阅读说明：本技术 使用糖基转移酶对甜菊糖苷类化合物进行糖基化方法 (Method for glycosylating stevioside compounds by using glycosyltransferase ) 是由 王靖 李皓然 王小艳 郭元亨 陈博 丁子元 祁飞 刘瑞敏 李晨晨 李帅朋 孙绍鹏 于 2020-03-24 设计创作，主要内容包括：本发明涉及糖基转移酶UGT-76用于在甜菊糖苷类化合物的O-(Glc)n的第一个糖基的C-3’添加β-葡萄糖苷的新用途。另外,本发明还涉及使用糖基转移酶UGT-76和/或糖基转移酶UGT-76和糖基转移酶UGT-91生产莱鲍迪苷A(RA)、莱鲍迪苷D(RD)和/或莱鲍迪苷M(RM)的方法。本发明在一定程度上解决了甜菊糖苷生物合成可用的糖基转移酶种类较少、方法较单一、且催化专一性及转化效率都较低的问题。本发明的转化率可达到100％,并且不需添加二甲亚砜或甲醛等不属于食品安全试剂的助溶剂和通透剂,因此更加安全,且方法操作简单,生产成本低,适合于工业化生产。(The invention relates to a new application of glycosyltransferase UGT-76 in adding β -glucoside to C-3' of the first glycosyl group of O- (Glc) n of stevioside compounds, and in addition, the invention also relates to a method for producing Rebaudioside A (RA), Rebaudioside D (RD) and/or Rebaudioside M (RM) by using glycosyltransferase UGT-76 and/or glycosyltransferase UGT-76 and UGT-91.)

1. A glycosylation method, the method comprising: glycosylation is carried out by adding glucoside to C-3' of first glucosyl group of O- (Glc) n of stevioside compound by using glycosyl transferase UGT-76 derived from sunflower, wherein n is an integer between 2 and 5.

2. The method of claim 1, which is a method of in vitro glycosylation comprising: step (1) of transferring a glycosyl group of a glycosyl donor onto C-3' of a first glycosyl group of O-glu (Glc) n of a stevioside compound in the presence of a first glucosyltransferase enzyme, wherein n is an integer between 2 and 5, which is a glycosyltransferase UGT-76 derived from sunflower, preferably having the amino acid sequence shown in SEQ id no: 1; preferably, the stevioside compound is one or more selected from the group consisting of: stevioside compounds present in natural plants, extracted stevioside compounds, and synthetic stevioside compounds.

3. The method of claim 2, further comprising the step of a: transferring a glycosyl group of a glycosyl donor onto C-2' of COO-Glc of the stevioside compound and/or the first glycosylation product in the presence of a second glucosyltransferase to obtain a second glycosylation product, wherein the second glucosyltransferase is a glycosyltransferase UGT-91 derived from Stalmoschus, preferably having an amino acid sequence as shown in SEQ ID No: 3.

4. The method of claim 2 or 3, wherein the stevioside compound is selected from one or more of the group consisting of: steviol bioside, stevioside rebaudioside D, and stevioside rebaudioside E; and/or, the glucose-based donor is selected from one or more of the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, or GDP-glucose, or a combination thereof.

5. The method of any one of claims 2 to 4, wherein the first glycosyltransferase and/or the second glycosyltransferase is/are used in an amount of 2000 to 10000U/L, preferably 3000 to 8000U/L, more preferably 3000 to 5000U/L0, most preferably 4000 to 5000U/L1, the initial concentration of the stevioside and/or the first glycosylation product is/are 0.1 to 100 g/L, preferably 1 to 60 g/L, more preferably 10 to 60 g/L, most preferably 30 to 60 g/L, and the initial concentration of the glucose donor is 0.065 to 65 g/L, preferably 0.065 to 40 g/L, more preferably 6.5 to 40 g/L, most preferably 20 to 40 g/L.

6. The method of any one of claims 2-5, wherein the conditions of in vitro glycosylation are one or more selected from the group consisting of:

(a) in an aqueous system having a pH of from 5.0 to 9.0, preferably from 6.0 to 7.5, more preferably from 6.5 to 7.0, said aqueous system being selected from one or more of the following: water, phosphate buffer, Tris-HCl buffer;

(b) the reaction temperature is 30-45 ℃, preferably 32-40 ℃, and more preferably 35-39 ℃; and/or

(c) The reaction time is 0.5 to 72 hours, preferably 5 to 48 hours, more preferably 1.5 to 36 hours, and most preferably 10 to 20 hours.

7. The method of any one of claims 2-6, wherein the method further comprises the step of isolating the first glycosylation product.

8. The method of claim 2, wherein, when the stevioside compound is a steviol glycoside, the first glycosylation product is stevioside rebaudioside a; and/or, when the stevioside compound is stevioside rebaudioside D, the first glycosylation product is stevioside rebaudioside M.

9. The method of claim 3, wherein when the stevioside compound is a stevioside, the first glycosylation product is stevioside rebaudioside A and/or stevioside rebaudioside M and the second glycosylation product is stevioside rebaudioside D.

Technical Field

The invention relates to the field of biochemical engineering, in particular to a method for glycosylating stevioside compounds by using glycosyl transferase UGT-76 derived from sunflower and/or the combination of glycosyl transferase UGT-76 derived from sunflower and glycosyl transferase UGT-91 derived from Staymus yeast, and a method for preparing glycosylated stevioside compounds.

Background

2016, the release of lancets in China has led to the fact that China has become the most obese world-wide country beyond the United states, healthy diet and low-sugar diet are receiving more and more attention and favor of the government and the people in China, and the demand of international markets for high-intensity sweeteners begins to enter an explosive growth stage along with the start of sugar tax collection policies in many countries in the world. The high-potency sweetener comprises a natural high-potency sweetener and an artificially synthesized high-potency sweetener, and successive published researches from nature and CellMetabolim in 2014 show that consumption of the artificially synthesized high-potency sweeteners such as aspartame, acesulfame potassium, sucralose and the like can change intestinal microbial flora to induce metabolic disorder (such as glucose intolerance) and can result in more food intake to increase body weight, so that the demand of consumers on the natural high-potency sweetener and the share of the natural high-potency sweetener in the market tend to rise year by year.

Stevioside compounds are natural high-power (the sweetness is 300 times of that of cane sugar) and zero-calorie sweeteners, and are known as the third most sugar source after sugarcane and beet. The structural formula of the currently known stevioside compounds is shown as follows, and different side chain modified stevioside compounds are generated along with the difference of R1 and R2, and are specifically shown in Table 1. With the continuous progress of research, it is found that in the stevioside product, high content of rare stevioside compounds such as stevioside rebaudioside a, stevioside rebaudioside D, stevioside rebaudioside M and the like can improve the bitter taste of the stevioside product, but the content of these rare components in stevia is extremely low, and it is difficult to obtain these rare stevioside rebaudiosides by the conventional method of extracting stevia as a raw material.

TABLE 1 stevioside compounds isolated from stevia rebaudiana

In order to break through the current limitation, related technologies of biological synthesis of rare stevioside and enzymatic conversion have been developed in recent years, and biological synthesis (a method for synthesizing stevioside from glucose in vivo by using microorganisms) is developed relatively quickly and is relatively hot, but no mature product is yet on the market at present.

Although a method for synthesizing stevioside compounds by partial enzyme method exists at present, the conversion efficiency of enzyme is low, generally 50% -90%; secondly, most methods need to add dimethyl sulfoxide or formaldehyde and other cosolvents and permeabilizers which do not belong to food safety reagents, and are not beneficial to the application of the method for producing the stevioside compounds; finally, most of the current methods need to adopt a buffer solution with a specific pH value as an enzyme catalysis substrate in consideration of the conversion rate of glycosyltransferase, so that the production cost is high, and the methods are not suitable for industrial production. Therefore, a new method for synthesizing stevioside compounds, which is safe, efficient and suitable for industrial production, is urgently needed.

Disclosure of Invention

Thus, in one aspect, the present invention provides the use of the glycosyltransferase UGT-76 derived from sunflower for catalyzing the addition of a glucoside at the C-3' of the first glycosyl group of O- (Glc) n of a stevioside compound.

In the present invention, glycosyltransferase UGT-76 derived from sunflower (Helianthus annuus) (hereinafter also referred to simply as UGT-76 enzyme) may have the activity of glycosyltransferase shown in NCBI ID: OTF 99622. For example, the sunflower-derived glycosyltransferase UGT-76 can have a sequence consisting of SEQ ID No: 1. O- (Glc) n corresponds to-O-R of the compound of formula 1₁and/or-O-R₂Wherein n may be selected from an integer between 2 and 5.

In another aspect, the present invention provides a method of in vitro glycosylation, the method comprising: step (1) of transferring a glycosyl group of a glycosyl donor to C-3' of a first glycosyl group of O- (Glc) n of a stevioside compound in the presence of a first glucosyltransferase to form a first glycosylation product, wherein n is an integer between 2 and 5, wherein the first glucosyltransferase is a UGT-76 enzyme as described above.

In some embodiments, the method further comprises the step of: transferring a glycosyl group of a glycosyl donor to C-2' of COO-Glc of the stevioside compound and/or the first glycosylation product in the presence of a first glucosyltransferase to obtain a second glycosylation product, wherein the second glucosyltransferase is a glycosyltransferase UGT-91 (hereinafter also referred to as UGT-91 enzyme) derived from Starmerella bombicola having the activity of a glycosyltransferase shown in NCBI ID: ADT 71703. For example, the second glucosyltransferase enzyme may have the amino acid sequence as shown in SEQ ID No: 3, or a pharmaceutically acceptable salt thereof.

In a further preferred embodiment, the method further comprises the step of isolating the first and/or second glycosylation product.

In one embodiment of the method of the present invention, when the stevioside compound is Stevioside (STV), the first glycosylation product is stevioside, rebaudioside a (ra); and/or the presence of a gas in the gas,

when the stevioside compound is stevioside Rebaudioside D (RD), the first glycosylation product is stevioside Rebaudioside M (RM).

In one embodiment of the method of the present invention, when the stevioside compound is stevioside, the first glycosylation product is RA and/or RM and the second glycosylation product is RD.

In another aspect, the invention provides a method for preparing RA, the method comprising catalyzing the production of RA from a steviol glycoside, a glycosyltransferase UGT-76 derived from sunflower, in the presence of a glucosyl donor.

In yet another aspect, the invention also provides a method of making one or more of RA and/or RD and/or RM, the method comprising catalyzing steviol glycosides by glycosyltransferase UGT-76 from sunflower and glycosyltransferase UGT-91 from saccharomyces stolonifera in the presence of a glucose group donor, thereby making one or more of RA and/or RD and/or RM.

The invention has the following beneficial technical effects: the invention relates to new application of UGT-76 derived from sunflower and UGT-91 derived from Staymoma yeast in biosynthesis of high-end stevioside compounds, and solves the problems of few types of glycosyltransferases, single method, low catalytic specificity and low conversion efficiency in stevioside biosynthesis to a certain extent. In addition, a high-efficiency enzyme catalysis system from stevioside to series stevioside compounds RA, RD, RM and the like is constructed on the basis of the substrate specificity of the two enzyme proteins, and a series of biosynthetic rare stevioside products can be developed and produced on the basis of the high-efficiency enzyme catalysis system.

Drawings

FIG. 1 is a schematic representation of the catalytic activity of UGT-76 and UGT-91 enzymes according to one embodiment of the present invention.

FIG. 2 shows the results of electrophoresis of the UGT-76 protein and the UGT-91 protein expressed.

FIGS. 3A and 3B are graphs showing the results of detection of HP L C of a substrate before conversion and a reaction product after conversion in the production of RA from STV using UGT-76 enzyme.

FIG. 4 is a graph showing the results of detection of HP L C on the reaction product when RD is synthesized from STV using UGT-76 enzyme and UGT-91 enzyme, where the substrate reaction is complete, the STV is 100% converted to RA, and then RA is 100% converted to RD.

FIG. 5 is a graph showing the result of detection of HP L C of the reaction product when RM was synthesized from STV using UGT-76 enzyme and UGT-91 enzyme, wherein the substrate reaction was complete, RA converted 100% to RD, and RD converted 95% or more to RM.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The inventor finds that the glycosyl transferase UGT-76 derived from sunflower can catalyze the saccharification reaction of adding β -glucoside to C-3' of the first glycosyl group of O- (Glc) n of a stevioside compound, wherein n can be an integer selected from 2-5, wherein the glycosyl transferase UGT-76 has a nucleotide sequence shown as SEQ ID No. 1, for example, the UGT-76 enzyme can have an amino acid sequence shown as SEQ ID No. 1.

For example, the UGT-76 enzyme can transfer the glycosyl of a glycosyl donor (e.g., UDP-glucose) to O- (Glc) of steviol glycoside₂To the C-3' of the first glycosyl group, thereby obtaining RA. Alternatively, the UGT-76 enzyme can transfer the glycosyl group of a glycosyl donor (e.g., UDP-glucose) to O- (Glc) of RD₂To the C-3' of the first glycosyl group, thereby obtaining RM.

Furthermore, the present inventors have also found that combining the glycosyltransferase UGT-76 derived from sunflower with the glycosyltransferase UGT-91 derived from Saccharomyces stevensis can catalyze the obtaining of further glycosylated stevioside compounds because the UGT-91 enzyme can catalyze a saccharification reaction in which β -glucoside is added at C-2' of COO-Glc of the stevioside compound, wherein the glycosyltransferase UGT-91 can have a nucleotide sequence as shown in SEQ ID No. 3.

Thus, in the present invention, the present inventors provide a method of in vitro glycosylation, the method comprising: step (1) of transferring a glycosyl group of a glycosyl donor onto C-3' of a first glycosyl group of O- (Glc) n of a stevioside compound in the presence of a first glucosyltransferase enzyme to form a first glycosylation product, wherein n is an integer between 2 and 5, the first glucosyltransferase being a glycosyl transferase having a sequence as set forth in SEQ ID No: 1, a glycosyltransferase UGT-76 derived from sunflower.

In a preferred embodiment, the method further comprises the step of a: transferring a glycosyl group of a glycosyl donor onto C-2' of COO-Glc of the stevioside compound and/or the first glycosylation product in the presence of a second glucosyltransferase to obtain a second glycosylation product, wherein the second glucosyltransferase is a glycosyl transferase having a sequence as set forth in SEQ ID No: 3, and a glycosyltransferase UGT-91 derived from saccharomyces stegandrum.

In some embodiments, UGT-76 enzyme catalyzed glycosylation reactions can be performed first, followed by UGT-91 enzyme catalyzed glycosylation reactions, or in reverse order. In some embodiments, the UGT-76 enzyme-catalyzed glycosylation reaction and the UGT-91 enzyme-catalyzed glycosylation reaction can be performed alternately multiple times or simultaneously. In some embodiments, the UGT-76 enzyme-catalyzed glycosylation reaction and the UGT-91 enzyme-catalyzed glycosylation reaction can be performed simultaneously.

For example, a combination of UGT-76 and UGT-91 enzymes can be prepared from steviol glycosides to produce stevioside compounds containing one or more of RA, RD, and RM. In another embodiment, glycosylation of steviol glycosides is first catalyzed by UGT-76 enzyme to give RA; then UGT-91 enzyme catalyzes glycosylation of RA to obtain RD. In another embodiment, glycosylation of steviol glycosides is first catalyzed by UGT-76 enzyme to give RA; catalyzing glycosylation of RA by UGT-91 enzyme to obtain RD; finally, glycosylation of RD is catalyzed by UGT-76 enzyme to give RM.

In the present invention, the main reaction raw material that can be glycosylated by the first and/or second glycosyltransferases may be stevioside compounds of various sources. Alternative stevioside compound sources include, but are not limited to: the stevioside compound extracted from natural plants and directly used in the method is obtained by taking stevia leaves as a raw material through the processes of leaching, impurity removal, decoloration, drying and the like; commercially available stevioside compounds; synthetic steviol glycoside compounds (e.g., steviol glycoside, stevioside rebaudioside a, and stevioside rebaudioside D) are synthesized, for example, by microbial fermentation (e.g., recombinant pichia pastoris, recombinant saccharomyces cerevisiae, recombinant escherichia coli, etc.). Stevioside compounds (e.g., stevioside rebaudioside a, and stevioside rebaudioside D) in the form of powder, crystals, solutions, etc. can be used in the reaction system of the present invention.

For example, the stevioside compound is selected from one or more of the group consisting of: steviolbioside, stevioside rebaudioside a, stevioside rebaudioside D, and stevioside rebaudioside E.

The glucose-based donor useful in the present invention is selected from one or more of the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, or GDP-glucose, or a combination thereof.

Glycosyltransferases are a class of enzymes that catalyze the attachment of an activated sugar to a variety of acceptor molecules, such as the steviol glycosides of the invention. In the present invention, the UGT-76 enzyme can have an amino acid sequence selected from SEQ ID NO: 1 or a functional derivative thereof. Meanwhile, the UGT-91 enzyme in the present invention may have a sequence selected from SEQ ID NO: 3 or a functional derivative thereof.

The term "functional derivative of a polypeptide" as used herein includes the polypeptides of SEQ ID NOs: 1 or 3, and derived polypeptides. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus.

These two enzymes of the invention may include, but are not limited to: an enzyme extracted from a natural source thereof, for example, a natural UGT-76 enzyme extracted from sunflower or a natural UGT-91 enzyme isolated from Staumotrichum, or an extract containing the enzyme; or a catalyst having UGT-76 enzyme and/or UGT-91 enzyme activity obtained by molecular biological methods and/or genetic engineering methods, as long as it has the desired catalytic activity.

The catalyst having the UGT-76 enzyme and/or UGT-91 enzyme activity obtained by the genetic engineering method described in the present invention includes, but is not limited to, microbial cells producing the UGT-76 enzyme and/or UGT-91 enzyme or treated matters (e.g., lysate) of the microbial cells, microbial extracts containing the UGT-76 enzyme and/or UGT-91 enzyme, and isolated UGT-76 enzyme and/or UGT-91 enzyme.

By way of example, the microbial host useful for preparing the above-described catalyst having UGT-76 enzyme and/or UGT-91 enzyme activity can be selected from the following: escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Pichia pastoris. Among them, the host cell is preferably Bacillus subtilis.

The person skilled in the art is aware of methods for expressing foreign proteins in the above-described host cells. For example, the method comprises the steps of: isolating a gene encoding the UGT-76 enzyme and/or the UGT-91 enzyme from a plant of natural origin of the UGT-76 enzyme and/or the UGT-91 enzyme, and/or artificially synthesizing a polynucleotide sequence encoding the UGT-76 enzyme and/or the UGT-91 enzyme based on a polypeptide sequence or a functional derivative thereof; transforming or transducing an expression module (e.g., a recombinant expression vector or recombinant DNA fragment comprising a coding sequence) comprising the above-described gene and/or polynucleotide sequence into a suitable host cell; a host cell cultured in a suitable medium; and isolating and purifying the protein from the culture medium or the cells.

The amino-or carboxy-terminus of the nucleotide sequences of the UGT-76 and UGT-91 enzymes of the invention may also contain one or more polypeptide fragments as protein tags.

In order to allow the translated protein to be expressed secretionally (e.g., extracellularly), a signal peptide sequence such as pelB signal peptide may be added to the amino terminus of the amino acid sequences of the UGT-76 enzyme and the UGT-91 enzyme. The signal peptide may be cleaved off during secretion of the polypeptide from the cell.

One skilled in the art can readily construct an expression vector comprising a polynucleotide sequence encoding the UGT-76 enzyme and UGT-91 enzyme DNA depending on the host chosen. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The polynucleotide sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, tetracycline or ampicillin resistance or Green Fluorescent Protein (GFP).

Regarding the recombinant DNA fragment containing the above-mentioned coding sequence, those skilled in the art can select an appropriate method according to the host selected, and it is clear to those skilled in the art how to select an appropriate promoter, terminator and host cell.

Transformation with recombinant DNAThe host cell may be transformed by conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptides in the above methods can be expressed intracellularly, or on cell membranes, or secreted extracellularly, if desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties, which are well known to those skilled in the art.

In the present invention, the UGT-76 enzyme and the UGT-91 enzyme may be expressed in different host cells or may be expressed in the same host cell.

The enzymatic catalytic reaction is carried out in a water phase system, stevioside compounds are used as acceptor substrates, and glycosylation reaction is carried out on a glucose group donor under the catalysis of UGT-76 enzyme and optionally UGT-91 enzyme to generate first and/or second glycosylated stevioside compounds.

The aqueous system of the present invention may comprise water (e.g., pure water, distilled water, ultrapure water, etc.), a phosphate buffer solution, or a Tris-HCl buffer solution, for example, the raw materials may be dissolved in water, and in the reaction system, the initial concentration of the raw materials (e.g., STV, RA, and/or RD) may be 0.1 to 100 g/L, preferably 1 to 60 g/L, and more preferably 10 to 60 g/L. when two or more raw materials are contained, the initial concentration refers to the respective concentrations of the two raw materials, and the initial concentration of the glucose-based donor in the reaction system may be 0.065 to 65 g/L, preferably 0.65 to 40 g/L, 6.5 to 40 g/L, and 26 to 40 g/L.

The final concentration of the UGT-76 enzyme and/or the UGT-91 enzyme in the reaction system may be 2000 to 10000U/L, preferably 3000 to 8000U/L, more preferably 3000 to 5000U/L, and particularly preferably 4000 to 5000U/L in the present invention, the ratio of the content of the UGT-76 enzyme and/or the UGT-91 enzyme in the reaction system to the content of the raw material (e.g., STV, RA, and/or RD) may be 1:1 to 10, preferably 1:3, more preferably 1: 5. for example, when the raw material is STV, the content of the UGT-76 enzyme in the reaction system is 50U/g STV, when the raw material is RD, the content of the UGT-76 enzyme in the reaction system is 100U/g RD., for example, when the raw material is STV, the content of the UGT-76 enzyme and the UGT-91 enzyme in the reaction system is 50U/g STV and 200U/g STV, respectively, and when the raw material is RA, the content of the UGT-76 enzyme in the reaction system is 100U/g STV and 500U/g.

According to the generation condition of the reaction product, the reaction temperature of the UGT-76 enzyme and/or the UGT-91 enzyme can be set to be 30-45 ℃, preferably 32-40 ℃, and more preferably 35-39 ℃, and the reaction temperature can be adjusted according to the specific enzyme used, industrial cost and the like. The pH of the UGT-76 enzyme and/or UGT-91 enzyme reaction system can be set to about the optimum pH for the enzyme, for example, a pH of 5.0 to 9.0, preferably 6.0 to 7.5, more preferably 6.5 to 7.0, which can be adjusted depending on the particular enzyme used. The time for the UGT-76 enzyme and/or UGT-91 enzyme reaction can be adjusted according to the reaction progress, for example, the reaction is carried out for 0.5 to 72 hours, preferably for 5 to 48 hours, more preferably for 1.5 to 36 hours, and most preferably for 10 to 20 hours.

After completion of the enzyme reaction, the enzyme reaction may be terminated in various ways (for example, it is simpler to terminate the reaction by denaturing the enzyme by boiling (e.g., boiling at 100 ℃ C. for 5 minutes)). Optionally, the resulting reaction product is centrifuged and the supernatant separated for the next reaction. The obtained reaction product can also be used in the next reaction without isolation and purification.

After the reaction is completed, the obtained reaction product can be further subjected to the steps of separation, drying, purification, identification and the like so as to obtain the required rebaudioside A/D/M.

For example, the reaction supernatant and precipitate can be separated by centrifugation, e.g., 12000rpm, 5 minutes, etc. for example, the reaction product can be separated by chromatography, e.g., using HP L C.

In another aspect of the invention, a method of making RA is also provided. The method includes catalyzing, by a UGT-76 enzyme, STV in the presence of a glucosyl donor to produce RA.

The reaction conditions are as defined above. In one embodiment, the STV is provided as a stevia extract comprising STV, wherein the content of STV is preferably above 50 wt%, e.g., 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt% or even 100 wt%. The glucose-based donor is selected from one or more of the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, or GDP-glucose, or a combination thereof; among them, UDP-glucose is preferable. In a preferred embodiment, the ratio of STV to UGT-76 enzyme in the reaction system is 50U/g STv or more, preferably 100U/g STv, which allows complete conversion of STV to RA. Wherein the content of the glucose group donor is provided in excess, and the ratio of the glucose group donor to the UGT-76 enzyme is preferably 35U/g or more, more preferably 70U/g or more.

In a preferred embodiment, the method for the preparation of RA further comprises the step of terminating the enzyme-catalyzed reaction and/or isolating RA.

In another aspect of the invention, methods of making RD are also provided. The method includes catalyzing the STV by a UGT-76 enzyme and a UGT-91 enzyme in the presence of a glucose-based donor to produce RD.

The reaction conditions are as defined above. In one embodiment, the STV is a stevia extract comprising STV, wherein the content of STV is preferably 50 wt% or more, e.g., 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt% or more, or even 100 wt%. The glucose-based donor is selected from one or more of the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, or GDP-glucose, or a combination thereof; among them, UDP-glucose is preferable.

In an embodiment of the present invention, the method for producing RD comprises the steps of: catalyzing STV by UGT-76 enzyme in the presence of a glucosyl donor to produce RA; optionally terminating the UGT-76 enzyme catalyzed reaction, and/or isolating RA; catalyzing RA by UGT-91 enzyme in the presence of a glucosyl donor to produce RD; and, optionally, terminating the enzyme-catalyzed reaction and/or isolating the RD.

In a preferred embodiment, the ratio of UGT-76 enzyme to STV is as described in the method for preparing RA, such that STV is completely converted to RA. In case the enzymatic reaction is not terminated or the step of isolation of RA is not performed, the ratio of UGT-76 enzyme to STV is preferably not more than 100U/g in order to avoid further conversion of RD to RM.

In some embodiments, the ratio of RA to UGT-91 enzyme in the reaction system is 200U/g RA, preferably 500U/g RA, which allows for a complete conversion of RA to RD. Wherein the glucose group donor is provided in excess, wherein the ratio of the glucose group donor to the UGT-91 enzyme is preferably 140U/g glucose group donor, more preferably 350U/g glucose group donor.

In an embodiment of the present invention, the method for preparing RD comprises the following steps: UGT-76 enzyme and UGT-91 enzyme are added simultaneously to catalyze STV to generate RD in one step in the presence of a glucosyl donor; and optionally terminating the catalytic reaction, and/or isolating the RD.

In a preferred embodiment, the ratio of UGT-76 enzyme to UGT-91 enzyme and STV is 100U UGT-76 enzyme/gSTv and 500U UGT-91 enzyme/g STv; and especially the content of UGT-76 enzyme and STV is not higher than 100U/g to avoid RM generation.

In another aspect of the present invention, a method of preparing an RM is also provided. The method comprises the following steps: catalyzing the STV by UGT-76 enzyme and UGT-91 enzyme in the presence of a glucosyl donor to produce RM; alternatively, RA is catalyzed by UGT-76 enzyme and UGT-91 enzyme in the presence of a glucosyl donor to produce RM; alternatively, RD is catalyzed by UGT-76 enzymes in the presence of a glucose-based donor to yield RM.

The reaction conditions are as defined above. In one embodiment, the STV is in the form of a stevia extract comprising STV, wherein the content of STV is preferably above 50 wt%, e.g., 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt% or above or even 100 wt%. The glucose-based donor is selected from one or more of the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, or GDP-glucose, or a combination thereof; among them, UDP-glucose is preferable.

The RA and RD may be prepared as described above or may be provided as a stevia extract comprising RA and/or RD, preferably in an amount of 50 wt% or more, e.g., 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%, 99 wt% or more, or even 100 wt%.

In a preferred embodiment, the method for generating RMs from STVs comprises the steps of: using the method described above, RD is generated from STV; optionally, terminating the catalytic reaction, and/or isolating the RD; and catalyzing the RD by the UGT-76 enzyme in the presence of a glucosyl donor to produce RM; and, optionally, terminating the catalytic reaction, and/or isolating the RM.

In the step of generating RM by catalyzing RD with UGT-76 enzyme, the ratio of UGT-76 enzyme to RD is 100U/g RD, preferably 200U/g RD; the amount of the glucose group donor was 140U/g glucose group donor.

In a preferred embodiment, the method for generating RM from RA comprises: catalyzing RA by UGT-91 enzyme in the presence of a glucosyl donor to produce RD; optionally, terminating the catalytic reaction, and/or isolating the RD; catalytic RD by UGT-76 enzyme in the presence of a glucosyl donor to yield RM; and, optionally, terminating the catalytic reaction, and/or isolating the RM.

In a preferred embodiment, the method for generating RM from RA comprises: catalyzing RA by UGT-91 enzyme and UGT-76 enzyme in the presence of a glucosyl donor to generate RM in one step; and, optionally, terminating the catalytic reaction, and/or isolating the RM.

Wherein the ratio of UGT-91 enzyme to UGT-76 enzyme to RA is preferably 200U UGT-91 enzyme to 50U UGT-76 enzyme: 1gRA, more preferably 500U UGT-91 enzyme 100U UGT-76 enzyme: 1g of RA.

The method for generating RM by RD comprises the step of catalyzing RD by UGT-76 enzyme to generate RM in the presence of a glucosyl donor; and, optionally, terminating the catalytic reaction, and/or isolating the RM. See the definitions above for details.

The invention will be further described by means of embodiments in the following paragraphs:

[1] a glycosylation method comprises adding β -glucoside to C-3' of the first glycosyl group of O- (Glc) n of stevioside compound by using glycosyl transferase UGT-76 derived from sunflower, wherein n is an integer between 2 and 5.

[2] The method of claim 1, which is a method of in vitro glycosylation comprising: step (1) of transferring a glycosyl group of a glycosyl donor onto C-3' of a first glycosyl group of O- (Glc) n of a stevioside compound in the presence of a first glucosyltransferase enzyme to form a first glycosylation product, wherein n is an integer between 2 and 5, the glucosyltransferase being a glycosyl transferase having an amino acid sequence as set forth in SEQ ID No: 1, a glycosyltransferase UGT-76 derived from sunflower.

[3] The method of paragraph [2], wherein the stevioside compound is selected from one or more of the group consisting of:

steviolbioside, stevioside rebaudioside D, and stevioside rebaudioside E.

[4] The method as recited in paragraphs [2] or [3], further comprising the step of a: transferring a glycosyl group of a glycosyl donor onto C-2' of COO-Glc of the stevioside compound and/or the first glycosylation product in the presence of a second glucosyltransferase to obtain a second glycosylation product, wherein the second glucosyltransferase is a glycosyl transferase having a sequence as set forth in SEQ ID No: 3, and a glycosyltransferase UGT-91 derived from saccharomyces stegandrum.

[5] The method of any of paragraphs [2] to [4], wherein the stevioside compound is one or more selected from the group consisting of: stevioside compounds present in natural plants, extracted stevioside compounds, and synthetic stevioside compounds; and/or, the glucose-based donor is selected from one or more of the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, or GDP-glucose, or a combination thereof.

[6] The process according to any of paragraphs [2] to [5], wherein the first and/or glycosyltransferase is used in an amount of 2000 to 10000U/L, preferably 3000 to 8000U/L, more preferably 3000 to 5000U/L, most preferably 4000 to 5000U/L.

[7] The method of any one of paragraphs [2] to [6], wherein the initial concentration of the stevioside compound and/or the first glycosylation product is 0.1-100 g/L, preferably 1-60 g/L, more preferably 10-60 g/L, and most preferably 30-60 g/L, and the initial concentration of the glucose-based donor is 0.065-65 g/L, preferably 0.65-40 g/L, more preferably 6.5-40 g/L, and most preferably 20-40 g/L.

[8] The method of any of paragraphs [2] to [7], wherein the glycosylation conditions are one or more selected from the group consisting of:

(a) in an aqueous system selected from one or more of the following: water, phosphate buffer, Tris-HCl buffer, pH 5.0-9.0, preferably 6.0-7.5, more preferably 6.5-7.0;

(b) the reaction temperature is 30-45 ℃, preferably 32-40 ℃ and more preferably 35-39 ℃; and/or

(c) The reaction time is 0.5 to 72 hours, preferably 5 to 48 hours, more preferably 1.5 to 36 hours, and most preferably 10 to 20 hours.

[9] The method of any of paragraphs [2] to [8], wherein the method further comprises a step of isolating the first glycosylation product.

[10] The method of paragraph [2], wherein,

when the stevioside compound is stevioside, the first glycosylation product is stevioside rebaudioside A; and/or the presence of a gas in the gas,

when the stevioside compound is stevioside rebaudioside D, the first glycosylation product is stevioside rebaudioside M.

[11] The method of paragraph [4], wherein,

when the stevioside compound is stevioside, the first glycosylation product is stevioside rebaudioside A and/or stevioside rebaudioside M, and the second glycosylation product is stevioside rebaudioside D.

[12] A method of preparing stevioside rebaudioside a, the method comprising catalyzing the production of the stevioside to the stevioside rebaudioside a by glycosyltransferase UGT-76 derived from sunflower in the presence of a glucose group donor.

[13] A method of preparing one or more of stevioside rebaudioside a and/or stevioside rebaudioside D and/or stevioside rebaudioside M, the method comprising catalyzing stevioside by glycosyltransferase UGT-76 from sunflower and glycosyltransferase UGT-91 derived from talmo yeast in the presence of a glucose group donor to produce one or more of stevioside rebaudioside a and/or stevioside rebaudioside D and/or stevioside rebaudioside M.

[14] A composition for preparing stevioside rebaudioside a, the composition comprising a recombinant bacterium comprising glycosyltransferase UGT-76 or a lysate thereof, an extract comprising glycosyltransferase UGT-76 or glycosyltransferase UGT-76, and a glucose group donor.

[15] A composition for preparing stevioside rebaudioside D, the composition comprising a recombinant bacterium comprising glycosyltransferase UGT-76 or a lysate thereof, an extract comprising glycosyltransferase UGT-76 or glycosyltransferase UGT-76, a recombinant bacterium comprising glycosyltransferase UGT-91 or a lysate thereof, an extract comprising glycosyltransferase UGT-91 or glycosyltransferase UGT-91, and a glucose group donor.

[16] A composition for preparing stevioside rebaudioside M, comprising a recombinant bacterium comprising glycosyltransferase UGT-76 or a lysate thereof, an extract comprising glycosyltransferase UGT-76 or glycosyltransferase UGT-76, a recombinant bacterium comprising glycosyltransferase UGT-91 or a lysate thereof, an extract comprising glycosyltransferase UGT-91 or glycosyltransferase UGT-91, and a glucose group donor.

[17] A composition for preparing any one or more of stevioside rebaudioside a and/or stevioside rebaudioside D and/or stevioside rebaudioside M, wherein said composition comprises a recombinant bacterium comprising glycosyltransferase UGT-76 or a lysate thereof, an extract comprising glycosyltransferase UGT-76 or glycosyltransferase UGT-76, a recombinant bacterium comprising glycosyltransferase UGT-91 or a lysate thereof, an extract comprising glycosyltransferase UGT-91 or glycosyltransferase UGT-91, and a glucose group donor.