resuspended in a lysis buffer, ultrasonically pulverized, and supernatant and insoluble pellets were separated by ultra-high-speed centrifugation after pulverization. The obtained supernatant was first purified using a Histrap HP 5ml column connected to FPLC to perform affinity chromatography. At this time, the target protein can be effectively separated because the His tag is present. Then, further separation and purification were performed using ion exchange chromatography (Resource Q 6ml, Anion exchange). After concentrating the purified UGT enzyme variant, the buffer was exchanged with Tris-HCl pH 7.5, 100 mM NaCl buffer, filtered through a 0.2 um syringe filter, the concentration was adjusted to 1 mg/ml, and Coomassie blue staining was performed after SDS-PAGE. and bleaching, and comparing the protein bands on the acrylamide gel to confirm the final purity of the purified UGT.

Experimental Example 3. Enzyme Reaction Conditions and Sample Preparation

Both stevioside and rebaudioside A, samples to be used for the enzyme reaction, were dissolved in 100% DMSO of HPLC grade to be 30mM, respectively, and UDP-Glucose was dissolved in 3rd water to prepare 100mM. At this time, 30mM Stevioside: 100mM UDP-Glucose: 3 order = 1: 1: 8 to make a 10x Substrate mix (3mM Stevioside, 10mM UDP-Glucose). The 4x Salt mix was made to have 100 mM NaCl and 20 mM Tris-HCl pH 7.5 at the time of final reaction using 400 mM NaCl and 80 mM Tris-HCl pH 7.5.

For the UGT enzyme reaction, mix 10x substrate mix 10ul, 10uM UGT 10ul, 4x Salt mix 25ul, and DDW 55ul to make the final volume 100ul, put it in a 0.3ml PCR tube, and incubate in an incubator set at 37 degrees, 45 degrees, and 55 degrees for 18 hours. It was reacted for a while (after making 4 times the amount, it was distributed by 100ul). To terminate the enzyme reaction, 100 ul of water-saturated n-butanol of the same volume as the reaction solution was mixed and centrifuged to separate the upper butanol layer and the lower aqueous layer. After removing the lower aqueous layer by pipetting, the butanol layer was taken and filtered through a 0.2um syringe filter, transferred to a 500ul glass bottle for HPLC, and the inlet was sealed with a crimp seal.

Experimental Example 4. Analysis of enzyme reaction products using HPLC

To prepare a mobile phase to be used in HPLC, HPLC grade 100% acetonitrile (ACN) and 10 mM sodium phosphate pH 2.7 were prepared. The mobile phase was 100% ACN: 10 mM sodium phosphate pH 2.7 = 50:50, which was prepared by mixing 396.2 g of ACN in 500 g of sodium phosphate buffer and then removing residual air by ultrasonic waves.

Specifically, Shimadzu's UPLC (Nexera XR) was used for HPLC analysis, and Sigma-Aldrich's Acclaim® Mixed- Mode WAX-1 Columns (4.6mmx150mm, 5μm) were used. Before, between, and after sample analysis, more than 15 ml of the mobile phase was flowed, and the baseline was analyzed in a stabilized state. The total analysis time was 25 minutes, the flow rate was 1.0 ml/min, the oven temperature was 40 degrees, the detection wavelength was 210 nm, and the injection volume was 5ul (Table 2). Before analyzing the reaction sample, stevioside, rebaudioside A, and the reaction product without enzyme (Control) were analyzed, and the retention time of each compound peak and the area under the curve (AUC) of the peak were determined. measured.

Experimental Example 5. Analysis of enzyme reaction products using a glycosyltransfer activity assay kit

For glycosyltransferase function confirmation (Glycosyltransferase assay), Promega's UDP-Glo™ Glycosyltransferase assay kit was used. This kit is specialized for measuring glycosyltransfer reactions and can quantitatively analyze the product by increasing the luminescence intensity according to the amount of UDP present in the sample. After converting UDP present in the sample to ATP, luciferase consumes ATP and generates a luminescence signal, so ATP-free water was used for all distilled water when using the kit. After preparing by putting 20ul of the reagent (UDP Detection Reagent, UDR) that causes a luminescence reaction according to the concentration of UDP in a 96-well white plate, put 20ul of the reaction sample to be measured and leave the plate at room temperature for 1 hour to stabilize the luminescence reaction. was repeated twice with a VICTOR™ X3 multiplate reader (Perkin Elmer). When UDP was added at 25uM to 0uM, a standard curve of UDP was calculated according to the degree of luminescence, and the amount of UDP corresponding to the luminescence intensity of the sample was quantitatively calculated through linear regression.

Experimental Example 6. Measurement of enzyme reaction kinetics

UDP-Glo™ Glycosyltransferase assay kit was used to compare the activity of natural UGT and enzyme model based on enzyme reaction kinetics. 50mg of stevioside hydrate was dissolved in 200ul of DMSO to prepare 30mM, and 61.03mg of UDP-Glucose was dissolved in 1ml of ATP-Free water to prepare 100mM. At this time, UDP-Glucose was set to 1 mM in all conditions during the reaction, and the final concentration of stevioside was 300uM, 200uM, 100uM, 50uM, 20uM, 10uM, 0uM, and natural UGT and 76_4 The reaction rate of was measured. After preparing by putting 20ul of UDR according to the concentration of UDP in each well, 20ul of the enzyme reaction was taken and mixed with UDR every 30 seconds after the start of the reaction (30 seconds, 1 minute, 1 minute 30 seconds, 2 minutes). In order to reduce the experimental error, the measurement was repeated 3 times. After mixing with UDR, the plate was left at room temperature for 1 hour and the luminescence reaction was measured twice with a VICTOR™ X3 multiplate reader (Perkin Elmer) in a stable state. The reaction of UGT76G1 was measured by shortening the reaction time since the sugar transfer reaction of converting stevioside to rebaudioside A was followed by the reaction of generating rebaudioside I from rebaudioside A (Table 3). In the last row, 25uM~0uM UDP-Glucose was put in to calculate the standard curve of UDP according to the degree of light emission. Based on the standard curve according to the concentration of UDP, the amount of light emitted in each well was converted into the amount of UDP, and then the Michaelis-Menten equation ( Kcat/Km was calculated according to Vo = (Vmax*[S])/(Km + [S])).

Example 1. Confirmation of Rebaudioside Production Efficiency of Enzyme Variants

Example 1-1. Confirmation of rebaudioside production efficiency of enzyme variants

The amount of reactant change, product change amount, rebaudioside A (RebA) production amount, and rebaudioside I (RebI) production amount of the WT (control group) and the selected 9 enzyme models were measured using the HPLC method of Experimental Example 4. 1uM UGT, 0.3mM Stevioside (ST) and 1mM UDPG (UDP-glucose) were reacted at 37°C for 18 hours as substrates, and 100mM NaCl and 20mM Tris-HCl pH 7.5 were used as buffers. The reactant change (ΔST/STo) is the AUC (area under the curve) of ST after the reaction/ AUC of ST before the reaction, the product change (ΔProduct/STo) is (AUC of Reb A + AUC of Reb I/AUC of initial ST), RebA production was calculated as AUC of the produced RebA/AUC of ST before reaction, and RebI production was calculated as AUC of produced RebI/AUC of ST before reaction (FIG. 3).

As a result, it was confirmed that 3 of the 9 enzyme models showed high changes in reactants and products compared to the control group, and among them, 76_4 and 76_7 showed excellent production efficiency.

Example 1-2. Stevioside (Stevioside) to Rebaudioside A (Rebaudioside A, RebA) conversion ratio and glycosyltransferase function confirmation (Glycosyltransferase assay)

In order to confirm the conversion rate of stevioside to rebaudioside A (RebA) and the functions of UDP-glycosyltransferases (UGTs) in the WT and the selected 9 enzyme models, the HPLC method of Experimental Example 4 was used. did 1uM UGT, 0.3mM stevioside and 1mM UDPG (UDP-glucose) were reacted at 37°C for 18 hours as substrates, and 100mM NaCl and 20mM Tris-HCl pH 7.5 were used as buffers. The conversion rate was calculated as the AUC of the generated RebA/AUC of ST before reaction, and the fold was calculated as the conversion rate of UGT to RebA/wT conversion rate (FIG. 4).

As a result, it was confirmed that 4 of the 9 enzyme models had a higher conversion rate than the control group, and among them, 76_4, 76_6 and 76_7 were converted from stevioside to rebaudioside A by about 2 to 2.6 times more than the control group. It was confirmed that the ratio was high.

Example 1-3. Confirmation of conversion ratio from stevioside to Rebaudioside A and Rebaudioside I (Rebaudioside Ⅰ, RebI) and function of glycosyltransferase

In order to confirm the rate of conversion from stevioside to rebaudioside A, from rebaudioside A to rebaudioside I and the glycosyltransferase function of WT and the selected 9 enzyme models, Experimental Example 4. HPLC method was used. 1uM UGT, 0.3mM Stevioside and 1mM UDPG were reacted at 37°C for 18 hours as substrates, and 100mM NaCl and 20mM Tris-HCl pH 7.5 were used as buffers. The conversion rate was calculated as the sum of the AUC of RebA and AUC of RebI/AUC of ST before reaction, and the fold was calculated as the sum of the conversion rates of the designed UGT to RebA and RebI/conversion of WT (FIG. 5).

As a result, it was confirmed that 76_4, 76_6, and 76_7 among the nine enzyme models had higher conversion rates than the control group. It was confirmed that the conversion rate to Side I was high.

In the following, 76_4, 76_6, and 76_7 enzyme models having excellent conversion rates to Rebaudioside A or Rebaudioside I and glycosyltransferase functions confirmed through Examples 1-2 were selected for stability at high temperatures (heat-resistant reaction). ) or an enzyme model with excellent rebaudioside A production efficiency was selected.

Example 2. Selection and evaluation of enzyme variants with enhanced glycosyltransferase function by heat resistance and temperature

Example 2-1. Determination of thermostable (Tm) of enzyme variants

WT and Example 1. Circular dichroism at the Korea Basic Science Institute (KBSI) to confirm the heat resistance of the three designed UGTs (76_4, 76_6, 76_7), which were excellent in glycosyltransferase function as a result of the experiment. ) Melting temperature (Tm) analysis was performed in the measurement mode. Specifically, after preparing 200ul of each enzyme model at a concentration of 0.5mg/ml, the temperature was increased by 0.1℃ every 5 seconds from 20℃ to 95℃ using JASCO J-1500 CD measuring equipment, and the enzyme protein secondary at 222nm Structural changes were measured (FIG. 6).

As a result, the Tm of WT was 59.9 ° C, but the 76_4, 76_6, and 76_7 enzyme models were about 63 ° C to 70 ° C, which increased by about 3 ° C to a maximum of 9.3 ° C compared to WT.

Through this, it was found that the 76_4, 76_6, and 76_7 enzyme models had excellent structural stability and high heat resistance.

Example 2-2. Check the conversion rate of stevioside to rebaudioside A by temperature

When the temperature was raised from 37 ° C to 45 ° C and 55 ° C, in order to confirm the conversion rate of stevioside to rebaudioside A (glycosyltransferase function), the HPLC method of Experimental Example 4 was used. did 1 uM UGT (76_4, 76_6, 76_7), 0.3 mM stevioside and 1 mM UDPG were reacted for 18 hours as substrates, and 100 mM NaCl and 20 mM Tris-HCl pH 7.5 were used as buffers. The fold was the conversion rate to RebA/WT conversion rate of the designed UGT (76_4, 76_6, 76_7), and the conversion rate was calculated as AUC of the produced RebA/AUC of ST before reaction (FIG. 7).

As a result, as the temperature increased, the WT, 76_4, and 76_6 enzyme models showed a decrease in rebaudioside A production, while the 76_7 enzyme model increased the temperature to 45°C and 55°C, and the production of rebaudioside A decreased. It was confirmed that 76_7 increased by about 52 times or more compared to WT.

Through this, it was found that the 76_7 enzyme model maintains stability even at high temperatures and has excellent glycosylation.

Example 2-3. Check the conversion rate from stevioside to rebaudioside A and rebaudioside I by temperature

When the temperature is raised from 37 °C to 45 °C and 55 °C, the conversion rate of stevioside to rebaudioside A and rebaudioside A back to rebaudioside I (total glycosyltransferase activity ), the HPLC method of Experimental Example 4 was used. 1 uM UGT (76_4, 76_6, 76_7), 0.3 mM stevioside and 1 mM UDPG were reacted for 18 hours as substrates, and 100 mM NaCl and 20 mM Tris-HCl pH 7.5 were used as buffers. The fold was calculated as the conversion rate of the designed UGT (76_4, 76_6, 76_7) / conversion rate of WT, and the conversion rate was calculated as the sum of the AUC of RebA and AUC of RebI / AUC of ST before reaction (FIG. 8).

As a result, as the temperature increased, the total glycosyltransferase activity (rebaudioside A and rebaudioside I production) decreased in the WT, 76_4, and 76_6 enzyme models, whereas in the 76_7 enzyme model, When the temperature was raised to °C, the production of Rebaudioside I increased, and the ratio of conversion from stevioside to Rebaudioside A and Rebaudioside I (total glycosyltransferase activity) was also higher than that of 76_7 WT. It was confirmed that it increased by about 71 times or more.

Through this, it was found that the 76_7 enzyme model maintains stability even at high temperatures and has excellent glycosyltransfer activity.

Example 2-4. Check the conversion rate of stevioside to rebaudioside A at high temperature

In order to clearly confirm the production of Rebaudioside A (conversion rate, glycosyltransferase function) from stevioside at 55 ° C, the 76_7 enzyme model, which maintained stability at high temperature and had the best glycosyltransfer activity, was tested in Experimental Example 4. Experiments were conducted using the HPLC method of Specifically, reactants (100mM NaCl, 20mM Tris-HCl pH 7.5, 0.3mM ST, 1mM UDPG, n-butanol) excluding the enzyme were set as Control, and the native UGT enzyme was set as WT UGT, and then 1uM UGT (Control) was used as the substrate. , WT UGT, 76_7), 0.3 mM stevioside and 1 mM UDPG were reacted at 37° C. for 18 hours, and 100 mM NaCl and 20 mM Tris-HCl pH 7.5 were used as buffers. Glycotransfer activity was calculated as AUC of the produced RebA/AUC of ST before reaction (FIG. 9).

As a result, it was confirmed that the production of Rebaudioside A was low in the control group and WT, whereas the production of 76_7 of the present invention was about 54 times higher than that of WT.

Through this, it was found that the 76_7 enzyme model maintains stability even at high temperatures, significantly increasing the production of Rebaudioside A.

Example 2-5. Determination of conversion rate from Rebaudioside D to Rebaudioside M

UDP-Glo™ Glycosyltransferase assay kit of Experimental Example 5 was used to confirm the rate of conversion from Rebaudioside D to Rebaudioside M (RebD→RebM) in the conversion reaction to Rebaudioside M. . 1uM UGT (76_4, 76_7), 0.5mM Rebaudioside D and 1mM UDPG were reacted at 37°C for 18 hours as substrates, and 100mM NaCl and 20mM Tris-HCl pH 7.5 were used as buffers. fold is the UDP production amount of the enzyme model / UDP production amount of WT, and UDP (uM) was calculated by converting the luminescence (RLU) measured by the UDP Glo™ kit according to the UDP standard curve (FIG. 10).

As a result, it was confirmed that 76_7 had excellent glycosyltransfer activity from Rebaudioside D to Rebaudioside M compared to WT.

Through this, it was found that the 76_7 enzyme model can be used for the reaction from Rebaudioside D to Rebaudioside M while maintaining a more stable enzyme reaction through improved heat resistance.

Example 2-6. Sequence analysis of selected enzyme variants

The selected 76_7 enzyme model was named UGT76G1-T (UGT76G1 Thermostable), and the sequence of the UGT76G1-T enzyme variant was analyzed.

Specifically, the percentage of the designed enzyme was first calculated using a multi-sequence comparison service such as Clustal Omega from the natural enzyme. Subsequently, as shown in FIG. 11, mutation information was extracted using the self-produced Python script of the present invention by using a natural type amino acid and a mutated amino acid sequence as a set (Table 4).

Name Mutation ratio Mutation sites 76_7 SEQ ID NO: 2 6.33%
(11+18 residues) Δ1-11(deletion), I29M / V35I / A111N / S119A / R140P / N151S / G181E / S192K / G205D / L246F / Q266S / S274N / R297W / L329P / G348E / L379G / L392W / V429C

It was confirmed that the amino acid sequence of 76_7 was a sequence (SEQ ID NO: 2) in which the N-terminal amino acid sequence of WT (UGT76G1, SEQ ID NO: 1) was removed and 18 amino acids were substituted.

Example 3. Selection and Evaluation of Enzyme Variants with Enhanced Rebaudioside A Production Efficiency

Example 3-1. Determination of conversion rate from stevioside to rebaudioside A

The HPLC method of Experimental Example 4 was used to confirm the amount of Rebaudioside A production (converted ratio, glycosyltransferase function) in stevioside. Specifically, 1uM UGT (Control, WT UGT, 76_4), 0.3mM Stevioside, and 1mM UDPG were reacted at 37° C. for 18 hours as substrates, and 100mM NaCl and 20mM Tris-HCl pH 7.5 were used as buffers. Glycotransfer activity was calculated as AUC of the produced RebA/AUC of ST before reaction (FIG. 12).

As a result, it was confirmed that 76_4 increased the production of Rebaudioside A by about 2.6 times or more compared to WT.

Example 3-2. Check the conversion rate from Stevioside to Rebaudioside A and Rebaudioside I

Experimental Example to confirm the rate of conversion to Rebaudioside I in the conversion reaction from Stevioside to Rebaudioside A and from Rebaudioside A to Rebaudioside I (ST → RebA → RebI) The HPLC method of 4. was used. 1uM UGT (76_4, 76_7), 0.3mM Stevioside and 1mM UDPG were reacted at 37°C for 18 hours as substrates, and 100mM NaCl and 20mM Tris-HCl pH 7.5 were used as buffers. The fold is the conversion rate of UGT to RebI (76_4, 76_7) / conversion rate of WT, and the conversion rate (the rate of conversion from ST to RebI via RebA) was calculated as AUC of RebI produced / AUC of ST before reaction (FIG. 13) .

As a result, in 76_4 (ST → RebA → RebI), the production of Rebaudioside I produced from stevioside decreased by more than 72% compared to WT.

Example 3-3. Determination of conversion rate from Rebaudioside A to Rebaudioside I

In order to more accurately confirm the rate of conversion from Rebaudioside A to Rebaudioside I (RebA→ RebI) in a single reaction, the HPLC method of Experimental Example 4 and the UDP Glo™ Glycosyltransferase assay kit of Experimental Example 5 were used. used

Specifically, the HPLC method was reacted with 1uM UGT (76_4, 76_7), 0.3mM Rebaudioside A and 1mM UDPG, 37 ° C as substrates for 18 hours, and used 100mM NaCl and 20mM Tris-HCl pH 7.5 as buffers. . The fold is the RebI conversion rate of the designed UGT (76_4, 76_7)/ the RebI conversion rate of WT, and the conversion rate (the ratio of RebA converted to RebI) is (RebA AUC-initial RebA AUC)/ initial RebA (control) AUC calculated (FIG. 14).

As a result of the HPLC analysis, since the Reb I and noise peaks overlap, cross-validation was performed using other experimental methods for a clear analysis. Specifically, the conversion reaction from RebA to RebI was measured again using UDP Glo™ Glycosyltransferase assay kit. At this time, 1uM UGT (76_4, 76_7), 0.3mM Rebaudioside A and 1mM UDPG were reacted at 37 °C for 18 hours as substrates, and 100mM NaCl and 20mM Tris-HCl pH 7.5 were used as buffers. UDP (uM) was calculated by converting luminescence (RLU) measured by UDP Glo kit™ according to the UDP standard curve. (FIG. 15).

As a result, it was confirmed that the 76_7 enzyme model increased the conversion rate from Rebaudioside A to Rebadioside I compared to the WT control group, but in contrast, the 76_4 enzyme model showed about 72% conversion rate from Rebaudioside A compared to WT, UDP When measured using the Glo™ kit, it was confirmed that the reduction was about 92% or more. Even when the ratio of conversion from RebA to RebI was measured using different experimental methods, a consistent tendency was shown.

Through this, it was found that the 76_4 enzyme model can produce rebaudioside A from stevioside with high efficiency by reducing the synthesis of rebaudioside I, a byproduct, and increasing the production efficiency of rebaudioside A. .

Example 3-4. Analysis of reaction characteristics of enzymes according to stevioside concentration

Enzyme kinetics experiments were performed to analyze the reaction characteristics of the enzyme of 76_4. Enzyme reaction rates for each concentration of various stevioside substrates were measured using the UDP-Glo™ Glycosyltransferase assay kit of Experimental Example 6. 0.165uM UGT as a substrate, 0 to 300mM Stevioside and 1mM UDPG, 30 seconds, 60 seconds, 90 seconds and 120 seconds at 18 ° C were reacted three times, and 20 mM Tris-HCl pH 7.5 was used as a buffer. The Michaelis-Menten equation (Vo = (Vmax*[S])/(Km + [S])) was used to calculate K _m and K _cat (FIG. 16).

As a result, it was confirmed that the K _cat /K _m value of 76_4 according to the stevioside concentration was 5.5 times higher than that of WT.

Through this, it was found that the 76_4 enzyme model of the present invention can be usefully used for the initial reaction because the reaction rate of producing rebaudioside A from stevioside is high without producing rebaudioside I, a bitter byproduct. .

Example 3-5. Sequence analysis of selected enzyme variants

The selected 76_4 enzyme model was named UGT76G1-LRI (UGT76G1 Low Reb I), and the sequence of the UGT76G1-LRI enzyme variant was analyzed.

Specifically, the percentage of the designed enzyme was initially calculated using a multi-sequence comparison service such as Clustal Omega from the natural enzyme. Subsequently, as shown in FIG. 11, mutation information was extracted by using the self-produced Python script of the present invention by using a natural type amino acid and a mutated amino acid sequence as a set (Table 5).

Name Mutation ratio Mutation sites 76_4 SEQ ID NO: 4 7.64%
(11+24 residues) Δ1-11(deletion), I29M / N34W / K52A / L101F / R103D / S119A / R140P / L149A / D162E / S180F / S192C / G205A / S241F / S253T / Q270K / S274N / D291E / I295M / R297W / V300K / S305P / G348Y / V366I / Q425E

It was confirmed that the amino acid sequence of 76_4 was a sequence (SEQ ID NO: 4) in which the N-terminal amino acid sequence of WT (UGT76G1, SEQ ID NO: 1) was removed and 24 amino acids were substituted.

From the above description, other people skilled in the art to which the present invention pertains will be able to understand that the present invention can be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the above detailed description included in the scope of the present invention.

Claims (15)

A UGT enzyme (UDP-glycosyltransferase) variant comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
The UGT enzyme variant according to claim 1, wherein the enzyme variant comprising the amino acid sequence of SEQ ID NO: 2 has heat resistance at 40 - 65 °C.
The UGT enzyme variant according to claim 1, wherein the variant comprising the amino acid sequence of SEQ ID NO: 2 has a conversion rate from Rebaudioside D to Rebaudioside M.
The UGT enzyme variant according to claim 1, wherein the enzyme variant comprising the amino acid sequence of SEQ ID NO: 4 has a conversion rate from Stevioside to Rebaudioside A.
The UGT enzyme variant according to claim 1, wherein the enzyme variant comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 has glycosyltransferase activity.
A polynucleotide encoding the UGT enzyme variant of claim 1.
The polynucleotide according to claim 6, wherein the polynucleotide consists of any one base sequence selected from the nucleotide sequences of SEQ ID NO: 3 or SEQ ID NO: 5.
An expression vector comprising the polynucleotide of claim 6.
A transformant into which the expression vector of claim 8 is introduced.
A composition for producing rebaudioside, comprising the UGT enzyme variant of claim 1 as an active ingredient.
11. The composition according to claim 10, wherein the rebaudioside is rebaudioside A, rebaudioside I or rebaudioside M.
11. The composition according to claim 10, wherein the composition further comprises stevioside, rebaudioside A or rebaudioside D.
(a) culturing the transformant of claim 9; and
(b) a method for producing a UGT enzyme variant comprising the step of isolating the UGT enzyme variant from the culture of the transformant.
A method for producing rebaudioside, comprising reacting the UGT enzyme variant of claim 1 or a transformant expressing the same with stevioside, rebaudioside A or rebaudioside D.
The method of claim 13, wherein the step of recovering rebaudioside from the reaction product of the UGT enzyme variant and stevioside, rebaudioside A or rebaudioside D; further comprising, rebaudioside production method.