Highly Purified Steviol Glycosides

Abstract

To provide a method of preparing highly purified steviol glycosides, particularly rebaudiosides A, D, and X.SOLUTION: The method comprises expression of UDP-glucosyl transferases from Stevia rebaudiana Bertoni, which are capable converting certain steviol glycosides to rebaudiosides A, D, and X. The highly purified rebaudiosides A, D, and X, are useful as non-caloric sweetener in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical Field The present invention relates to biocatalytic processes for preparing compositions containing steviol glycosides, including highly purified steviol glycoside compositions.

[0002] Incorporation by reference of sequence listing The "PureCircle" file, created on March 12, 2013, has 5 KB (kilobytes) of data and was submitted with this specification. 22PCT The text file entitled "ST25.txt" is incorporated by reference in its entirety into this application. [Background technology]

[0003] High-intensity sweeteners have a sweetness level several times higher than that of sucrose.They are essentially non-caloric and are commonly used in diet and low-calorie products, including foods and beverages.High-intensity sweeteners do not induce a glycemic response, making them suitable for use in products targeted at diabetics and those concerned about limiting carbohydrate intake.

[0004] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub in the Asteraceae (Compositae) family native to certain regions of South America. They are structurally characterized by a single backbone, steviol, and differ by the presence of carbohydrate residues at positions C13 and C19. They accumulate in Stevia leaves (approximately 10%–20% of the total dry weight). On a dry weight basis, the four major glycosides found in Stevia leaves typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6–1.0%), and dulcoside A (0.3%). Other known steviol glycosides include rebaudiosides B, C, D, E, F, and X, steviolbioside, and rubusoside. Summary of the Invention [Problem to be solved by the invention]

[0005] Although methods for preparing steviol glycosides from Stevia rebaudiana are known, many of these methods are unsuitable for commercial use.

[0006] Thus, there remains a need for simple, efficient, and economical methods for preparing compositions containing steviol glycosides, including highly purified steviol glycoside compositions. [Means for solving the problem]

[0007] The present invention provides a biocatalytic process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising a steviol glycoside substrate with a UDP-glucosyltransferase, thereby producing a composition comprising a target steviol glycoside that comprises one or more additional glucose units than the steviol glycoside substrate.

[0008] The starting composition can be any composition comprising at least one steviol glycoside substrate. In one embodiment, the steviol glycoside substrate is selected from the group consisting of steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside X, rebaudioside D, rebaudioside N, rebaudioside O, synthetic steviol glycosides, or combinations thereof. The starting composition may be commercially available or prepared. The starting composition may include purified or partially purified steviol glycoside substrates.

[0009] In one embodiment, the steviol glycoside substrate is rubusoside.

[0010] In another embodiment, the steviol glycoside substrate is stevioside.

[0011] In yet another embodiment, the steviol glycoside substrate is rebaudioside A.

[0012] In yet another embodiment, the steviol glycoside substrate is rebaudioside D.

[0013] The target steviol glycoside can be any known steviol glycoside. In one embodiment, the target steviol glycoside is a steviol monoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside X, rebaudioside D, rebaudioside N, rebaudioside O, or a synthetic steviol glycoside.

[0014] In one embodiment, the target steviol glycoside is stevioside.

[0015] In another embodiment, the target steviol glycoside is rebaudioside A.

[0016] In yet another embodiment, the target steviol glycoside is rebaudioside D.

[0017] In yet another embodiment, the target steviol glycoside is rebaudioside X.

[0018] The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol glycoside substrate to yield the target steviol glycoside. In one embodiment, the UDP-glucosyltransferase is produced in a host. The host can be, for example, E. coli, a Saccharomyces sp., an Aspergillus sp., or a Pichia sp. In another embodiment, the UDP-glucosyltransferase is synthesized.

[0019] The UDP-glucosyltransferase can be provided in any suitable form, including free, immobilized, or whole cell systems. The purity of the UDP-glucosyltransferase may vary, for example, it may be provided as crude, semi-purified, or purified enzyme preparation(s).

[0020] In one embodiment, the UDP-glucosyltransferase is free. In another embodiment, the UDP-glucosyltransferase is immobilized, for example, on an inorganic or organic support. In yet another embodiment, the UDP-glucosyltransferase is provided in the form of a whole cell system (e.g., as living microbial cells) or in the form of a cell lysate.

[0021] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGT91D2.

[0022] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1.

[0023] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside A to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGT91D2.

[0024] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside D to form rebaudioside X. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1.

[0025] The methods of the invention optionally further comprise recycling UDP to obtain UDP-glucose. In one embodiment, the methods comprise recycling UDP by obtaining a recycled catalyst and a recycled substrate, whereby bioconversion of a steviol glycoside substrate to a target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose (FIG. 3).

[0026] In one embodiment, the recycled catalyst is sucrose synthase.

[0027] In one embodiment, the recycled substrate is sucrose.

[0028] The methods of the present invention optionally further comprise separating the target steviol glycosides from the starting composition. The target steviol glycosides can be separated by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.

[0029] In one embodiment, the separation yields greater than about 80% by weight of the target steviol glycoside as anhydrate. The separation produces a composition containing the target steviol glycoside, i.e., a highly purified steviol glycoside composition. In another embodiment, the separation produces a composition containing greater than about 90% by weight of the target steviol glycoside. In a particular embodiment, the composition contains greater than about 95% by weight of the target steviol glycoside.

[0030] The target steviol glycoside may be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0031] The purified target steviol glycosides can be used as sweeteners in consumer products, including, but not limited to, foods, beverages, pharmaceutical compositions, tobacco products, nutritional supplement compositions, oral hygiene compositions, and cosmetic compositions.

[0032] The accompanying drawings are included to provide a further understanding of the invention and illustrate embodiments of the invention, together with the description to explain the principles of embodiments of the invention. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 shows the structure of reb X. [Figure 2] FIG. 1 shows the biocatalytic production of reb X from stevioside. [Figure 3] FIG. 1 shows the biocatalytic production of reb A from stevioside using the enzyme UGT76G1 with simultaneous recycling of UDP to UDP-glucose via sucrose synthase. [Figure 4] FIG. 1 shows the IR spectrum of reb X. [Figure 5] Figure 1 shows an HPLC chromatogram of the products of the biocatalytic production of reb X from reb D, as detailed in Example 14. The peak at retention time 24.165 minutes corresponds to unreacted reb D. The peak at retention time 31.325 minutes corresponds to reb X. [Figure 6] FIG. 1 shows an HPLC chromatogram of purified reb X biocatalytically produced from reb D. [Figure 7] FIG. 1 shows an HPLC chromatogram of reb X standard. [Figure 8] FIG. 1 shows HPLC chromatograms of co-injection of reb X standards and purified reb X derived from biotransformation from reb D. [Figure 9] FIG. 1 shows an overlay of 1H NMR spectra of authentic reb X and purified reb X after biosynthesis from reb D. [Figure 10] FIG. 1 shows the HRMS spectrum of purified reb X after biocatalytic production from reb D. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention provides a biocatalytic process for the preparation of a composition comprising a target steviol glycoside from a starting composition comprising a steviol glycoside substrate, where the target steviol glycoside comprises one or more additional glucose units than the steviol glycoside substrate.

[0035] One object of the present invention is to provide an efficient biocatalytic method for preparing steviol glycosides (particularly stevioside, reb A, reb D and reb X) from other steviol glycosides and / or combinations thereof.

[0036] As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural catalysts, such as protein enzymes, to carry out chemical transformations on organic compounds. The process is alternatively known as biotransformation or biosynthesis. Both isolated and whole cell biocatalytic processes are known in the art. Biocatalytic protein enzymes can be naturally occurring or recombinant proteins.

[0037] As used herein, the term "steviol glycoside(s)" refers to glycosides of steviol, including, but not limited to, naturally occurring steviol glycosides (e.g., steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside X, rebaudioside D, rebaudioside N, rebaudioside O), synthetic steviol glycosides (e.g., enzymatically glycosylated steviol glycosides), and combinations thereof.

[0038] Chemical structures of steviol and its glycosides

[0039] [ka]

[0040] [Table 1]

[0041] Starting Composition As used herein, "starting composition" refers to any composition (generally an aqueous solution) containing one or more steviol glycosides, which act as substrates for biotransformation.

[0042] In one embodiment, the starting composition comprises one or more steviol glycosides selected from the group consisting of steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside X, rebaudioside D, rebaudioside N, rebaudioside O, or a synthetic steviol glycoside. In certain embodiments, the starting composition comprises two or more steviol glycosides.

[0043] In one embodiment, the starting composition includes the steviol glycoside substrate rubusoside.

[0044] In one embodiment, the starting composition includes the steviol glycoside substrate stevioside.

[0045] In another embodiment, the starting composition includes the steviol glycoside substrate rebaudioside A.

[0046] In yet another embodiment, the starting composition includes the steviol glycoside substrate rebaudioside D.

[0047] The starting composition may be synthetic or purified (partially or fully purified), commercially available, or prepared. One example of a starting composition useful in the methods of the present invention is an extract obtained from the purification of Stevia rebaudiana plant material (e.g., leaves). Another example of a starting composition is a commercially available Stevia extract in a solvent solution. Yet another example of a starting composition is a commercially available mixture of steviol glycosides in a solvent solution. Other suitable starting compositions include by-products of processes for isolating and purifying steviol glycosides.

[0048] In one embodiment, the starting composition comprises a purified steviol glycoside substrate. For example, the starting composition may comprise greater than about 99% by weight of a particular substrate steviol glycoside on a dry matter basis.

[0049] In another embodiment, the starting composition comprises a partially purified substrate steviol glycoside composition, e.g., the starting composition contains greater than about 50%, about 60%, about 70%, about 80%, or about 90% by weight of a particular substrate steviol glycoside on a dry matter basis.

[0050] In one embodiment, the starting composition comprises purified rubusoside. In certain embodiments, the starting composition contains >99% rubusoside by weight on a dry matter basis. In another embodiment, the starting composition comprises partially purified rubusoside. In certain embodiments, the starting composition contains greater than about 50%, about 60%, about 70%, about 80%, or about 90% rubusoside by weight on a dry matter basis.

[0051] In one embodiment, the starting composition comprises purified stevioside. In certain embodiments, the starting composition contains >99% stevioside by weight on a dry matter basis. In another embodiment, the starting composition comprises partially purified stevioside. In certain embodiments, the starting composition contains more than about 50%, about 60%, about 70%, about 80%, or about 90% stevioside by weight on a dry matter basis.

[0052] In another embodiment, the starting composition comprises purified rebaudioside A. In certain embodiments, the starting composition contains greater than about 99% rebaudioside A by weight on a dry matter basis. In another embodiment, the starting composition comprises partially purified rebaudioside A. In certain embodiments, the starting composition contains greater than about 50%, about 60%, about 70%, about 80%, or about 90% rebaudioside A by weight on a dry matter basis.

[0053] In yet another embodiment, the starting composition comprises purified rebaudioside D. In certain embodiments, the starting composition contains greater than about 99% rebaudioside D by weight on a dry matter basis. In another embodiment, the starting composition comprises partially purified rebaudioside D. In certain embodiments, the starting composition contains greater than about 50%, about 60%, about 70%, about 80%, or about 90% rebaudioside D by weight on a dry matter basis.

[0054] The steviol glycoside component(s) of the starting composition serve as substrate(s) for the production of target steviol glycoside(s) as described herein. The target steviol glycoside target(s) differ chemically from their corresponding steviol glycoside substrate(s) by the addition of one or more glucose units.

[0055] Target Steviol Glycosides The target steviol glycosides of this method can be prepared by the processes disclosed herein. In one embodiment, the target steviol glycoside is selected from the group consisting of steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside X, rebaudioside D, rebaudioside N, or rebaudioside O.

[0056] In one embodiment, the target steviol glycoside is stevioside. In another embodiment, the target steviol glycoside is reb A. In yet another embodiment, the target steviol glycoside is reb D. In yet another embodiment, the target steviol glycoside is reb X.

[0057] The target steviol glycoside may be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0058] In one embodiment, the invention is a biocatalytic process for the production of stevioside from rubusoside, where the starting composition comprises the steviol glycoside substrate rubusoside. In a particular embodiment, the invention is a biocatalytic process for the production of stevioside from rubusoside, where the starting composition comprises partially purified rubusoside. In another particular embodiment, the invention is a biocatalytic process for the production of stevioside from rubusoside, where the starting composition comprises purified rubusoside.

[0059] In one embodiment, the invention is a biocatalytic process for the production of reb A from stevioside, wherein the starting composition comprises the steviol glycoside substrate stevioside. In a particular embodiment, the invention is a biocatalytic process for the production of reb A from stevioside, wherein the starting composition comprises partially purified stevioside. In another particular embodiment, the invention is a biocatalytic process for the production of reb A from stevioside, wherein the starting composition comprises purified stevioside.

[0060] In another embodiment, the invention is a biocatalytic process for the production of reb D from reb A, where the starting composition comprises the steviol glycoside substrate reb A. In a particular embodiment, the invention is a biocatalytic process for the production of reb D from reb A, where the starting composition comprises partially purified reb A. In another particular embodiment, the invention is a biocatalytic process for the production of reb D from reb A, where the starting composition comprises purified reb A.

[0061] In yet another embodiment, the invention is a biocatalytic process for the production of reb X from reb D, wherein the starting composition comprises the steviol glycoside substrate reb D. In a particular embodiment, the invention is a biocatalytic process for the production of reb X from reb D, wherein the starting composition comprises partially purified reb D. In another particular embodiment, the invention is a biocatalytic process for the production of reb X from reb D, wherein the starting composition comprises purified reb D.

[0062] In certain embodiments, the target steviol glycoside is present in a mixture. For example, in one embodiment, the target steviol glycoside is reb X present in the mixture. In one embodiment, the purity of the target steviol glycoside is increased compared to the purity of the target steviol glycoside present in the starting composition. For example, the purity of reb X present in the starting composition is increased as a result of carrying out the method of the present invention.

[0063] The methods of the present invention optionally further comprise separating the target steviol glycosides from the starting composition. The target steviol glycosides can be separated by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.

[0064] In certain embodiments, the processes described herein result in highly purified target steviol glycoside compositions. As used herein, the term "highly purified" refers to a composition containing greater than about 80% by weight of the target steviol glycoside on an anhydrous basis. In one embodiment, a highly purified target steviol glycoside composition contains greater than about 90% by weight of the target steviol glycoside on an anhydrous basis, such as, for example, a target steviol glycoside content of 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% on a dry matter basis.

[0065] In more specific embodiments, when the target steviol glycoside is reb X, the processes described herein provide compositions having a reb X content of greater than about 90% by weight on a dry matter basis. In another specific embodiment, when the target steviol glycoside is reb X, the processes described herein provide compositions having a reb X content of greater than about 95% by weight on a dry matter basis.

[0066] In another specific embodiment, when the target steviol glycoside is reb D, the processes described herein provide compositions with a reb D content of greater than about 90% by weight on a dry matter basis. In another specific embodiment, when the target steviol glycoside is reb D, the processes described herein provide compositions with a reb D content of greater than about 95% by weight on a dry matter basis.

[0067] In yet another specific embodiment, when the target steviol glycoside is reb A, the processes described herein provide compositions having a reb A content of greater than about 90% by weight on a dry matter basis. In another specific embodiment, when the target steviol glycoside is reb A, the processes described herein provide compositions having a reb A content of greater than about 95% by weight on a dry matter basis.

[0068] In yet another specific embodiment, when the target steviol glycoside is stevioside, the processes described herein provide compositions having a stevioside content of greater than about 90% by weight on a dry matter basis. In another specific embodiment, when the target steviol glycoside is stevioside, the processes described herein provide compositions having a stevioside content of greater than about 95% by weight on a dry matter basis.

[0069] In one embodiment, the biocatalytic methods of the invention are carried out more than once, with the target steviol glycoside produced by a first biocatalytic process serving as a steviol glycoside substrate (also considered to be an intermediate target steviol glycoside) for a second biocatalytic process in which the target steviol glycoside is produced.

[0070] In certain embodiments, the present invention provides a biocatalytic process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising a steviol glycoside substrate with a UDP-glucosyltransferase, thereby producing a composition comprising an intermediate target steviol glycoside that comprises one or more additional glucose units than the steviol glycoside substrate; and contacting the composition comprising the intermediate target steviol glycoside with a UDP-glucosyltransferase, thereby producing a target steviol glycoside that comprises one or more additional glucose units than the intermediate target steviol glycoside. Depending on the number of times the method is performed, there may be one or more intermediate target steviol glycosides (e.g., a first intermediate target steviol glycoside, a second intermediate target steviol glycoside, a third intermediate target steviol glycoside) involved in producing the target steviol glycoside.

[0071] UDP-glucosyltransferase The method is biocatalytic, i.e., utilizes a biological catalyst. In one embodiment, the biocatalyst is a protein enzyme. In a particular embodiment, the biocatalyst is a UDP-glucosyltransferase. The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol glycoside substrate to yield the target steviol glycoside.

[0072] In one embodiment, the UDP-glucosyltransferase is produced in a host such as a microorganism. For example, a DNA sequence encoding the UDP-glucosyltransferase is cloned into an expression vector and transferred into a production host such as a microorganism (e.g., a bacterium). Non-limiting examples of suitable hosts include Escherichia coli, yeast species, Aspergillus species, and Pichia species. The overexpressed protein can be isolated from cell extracts based on its physical and chemical properties using techniques known in the art. Exemplary, non-limiting techniques for isolating the UDP-glucosyltransferase from a host include centrifugation, electrophoresis, liquid chromatography, ion exchange chromatography, gel filtration chromatography, or affinity chromatography.

[0073] UDP-glucosyltransferase can be provided as crude, semi-purified and purified enzyme preparation(s).

[0074] In one embodiment, the UDP-glucosyltransferase is free. In another embodiment, the UDP-glucosyltransferase is immobilized. For example, the UDP-glucosyltransferase may be immobilized on a solid support made of inorganic or organic materials. Non-limiting examples of solid supports suitable for immobilizing the UDP-glucosyltransferase include derivatized cellulose or glass, ceramics, metal oxides, or membranes. The UDP-glucosyltransferase may be immobilized on the solid support by, for example, covalent bonding, adsorption, cross-linking, entrapment, or encapsulation.

[0075] The reaction medium for the conversion is generally aqueous (e.g., purified water, a buffer solution, or a combination thereof). In certain embodiments, the reaction medium is a buffer solution. Suitable buffer solutions include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In certain embodiments, the reaction medium is a phosphate buffer solution. The reaction medium may alternatively be an organic solvent.

[0076] In one embodiment, the UDP-glucosyltransferase is provided in the form of a whole cell system, such as a living microbial cell, which may optionally be similarly immobilized using the techniques identified above for immobilizing the enzyme.

[0077] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside, thereby producing stevioside. The UDP-glucosyltransferase may be, for example, UGT91D2.

[0078] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside, thereby producing rebaudioside A. The UDP-glucosyltransferase may be, for example, UGT76G1.

[0079] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside A, thereby producing rebaudioside D. The UDP-glucosyltransferase can be, for example, UGT91D2.

[0080] In still yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside D to form rebaudioside X. The UDP-glucosyltransferase can be, for example, UGT76G1.

[0081] The method of the present invention optionally further comprises recycling UDP to obtain UDP-glucose. In one embodiment, the method comprises recycling UDP by obtaining a recycled catalyst (i.e., a biocatalyst capable of overproducing UDP-glucose) and a recycled substrate, whereby conversion of the substrate steviol glycoside to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose (FIG. 3).

[0082] In one embodiment, the UDP-glucose recycling catalyst is sucrose synthase.

[0083] In one embodiment, the recycled substrate is sucrose.

[0084] Conversion of rubusoside to stevioside In one embodiment, a starting composition comprising rubusoside is contacted with a UDP-glucosyltransferase capable of catalyzing the reaction of UDP-glucose with stevioside to produce stevioside. In one embodiment, the starting composition comprises partially purified rubusoside. In another embodiment, the starting composition comprises purified rubusoside. In certain embodiments, the starting composition comprises >99% rubusoside. In certain embodiments, the starting composition comprises greater than about 50%, about 60%, about 70%, about 80%, or about 90% rubusoside.

[0085] In certain embodiments, the UDP-glucosyltransferase is UGT91D2, described by Joseph et al. (Genbank accession number ACE87855). It should be noted that a similar sequence was later described in patent application PCT / US2011 / 038967 and designated UGT91D2e. UGT91D2e shares >95% identity with UGT91D11 (Genbank accession number AAR06918) and >99% identity with the UGT of Joseph et al. (Genbank accession number ACE87855).

[0086] In some embodiments, UDP-glucosyltransferases, such as UGT91D2, are prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, Escherichia coli, yeast species, Aspergillus species, and Pichia species. In certain embodiments, UGT91D2 is expressed in E. coli.

[0087] The UDP-glucosyltransferase, such as UGT91D2, may be provided in free or immobilized form. The enzyme preparation may be crude, semi-purified, or purified. In one embodiment, the UDP-glucosyltransferase is provided as a whole cell system (e.g., live or whole microbial cells), a cell lysate, and / or any other form known in the art.

[0088] The reaction medium for the conversion is generally aqueous and may be purified water, a buffer solution, or a combination thereof. In certain embodiments, the reaction medium is a buffer solution. Suitable buffer solutions include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In one embodiment, the reaction medium is a phosphate buffer solution.

[0089] In one embodiment, the conversion of rubusoside to stevioside further comprises the addition of a compound other than UDP-glucose, rubusoside, and UDP-glucosyltransferase. For example, in some embodiments, the reaction medium comprises MgCl and / or MnCl.

[0090] The reaction can be carried out at a temperature between about 0° C. and about 60° C., such as, for example, about 10° C., about 20° C., about 30° C., about 40° C., about 50° C., or about 60° C. In certain embodiments, the reaction is carried out at about 30° C.

[0091] The reaction can proceed for a period of between 1 hour and 1 week, such as, for example, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 120 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In certain embodiments, the reaction is carried out for about 120 hours.

[0092] The UDP-glucose used as the glucose donor can optionally be recycled by use of the enzyme sucrose synthase (Figure 3). Rubusoside is converted to stevioside with the recycled UDP-glucose by a reaction between sucrose and UDP. As a result, rubusoside and sucrose are used in stoichiometric amounts, while UDP is present in catalytic amounts.

[0093] Reactions can be monitored by any suitable method, including but not limited to, HPLC, LCMS, TLC, IR or NMR.

[0094] In one embodiment, the conversion of rubusoside to stevioside is at least about 2% complete, as determined by any of the methods described above. In certain embodiments, the conversion of rubusoside to stevioside is at least about 10% complete, at least about 20% complete, at least about 30% complete, at least about 40% complete, at least about 50% complete, at least about 60% complete, at least about 70% complete, at least about 80% complete, or at least about 90% complete. In certain embodiments, the conversion of rubusoside to stevioside is at least about 95% complete.

[0095] Conversion of stevioside to reb A In one embodiment, a starting composition comprising stevioside is contacted with a UDP-glucosyltransferase capable of catalyzing the reaction of UDP-glucose with stevioside to produce reb A. A glucose unit is chemically added to the disaccharide at the C13 position of stevioside to yield reb A. In one embodiment, the starting composition comprises partially purified stevioside. In another embodiment, the starting composition comprises purified stevioside. In certain embodiments, the starting composition comprises >99% stevioside. In certain embodiments, the starting composition comprises greater than about 50%, about 60%, about 70%, about 80%, or about 90% stevioside.

[0096] In certain embodiments, the UDP-glucosyltransferase is UGT76G1. UGT76G1 was described by Richman et al. (Richman, A., Swanson, A., Humphrey, T., Chapman, R., McGarvey, B., Pocs, R., Brandle, J. Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana. The Plant Journal, 2005, 41, 56-67) and is available in Genbank (ACT33422.1) and Uniprot (C7EA09). The enzyme was overexpressed in Escherichia coli and shown to convert stevioside to reb A.

[0097] In some embodiments, UDP-glucosyltransferases such as UGT76G1 can be prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, Escherichia coli, yeast species, Aspergillus species, and Pichia species. In particular embodiments, UGT76G1 is expressed in E. coli.

[0098] The UDP-glucosyltransferase, such as UGT76G1, may be free or immobilized. It may be in the form of crude, semi-purified, and purified enzyme preparation(s). The UDP-glucosyltransferase may also be provided as a whole cell system (e.g., living microbial cells), whole microbial cells or cell lysates, and / or any other form known in the art.

[0099] The reaction medium for the conversion is generally aqueous and may be purified water, a buffer solution, or a combination thereof. In certain embodiments, the reaction medium is a buffer solution. Suitable buffer solutions include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In one embodiment, the reaction medium is a phosphate buffer solution.

[0100] In one embodiment, the conversion of stevioside to reb A further comprises the addition of a compound other than UDP-glucose, stevioside, and UDP-glucosyltransferase. For example, in some embodiments, the reaction medium comprises MgCl and / or MnCl.

[0101] The reaction can be carried out at a temperature between about 0° C. and about 60° C., such as, for example, about 10° C., about 20° C., about 30° C., about 40° C., about 50° C., or about 60° C. In certain embodiments, the reaction is carried out at about 30° C.

[0102] The reaction can proceed for a period of between 1 hour and 1 week, such as, for example, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 120 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In certain embodiments, the reaction is carried out for about 120 hours.

[0103] Optionally, the UDP-glucose used as the glucose donor can be recycled by use of the enzyme sucrose synthase (Figure 3). Stevioside is converted to reb A with the recycled UDP-glucose by reaction of sucrose with UDP. As a result, stevioside and sucrose are used in stoichiometric amounts, but UDP is present in catalytic amounts.

[0104] Reactions can be monitored by any suitable method, including but not limited to, HPLC, LCMS, TLC, IR or NMR.

[0105] In one embodiment, the biocatalytic or bioconversion of stevioside to reb A is at least about 50% complete, as determined by any of the methods described above. In certain embodiments, the conversion of stevioside to reb A is at least about 60% complete, at least about 70% complete, at least about 80% complete, or at least about 90% complete. In certain embodiments, the conversion of stevioside to reb A is at least about 95% complete.

[0106] Conversion of reb A to reb D In one embodiment, a starting composition comprising reb A is contacted with a UDP-glucosyltransferase capable of catalyzing the reaction of UDP-glucose with reb A to produce reb D. A glucose unit is chemically added to the monosaccharide at the C19 position of reb A to yield reb D. In one embodiment, the starting composition comprises partially purified reb A. In certain embodiments, the starting composition comprises purified reb A. In certain embodiments, the starting composition comprises >99% reb A. In certain embodiments, the starting composition comprises greater than about 50%, about 60%, about 70%, about 80%, or about 90% reb A.

[0107] In certain embodiments, the UDP-glucosyltransferase is UGT91D2, described by Joseph et al. (Genbank accession number ACE87855). It should be noted that a similar sequence was later described in patent application PCT / US2011 / 038967 and designated UGT91D2e. UGT91D2e shares >95% identity with UGT91D11 (Genbank accession number AAR06918) and >99% identity with the UGT of Joseph et al. (Genbank accession number ACE87855).

[0108] In some embodiments, UDP-glucosyltransferases, such as UGT91D2, are prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, Escherichia coli, yeast species, Aspergillus species, and Pichia species. In certain embodiments, UGT91D2 is expressed in E. coli.

[0109] The UDP-glucosyltransferase, such as UGT91D2, may be provided in free or immobilized form. The enzyme preparation may be crude, semi-purified, or purified. In one embodiment, the UDP-glucosyltransferase is provided as a whole cell system (e.g., live or whole microbial cells), a cell lysate, and / or any other form known in the art.

[0110] The reaction medium for the conversion is generally aqueous and may be purified water, a buffer solution, or a combination thereof. In certain embodiments, the reaction medium is a buffer solution. Suitable buffer solutions include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In one embodiment, the reaction medium is a phosphate buffer solution.

[0111] In one embodiment, the conversion of reb A to reb D further comprises the addition of a compound other than UDP-glucose, reb A, and UDP-glucosyltransferase. For example, in some embodiments, the reaction medium comprises MgCl and / or MnCl.

[0112] The reaction can be carried out at a temperature between about 0° C. and about 60° C., such as, for example, about 10° C., about 20° C., about 30° C., about 40° C., about 50° C., or about 60° C. In certain embodiments, the reaction is carried out at about 30° C.

[0113] The reaction can proceed for a period of between 1 hour and 1 week, such as, for example, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 120 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In certain embodiments, the reaction is carried out for about 120 hours.

[0114] Optionally, the UDP-glucose used as the glucose donor can be recycled by use of the enzyme sucrose synthase (Figure 3). Reb A is converted to reb D with the recycled UDP-glucose by reaction of sucrose with UDP. As a result, reb A and sucrose are used in stoichiometric amounts, but UDP is present in catalytic amounts.

[0115] Reactions can be monitored by any suitable method, including but not limited to, HPLC, LCMS, TLC, IR or NMR.

[0116] In one embodiment, the conversion of reb A to reb D is at least about 2% complete, as determined by any of the methods described above. In certain embodiments, the conversion of reb A to reb D is at least about 10% complete, at least about 20% complete, at least about 30% complete, at least about 40% complete, at least about 50% complete, at least about 60% complete, at least about 70% complete, at least about 80% complete, or at least about 90% complete. In certain embodiments, the conversion of reb A to reb D is at least about 95% complete.

[0117] Conversion of reb D to reb X In one embodiment, the starting composition comprises reb D and is contacted with a UDP-glucosyltransferase capable of catalyzing the reaction of UDP-glucose with reb D to produce reb X. A glucose unit is chemically added to the disaccharide at the C19 position of reb D to yield reb X. In one embodiment, the starting composition comprises partially purified reb D. In another embodiment, the starting composition comprises purified reb D. In certain embodiments, the starting composition comprises >99% reb D. In certain embodiments, the starting composition comprises greater than about 50%, about 60%, about 70%, about 80%, or about 90% reb D. In certain embodiments, the UDP-glucosyltransferase is UGT76G1.

[0118] In some embodiments, UDP-glucosyltransferases, such as UGT91D2, can be prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, Escherichia coli, yeast species, Aspergillus species, and Pichia species. In particular embodiments, UGT91D2 is expressed in E. coli.

[0119] The UDP-glucosyltransferase, such as UGT91D2, may be provided free or immobilized. The enzyme preparation may be crude, semi-purified, or purified. In one embodiment, the UDP-glucosyltransferase is provided as a whole cell preparation (e.g., in the form of live or whole microbial cells), a cell lysate, and / or any other form known in the art.

[0120] The reaction medium for the conversion is generally aqueous and may be purified water, a buffer solution, or a combination thereof. In certain embodiments, the reaction medium is a buffer solution. Suitable buffer solutions include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In one embodiment, the reaction medium is a phosphate buffer solution.

[0121] In one embodiment, the conversion of reb D to reb X uses compounds in addition to UDP-glucose, reb D, and UDP-glucosyltransferase. For example, in some embodiments, the reaction medium includes MgCl and / or MnCl.

[0122] The reaction can be carried out at a temperature between about 0° C. and about 60° C., such as, for example, about 10° C., about 20° C., about 30° C., about 40° C., about 50° C., or about 60° C. In certain embodiments, the reaction is carried out at about 30° C.

[0123] The reaction can proceed for a period of between 1 hour and 1 week, such as, for example, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 120 hours, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In certain embodiments, the reaction is carried out for about 120 hours.

[0124] Optionally, the UDP-glucose used as the glucose donor can be recycled by use of the enzyme sucrose synthase (Figure 3). Reb D is converted to reb X with the recycled UDP-glucose by reaction of sucrose with UDP. As a result, reb D and sucrose are used in stoichiometric amounts, but UDP is present in catalytic amounts.

[0125] Reactions can be monitored by any suitable method, including but not limited to, HPLC, LCMS, TLC, IR or NMR.

[0126] In one embodiment, conversion of reb D to reb X is at least about 50% complete as determined by any of the methods described above. In certain embodiments, conversion of reb D to reb X is at least about 60% complete, at least about 70% complete, at least about 80% complete, or at least about 90% complete. In certain embodiments, conversion of reb D to reb X is at least about 95% complete.

[0127] The target steviol glycoside is optionally purified from the resulting composition. Purification of the target steviol glycoside from the reaction medium can be accomplished by any suitable method to provide a highly purified target steviol glycoside composition. Suitable methods include crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or a combination of such methods.

[0128] In one embodiment, certain biocatalytic transformations may be quenched to stop the reaction. The resulting mixture is then centrifuged. The supernatant generally contains the target steviol glycoside and may then be further purified, if desired. For example, analytical or preparative HPLC can be used to separate the remaining target or starting steviol glycoside(s) or reaction by-products from the target steviol glycoside. In one embodiment, separation is achieved by analytical HPLC. In another embodiment, separation is achieved by preparative HPLC. One of skill in the art will recognize that the particular HPLC method used can vary based on the particular system, solvent, and column. A suitable system for separating reb X from reb D is provided in Example 20.

[0129] It is anticipated that the methods provided herein can be repeated, and the composition resulting from an initial process (i.e., a composition comprising a target steviol glycoside) can then be used as the starting composition when the method is run a second time, and the target steviol glycoside can optionally be purified from the composition comprising the target steviol glycoside to yield a highly purified target steviol glycoside or steviol glycoside composition. According to this embodiment, the target steviol glycoside produced in the first run of the method is considered a first target steviol glycoside or intermediate target steviol glycoside and is useful as a substrate for producing a second target steviol glycoside, a second intermediate target steviol glycoside, or a final target steviol glycoside. The process can be repeated as many times as necessary to arrive at the final target steviol glycoside. In one embodiment, the method is repeated once. In another embodiment, the method is repeated twice. In yet another embodiment, the method is repeated three times. In yet another embodiment, the method is repeated four times. The reaction is repeated 5, 6, 7, 8, or 9 times. One of skill in the art will recognize that the particular UDP-glucosyltransferase used in each reaction can be the same or different, depending on the particular site on the steviol glycoside substrate to which glucose is added.

[0130] Thus, in one embodiment, the method is repeated once, wherein the starting composition of the first method comprises reb A and the target steviol glycoside is reb D, and the starting composition of the second method comprises reb D and the target steviol glycoside is reb X.

[0131] In another embodiment, the method is repeated twice, with the starting composition of the first method comprising stevioside and the target steviol glycoside being reb A; the starting composition of the second method comprising reb A and the target steviol glycoside being reb D; and the starting composition of the third method comprising reb D and the target steviol glycoside being reb X.

[0132] In yet another embodiment, the method is repeated three times, where the starting composition of the first method comprises rubusoside and the target steviol glycoside is stevioside; the starting composition of the second method comprises stevioside and the target steviol glycoside is reb A; the starting composition of the third method comprises reb A and the target steviol glycoside is reb D; and the starting composition of the fourth method comprises reb D and the target steviol glycoside is reb X.

[0133] In one embodiment, a method for producing a highly purified target steviol glycoside composition comprises: a. contacting a first starting composition comprising a steviol glycoside substrate with a first UDP-glucosyltransferase to produce a composition comprising a first target steviol glycoside; b. optionally, separating the first target steviol glycoside from the culture medium to obtain a highly purified first target steviol glycoside composition; c. contacting the composition comprising the first target steviol glycoside or the highly purified first target steviol glycoside composition with a second UDP-glucosyltransferase to produce a composition comprising a second target steviol glycoside; d. Optionally, separating the second target steviol glycoside from the culture medium to obtain a highly purified second target steviol glycoside composition; e. contacting the second target steviol glycoside-containing composition or the highly purified second target steviol glycoside composition with a third UDP-glucosyltransferase to produce a third target steviol glycoside-containing composition; and f. Optionally, separating the third target steviol glycoside from the culture medium to obtain a highly purified third target steviol glycoside composition. Includes:

[0134] In one embodiment, the first starting composition comprises stevioside, the first target steviol glycoside is reb A, and the first UDP-glucosyltransferase is UGT76G1.

[0135] In a further embodiment, the second UDP-glucosyltransferase is UGT91D2 and the second target steviol glycoside is reb D.

[0136] In still further embodiments, the third UDP-glucosyltransferase is UGT91D2 and the third target steviol glycoside is reb X.

[0137] In one embodiment, one or more of the steps of contacting a composition comprising a steviol glycoside substrate with a UDP-glucosyltransferase further comprises providing a biocatalyst capable of overproducing and recycling UDP and a substrate for said recycling.

[0138] In more particular embodiments, a method for producing a highly purified target steviol glycoside composition comprises: a. contacting a first starting composition comprising a steviol glycoside substrate with a first UDP-glucosyltransferase to produce a composition comprising a first target steviol glycoside; b. optionally providing a biocatalyst capable of overproducing and recycling UDP and a substrate for said recycling; c. optionally, separating the first target steviol glycoside from the culture medium to obtain a highly purified first target steviol glycoside composition; d. contacting the composition comprising the first target steviol glycoside or the highly purified first target steviol glycoside composition with a second UDP-glucosyltransferase to obtain a composition comprising a second target steviol glycoside; e. optionally providing a biocatalyst capable of overproducing and recycling UDP and a substrate for said recycling; f. optionally, separating the second target steviol glycoside from the culture medium to obtain a highly purified second target steviol glycoside composition; g. contacting the composition comprising the second target steviol glycoside or the highly purified second target steviol glycoside composition with a third UDP-glucosyltransferase to obtain a composition comprising the third target steviol glycoside; and h. Optionally, separating the third target steviol glycoside from the culture medium to obtain a highly purified third target steviol glycoside composition. Includes:

[0139] In one embodiment, the first starting composition comprises stevioside, the first target steviol glycoside is reb A, and the first UDP-glucosyltransferase is UGT76G1.

[0140] In a further embodiment, the second UDP-glucosyltransferase is UGT91D2 and the second target steviol glycoside is reb D.

[0141] In still further embodiments, the third UDP-glucosyltransferase is UGT91D2 and the third target steviol glycoside is reb X.

[0142] In another specific embodiment, a method for producing a highly purified target steviol glycoside composition comprises: a. contacting a first starting composition comprising a steviol glycoside substrate with a first UDP-glucosyltransferase to produce a composition comprising a first target steviol glycoside; b. optionally providing a biocatalyst capable of overproducing and recycling UDP and a substrate for said recycling; c. optionally, separating the first target steviol glycoside from the culture medium to obtain a highly purified first target steviol glycoside composition; d. contacting the composition comprising the first target steviol glycoside or the highly purified first target steviol glycoside composition with a second UDP-glucosyltransferase to produce a composition comprising a second target steviol glycoside; e. optionally providing a biocatalyst capable of overproducing and recycling UDP and a substrate for said recycling; f. optionally, separating the second target steviol glycoside from the culture medium to obtain a highly purified second target steviol glycoside composition; g. contacting the composition comprising the second target steviol glycoside or the highly purified second target steviol glycoside composition with a third UDP-glucosyltransferase to obtain a composition comprising the third target steviol glycoside; and h. optionally providing a biocatalyst capable of overproducing and recycling UDP and a substrate for said recycling; i. optionally, separating the third target steviol glycoside from the culture medium to obtain a highly purified third target steviol glycoside composition; j. contacting the composition comprising the third target steviol glycoside or the highly purified third target steviol glycoside composition with a fourth UDP-glucosyltransferase to produce a composition comprising the fourth target steviol glycoside; and k. optionally providing a biocatalyst capable of overproducing and recycling UDP and a substrate for said recycling; l. Optionally, separating the fourth target steviol glycoside from the culture medium to obtain a highly purified fourth target steviol glycoside composition. Includes:

[0143] In one embodiment, the first starting composition comprises rubusoside, the first target steviol glycoside is stevioside, and the first UDP-glucosyltransferase is UGT91D2.

[0144] In a further embodiment, the second UDP-glucosyltransferase is UGT76G1 and the second target steviol glycoside is reb A.

[0145] In a further embodiment, the third UDP-glucosyltransferase is UGT91D2 and the third target steviol glycoside is reb D.

[0146] In yet a further embodiment, the fourth UDP-glucosyltransferase is UGT91D2 and the fourth target steviol glycoside is reb X.

[0147] Purified steviol glycosides prepared according to the present invention can be used in a variety of products, including, but not limited to, foods, beverages, pharmaceutical compositions, tobacco products, nutritional supplement compositions, oral hygiene compositions, and cosmetic compositions.

[0148] The high-purity reb X obtained in this invention has a molecular weight of 1291.29, a molecular formula of C56H90O33, and the structure shown in Figure 1. It is in the form of a white, odorless powder. The compound is approximately 200 times sweeter than sugar when compared to a 10% sucrose solution. The infrared absorption spectrum is shown in Figure 4.

[0149] Other properties of the pure reb X compound include a melting point of 249-250°C and a specific optical rotation [α]D of -19.0° in 50% ethanol (C = 1.0). The solubility of reb X in water is approximately 0.3% and increases with increasing temperature.

[0150] Reb X is soluble in dilute solutions of methanol, ethanol, n-propanol, and isopropanol, but is insoluble in acetone, benzene, chloroform, and ether.

[0151] The reb X obtained according to the present invention is heat and pH stable.

[0152] The highly purified target glycoside(s) obtained by the present invention, particularly reb D and / or reb X, can be used directly in combination with other sweeteners, flavors, and food ingredients.

[0153] Non-limiting examples of flavorings include lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon, sugar, cotton candy, and vanilla flavoring.

[0154] Non-limiting examples of other food ingredients include flavorings, acidulants, organic acids and amino acids, colorings, bulking agents, modified starches, gums, texturizing agents, preservatives, antioxidants, emulsifiers, stabilizers, thickeners and gelling agents.

[0155] The highly purified target glycoside(s), particularly reb D and / or reb X, obtained by the present invention can be prepared in a variety of different forms, including but not limited to, hydrates, solvates, anhydrates, amorphous forms, and / or combinations thereof.

[0156] The highly purified target steviol glycoside(s) obtained by the present invention, particularly reb D and / or reb X, may be included as high-intensity natural sweeteners in foods, beverages, pharmaceutical compositions, cosmetics, chewing gum, tableware, cereals, dairy products, toothpaste, and other oral compositions, etc.

[0157] The highly purified target steviol glycoside(s) as sweetening compounds, particularly reb D and / or reb X, may be used alone as a sweetener, or may be combined with stevioside, reb A, reb B, reb C, reb D, reb E, reb F, steviolbioside, dulcoside A, rubusoside, mogroside, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusoside, periandrin, carnosifloside, cyclocarioside, pterocaryoside, polypodoside A, brazilin, herandalucine nandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydrofuronol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo It may also be used in conjunction with other naturally occurring high-intensity sweeteners such as Han Guo sweetener, mogroside V, and siamenoside.

[0158] The highly purified target steviol glycoside(s), particularly reb D and / or reb X, may also be used in combination with synthetic high-intensity sweeteners such as sucralose, acesulfame potassium, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamic acid, neotame, dulcin, suosan, N-[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester, N-[N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester, N-[N-[3-(3-methoxy-4-hydroxyphenyl)propyl]-L-α-aspartyl]-L-phenylalanine 1-methyl ester, or salts thereof.

[0159] Additionally, highly purified target steviol glycoside(s), particularly reb D and / or reb X, can be used in combination with natural sweetness suppressors such as gymnemic acid, phodulcin, digiphin, and lactisol. Reb D and / or reb X can also be combined with various umami enhancers. Reb D and / or reb X can be mixed with umami and sweet amino acids such as glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, glutamic acid, and tryptophan.

[0160] The highly purified target steviol glycoside(s), particularly reb D and / or reb X, are also combined with a polyol or sugar alcohol. The term "polyol" refers to a molecule containing multiple hydroxyl groups. Polyols can be diols, triols, or tetraols, containing 2, 3, and 4 hydroxyl groups, respectively. Polyols can also contain more than four hydroxyl groups, such as pentaols, hexaols, and heptaols, containing 5, 6, or 7 hydroxyl groups, respectively. Furthermore, polyols can be sugar alcohols, polyhydric alcohols, or polyalcohols, which are reduced forms of carbohydrates, in which the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycitol and sugar alcohols, or any other carbohydrate that can be reduced without adversely affecting the taste of the sweetener composition.

[0161] Highly purified target steviol glycoside(s), particularly reb D and / or reb X, can be combined with low-calorie sweeteners such as D-tagatose, L-sugars, L-sorbose, and L-arabinose.

[0162] The highly purified target steviol glycoside(s), particularly reb D and / or reb X, can be combined with a variety of carbohydrates. The term "carbohydrate" refers to carbohydrates having the general formula (CHO) n(where n is 3-30) and generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, as well as their oligomers and polymers. Furthermore, the carbohydrates of the present invention may be substituted or deoxygenated at one or more positions. As used herein, carbohydrate encompasses unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivative," "substituted carbohydrate," and "modified carbohydrate" are synonymous. Modified carbohydrate refers to any carbohydrate in which at least one atom has been added, removed, or substituted, or a combination thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivative or substituted carbohydrate can be optionally deoxygenated at any corresponding C-position and / or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonate, or any other practical functional group, so long as the carbohydrate derivative or substituted carbohydrate functions to improve the sweetness of the sweetener composition.

[0163] Examples of carbohydrates that can be used in accordance with the present invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, and isotrehalose. , neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abequose, galactosamine, beet Oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylo-oligosaccharides (xylotriose, xylobiose, etc.), xylo-terminal oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose) Such carbohydrates include maltotetraol, maltotriol, malto-oligosaccharides (such as maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose), starch, inulin, inulooligosaccharides, lactulose, melibiose, raffinose, ribose, and isomerized liquid sugars (such as high fructose corn syrup, coupling sugar, and soybean oligosaccharides). Moreover, the carbohydrates used herein may be in either the D- or L-configuration.

[0164] The highly purified target steviol glycoside(s), particularly reb D and / or reb X, obtained by the present invention can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients are generally classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols); polyols; prebiotics, probiotics; phytoestrogens; soy proteins; sulfides / thiols; amino acids; proteins; vitamins; and minerals. Functional ingredients can also be classified based on their health benefits, such as cardiovascular, cholesterol-lowering, and anti-inflammatory properties.

[0165] The highly purified target steviol glycoside(s), particularly reb D and / or reb X, obtained by the present invention can be applied as a high-intensity sweetener to produce zero-calorie, low-calorie, or diabetic beverages and foods with improved taste characteristics. It can also be used in beverages, food, pharmaceuticals, and other products where sugar cannot be used. Moreover, the highly purified target steviol glycoside(s), particularly reb D and / or reb X, can be used as a sweetener in beverages, food, and other products for human consumption, as well as in animal feed and feed with improved characteristics.

[0166] Examples of products in which highly purified target steviol glycoside(s), particularly reb D and / or reb X, can be used as sweetening compounds include, but are not limited to, alcoholic beverages such as vodka, wine, beer, sake, and Japanese sake; natural fruit juices; soft drinks; carbonated soft drinks; diet drinks; zero-calorie drinks; low-calorie beverages and foods; yogurt drinks; instant fruit juices; instant coffee; powdered instant beverages; canned goods; syrups; fermented soybean paste; soy sauce; wine vinegar; dressings; mayonnaise; ketchup; curry; soups; instant bouillon; and powdered soy sauce. ;Vinegar powder;Biscuits;Rice crackers;Crackers;Bread;Chocolate;Caramel;Candy;Chewing gum;Jelly;Pudding;Preserved fruits and vegetables;Fresh cream;Jam;Marmalade;Flour paste;Milk powder;Ice cream;Sherbet;Bottled vegetables and fruits;Canned boiled beans;Meat and foods cooked in sweet sauces;Agricultural plant foods;Seafood;Ham;Sausage;Fish ham;Fish sausage;Fish paste;Fried fish products;Dried seafood;Frozen foods;Preserved seaweed;Preserved meat;Tobacco;Medicines; etc.In principle, it is applicable unconditionally.

[0167] Conventional methods such as mixing, kneading, dissolving, rinsing, percolating, leaching, sprinkling, atomizing, injecting, and other methods can be used in the manufacture of products such as food, beverages, pharmaceuticals, cosmetics, tableware, and chewing gum.

[0168] Furthermore, the highly purified target steviol glycoside(s), particularly reb D and / or reb X, obtained in the present invention can be used in dry or liquid form. It can be added before or after the heat treatment of food. The amount of highly purified target steviol glycoside(s), particularly reb D and / or reb X, depends on the intended use. As discussed above, it can be added alone or in combination with other compounds.

[0169] The following examples illustrate preferred embodiments of the present invention for the preparation of highly purified target steviol glycoside(s), particularly reb D and / or reb X. It is understood that the present invention is not limited to the materials, proportions, conditions, and procedures set forth in the examples, which are merely illustrative. [Example]

[0170] Example 1 In vivo production of UGT76G1 NcoI and NdeI restriction sites were added to the original nucleic acid sequence as described in Genbank accession number AAR06912.1. After codon optimization, the following nucleic acid sequence (SEQ ID NO: 1) was obtained: Sequence Listing Free Text

[0171] [ka]

[0172] After gene synthesis and subcloning into the pET30A+ vector using the NdeI and XhoI cloning sites, UGT76G1 The pET30a+ plasmid was introduced into E. coli B121(DE3) and E. coli EC100 by electroporation. The resulting cells were grown in Petri dishes in the presence of kanamycin, and suitable colonies were selected and expanded in liquid LB medium (Erlenmeyer flasks). Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0173] pET30A+ A stock aliquot of E. coli BL21(DE3) containing the UGT76G1 plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). The culture was shaken at 135 rpm at 30°C for 8 hours.

[0174] The production medium contained 60 g / L overnight express instant TB medium (Novagen), 10 g / L glycerol, and 50 mg / L kanamycin. The medium was stirred at 20°C while samples were taken to measure OD and pH. The culture showed significant growth and a good OD was obtained. After 40 hours, the cells were harvested by centrifugation and frozen, yielding a wet weight of 12.7 g of cells.

[0175] Lysis was performed by adding Bugbuster Master mix (Novagen), and the lysates were collected by centrifugation and stored frozen. Activity tests were performed using the thawed lysates.

[0176] Example 2 In vitro production of UGT76G1 The S30 T7 High Yield Protein Expression System kit from Promega was used. UGT76G1 from E. coli EC100 4 μg of pET30a+ plasmid was mixed with 80 μL of S30 premix plus, and 72 μL of S30 T7 extract was added. Nuclease-free water was added to bring the total volume to 200 μL, and the resulting solution was incubated for 2 hours at 30°C. 180 μL was used for the catalytic test reaction.

[0177] Example 3 In vitro production of UGT91D2 NcoI and NdeI restriction sites were added to the original nucleic acid sequence as set forth in Genbank accession number ACE87855.1. After codon optimization, the following nucleic acid sequence (SEQ ID NO: 2) was obtained: Sequence Listing Free Text

[0178] [ka]

[0179] After gene synthesis and subcloning into the pET30A+ vector using the NcoI and XhoI cloning sites, UGT91D2 The pET30a+ plasmid was introduced into E. coli EC100 by electroporation. The resulting cells were grown in the presence of kanamycin, and suitable colonies were selected and expanded in liquid LB medium (Erlenmeyer flasks). Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0180] The S30 T7 High Yield Protein Expression System kit from Promega was used for in-vitro protein synthesis.

[0181] UGT91D2 Four micrograms of pET30a+ plasmid was mixed with 80 μL of S30 premix plus, and 72 μL of S30 T7 extract was added. Nuclease-free water was added to bring the total volume to 200 μL, and the resulting solution was incubated for 2 hours at 30°C. Five μL was used for SDS-page analysis, and the remaining 45 μL was used for the catalytic test reaction.

[0182] Example 4 Catalysis using in vivo produced UGT76G1 The total reaction volume was 5.0 mL and had the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl, 2.5 mM UDP-glucose, 0.5 mM stevioside, and 500 μL of UGT76G1 melt lysate. Reactions were performed at 30°C on an orbital shaker at 135 rpm. For each sample, 460 μL of the reaction mixture was quenched with 40 μL of 2N HSO and 420 μL of methanol / water (6 / 4). Samples were immediately centrifuged and stored at 10°C until analysis by HPLC (CAD). HPLC showed nearly complete conversion of stevioside to rebaudioside A.

[0183] [ka]

[0184] Example 5 Catalysis using in vitro produced UGT91D2 The total reaction volume was 0.5 mL and had the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl, 3.8 mM UDP-glucose, 0.1 mM rebaudioside A, and 180 μL of in-vitro produced UGT91D2. The reactions were carried out at 30 °C on an orbital shaker at 135 rpm. For each sample, 450 μL of the reaction mixture was quenched with 45 μL of 2N HSO and 405 μL of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC showed 4.7% conversion of rebaudioside A to rebaudioside D after 120 h.

[0185] Example 6 Catalysis using in vitro produced UGT76G1 The total reaction volume was 2 mL and had the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl, 3.8 mM UDP-glucose, 0.5 mM rebaudioside D, and 180 μL of in-vitro produced UGT76G1. The reactions were carried out at 30 °C on an orbital shaker at 135 rpm. For each sample, 400 μL of the reaction mixture was quenched with 40 μL of 2N HSO and 360 μL of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC showed 80% conversion of rebaudioside D to rebaudioside X after 120 h.

[0186] [ka]

[0187] For Examples 7-12, the following abbreviations were used: LBGKP medium: 20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin or ampicillin LB medium: (20g / L Luria Broth Lennox)

[0188] Example 7 Preparation and activity of UGT76G1 prepared by pET30a+ plasmid and BL21(DE3) expression strain pET30a+ The UGT76G1 plasmid was transformed into the BL21(DE3) expression strain (Lucigen E. Cloni® EXPRESS Electrocompetent Cells). The resulting cells were grown in LB Agar medium in Petri dishes in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium containing kanamycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0189] A stock aliquot was thawed and added to 30 mL of LBGKP medium. This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L of "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L glycerol, and 50 mg / L kanamycin. The medium was stirred at 20°C while samples were taken to measure OD (600 nm) and pH. After 40 hours, the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 10.58 g.

[0190] 3.24 g of the resulting precipitate were dissolved by adding 8.1 mL of "Bugbuster Master mix" (Novagen, ref. 71456) and 3.5 mL of water. The lysate was collected by centrifugation and stored frozen.

[0191] Example 8 Preparation and activity of UGT76G1 expressed by pET30a+ plasmid and Tuner(DE3) expression strain pET30a+ The UGT76G1 plasmid was transformed into the Tuner (DE3) expression strain (Novagen Tuner™ (DE3) competent cells) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium (containing kanamycin). Glycerol was added, and 400 μL aliquots were stored at −20°C and −80°C.

[0192] A stock aliquot was thawed and added to 100 mL of LB medium containing 50 mg / L kanamycin. The culture was shaken at 30°C for 15 hours. 4.4 mL of this culture was used to inoculate 200 mL of production medium containing LB. The medium was stirred at 37°C until an OD (600 nm) of 0.9 was obtained, after which 400 μL of 100 mM IPTG solution was added and the medium was stirred at 30°C for 4 hours. The cells were harvested by centrifugation and frozen. The resulting wet cell weight was 1.38 g.

[0193] The resulting precipitate was dissolved by adding 4.9 mL of "Bugbuster Master mix" (Novagen, ref. 71456) and 2.1 mL of water. The lysate was collected by centrifugation and stored frozen.

[0194] Example 9 Preparation and activity of UGT76G1 produced by pMAL plasmid and BL21 expression strain After subcloning of the synthetic UGT76G1 gene into the pMAL plasmid using the Nde1 and Sal1 cloning sites, The UGT76G1 plasmid was transformed into the BL21 expression strain (New England Biolabs BL21 competent E. coli) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium (containing ampicillin). Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0195] A stock aliquot was thawed and added to 30 mL of LBGKP medium. This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L of "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 20°C while samples were taken to measure OD and pH. After 40 hours, the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 5.86 g.

[0196] 2.74 g of the resulting precipitate were dissolved by adding 9.6 mL of "Bugbuster Master Mix" (Novagen, ref. 71456) and 4.1 mL of water. The lysate was collected by centrifugation and stored frozen.

[0197] Example 10 Preparation and activity of UGT76G1 expressed by pMAL plasmid and ArcticExpress expression strain pMAL The UGT76G1 plasmid was transformed into the ArcticExpress expression strain (Agilent ArcticExpress Competent Cells) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of ampicillin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin and geneticin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0198] A stock aliquot was thawed and added to 30 mL of LBGKP medium (containing ampicillin and geneticin). This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L of "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 12°C while samples were taken to measure OD (600 nm) and pH. After 68 hours, the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 8.96 g.

[0199] 2.47 g of the resulting precipitate were dissolved by adding 8.73 mL of "Bugbuster Master Mix" (Novagen, ref. 71456) and 3.79 mL of water. The lysate was collected by centrifugation and stored frozen.

[0200] Example 11 Preparation and activity of UGT76G1 expressed by pCOLDIII plasmid and ArcticExpress expression strain After subcloning of the synthetic UGT76G1 gene into the pCOLDIII plasmid using the Nde1 and Xho1 cloning sites, The UGT76G1 plasmid was transformed into the ArcticExpress expression strain (Agilent ArcticExpress Competent Cells) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of ampicillin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin and geneticin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0201] A stock aliquot was thawed and added to 30 mL of LBGKP medium (containing ampicillin and geneticin). This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L of "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L of glycerol, and 50 mg / L of kanamycin. The medium was stirred at 12°C while samples were taken to measure OD (600 nm) and pH. After 63 hours, the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 6.54 g.

[0202] 2.81 g of the resulting precipitate was dissolved by adding 9.8 mL of "Bugbuster Master Mix" (Novagen, ref. 71456) and 4.2 mL of water. The lysate was collected by centrifugation and stored frozen.

[0203] Example 12 Preparation and activity of UGT76G1 prepared by pCOLDIII plasmid and Origami2(DE3) expression strain pCOLDIII The UGT76G1 plasmid was transformed into the Origami2(DE3) expression strain (Novagen Origami™2(DE3) competent cells) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0204] A stock aliquot was thawed and added to 30 mL of LBGKP medium (containing ampicillin). This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L of "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L glycerol, and 50 mg / L kanamycin. The medium was stirred at 12°C while samples were taken to measure OD (600 nm) and pH. After 68 hours, the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 2.53 g.

[0205] 1.71 g of the resulting precipitate was dissolved by adding 6.0 mL of "Bugbuster Master mix" (Novagen, ref. 71456) and 1.9 mL of water. The lysate was collected by centrifugation and stored frozen.

[0206] Example 13 Determination of activity Activity tests were performed at a 5 mL scale on 500 μL of thawed lysate for the conversion of stevioside to rebaudioside A and rebaudioside D to rebaudioside X using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl in 50 mM sodium phosphate buffer pH 7.2. Samples were taken and analyzed by HPLC. The results for various preparations of UGT76G1 are summarized in the following table.

[0207] [Table 2]

[0208] Example 14 50 mL Scale Reaction for the Conversion of Rebaudioside D to Rebaudioside X Five mL of the solubilized material from Example 12 was used in a 50 mL scale for the conversion of rebaudioside D to rebaudioside X. The reaction medium consisted of 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl, 2.5 mM UDP-glucose, and 0.5 mM rebaudioside D. After shaking the reaction mixture at 30°C for 90 hours, 50 mL of ethanol was added, and the resulting mixture was stirred at -20°C for 1 hour. After centrifugation at 5000g for 10 minutes, the supernatant was purified via ultrafiltration (Vivaflow MWCO 30000). 78 mL of permeate was obtained, and the remaining 9 mL was diluted with 9 mL of ethanol and subjected to ultrafiltration again (Vivaflow MWCO 30000). Another 14 mL of filtrate was obtained and combined with the first permeate. The combined permeates were concentrated under reduced pressure at 30°C until 32 mL of a clear solution was obtained.

[0209] The HPLC trace of the product mixture is shown in Figure 5. HPLC was performed on an Agilent 1200 series equipped with a binary pump, autosampler, and thermostatted column compartment. The method was isocratic with a mobile phase consisting of 70% water (0.1% formic acid): 30% acetonitrile. The flow rate was 0.1 μL / min. The column used was a Phenomenex Prodigy 5μ ODS(3) 100A; 250 x 2 mm. The column temperature was maintained at 40 °C. The injection volume was 20–40 μL.

[0210] Example 15 Preparation of UGT91D2 using pMAL plasmid and BL21 expression strain The synthetic UGT91D2 gene was subcloned into the pMAL plasmid using the Nde1 and Sal1 cloning sites, followed by pMAL The UGT91D2 plasmid was transformed into the BL21 expression strain (New England Biolabs BL21 competent E. coli) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0211] A stock aliquot was thawed and added to 30 mL of LBGKP medium. This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L of "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L of glycerol, and 50 mg / L of ampicillin. The medium was stirred at 20°C while samples were taken to measure OD and pH. After 40 hours, the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 12.32 g.

[0212] 2.18 g of the resulting precipitate were dissolved by adding 7.7 mL of "Bugbuster Master Mix" (Novagen, ref. 71456) and 3.2 mL of water. The solubilisate was collected by centrifugation and used immediately for activity testing.

[0213] Example 16 Preparation of UGT91D2 using pMAL plasmid and ArcticExpress expression strain pMAL The UGT91D2 plasmid was transformed into the ArcticExpress expression strain (Agilent ArcticExpress Competent Cells) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of ampicillin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin and geneticin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0214] A stock aliquot was thawed and added to 30 mL of LBGKP medium (containing ampicillin and geneticin). This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 20°C for 16 hours, then at 12°C for an additional 50 hours, while samples were taken to measure OD (600 nm) and pH. Cells were harvested by centrifugation and frozen. The resulting wet cell weight was 15.77 g.

[0215] 2.57 g of the resulting precipitate was dissolved by adding 9.0 mL of "Bugbuster Master Mix" (Novagen, ref. 71456) and 3.8 mL of water. The solubilisate was collected by centrifugation and used immediately for activity testing.

[0216] Example 17 Preparation of UGT91D2 using pET30a+ plasmid and Tuner(DE3) expression strain pET30a+ The UGT91D2 plasmid was transformed into the Tuner (DE3) expression strain (Novagen Tuner™ (DE3) competent cells) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium (containing kanamycin). Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0217] A stock aliquot was thawed and added to 100 mL of LB medium containing 50 mg / L kanamycin. The culture was shaken at 30°C for 15 hours. 6.2 mL of this culture was used to inoculate 500 mL of production medium containing LB. The medium was stirred at 37°C until an OD (600 nm) of 0.9 was obtained, after which 500 μL of 100 mM IPTG solution was added (IPTG concentration in the medium was 100 μM), the medium was stirred at 30°C for 4 hours, and the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 4.02 g.

[0218] 1.92 g of the resulting precipitate was dissolved by adding 6.8 mL of "Bugbuster Master mix" (Novagen, ref. 71456) and 2.8 mL of water. The solubilisate was collected by centrifugation and immediately tested for activity.

[0219] Example 18 Preparation of UGT91D2 using pET30a+ plasmid and ArcticExpress expression strain pET30a+ The UGT91D2 plasmid was transformed into the ArcticExpress(DE3) expression strain (Agilent ArcticExpress competent cells) by heat shock treatment. The resulting cells were grown in LB Agar medium in Petri dishes in the presence of kanamycin and geneticin. Suitable colonies were selected and grown in liquid LBGKP medium containing kanamycin and geneticin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0220] A stock aliquot was thawed and added to 30 mL of LBGKP medium (containing kanamycin and geneticin). This culture was shaken at 30°C for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L of "Overnight express instant TB medium" (Novagen, ref. 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 20°C for 16 hours and then at 12°C for an additional 50 hours, during which time samples were taken to measure OD (600 nm) and pH. After 60 hours, the cells were harvested by centrifugation and frozen. The resulting wet cell weight was 16.07 g.

[0221] 3.24 g of the resulting precipitate was dissolved by adding 11.4 mL of "Bugbuster Master mix" (Novagen, ref. 71456) and 4.8 mL of water. The solubilisate was collected by centrifugation and used immediately for activity testing.

[0222] Example 19 Determination of the activity of in vivo preparations of UGT91D2 Activity tests were performed on 1000 μL of lysate at a 5 mL scale for the conversion of rubusoside to stevioside using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl in 50 mM sodium phosphate buffer, pH 7.2. Samples were taken and analyzed by HPLC. The results for various preparations of UGT91D2 are summarized in the following table.

[0223] [Table 3]

[0224] Example 20 Isolation of Rebaudioside X The amount of the product mixture in Example 14 was not sufficient for separation via preparative HPLC. Therefore, analytical HPLC was used to separate the components of the mixture in a series of injections. The separation was performed according to the method described in Example 14 above and resulted in two fractions that correspond to the two major peaks in the HPLC trace in Figure 5: Fraction A (retention time 24.165 min) and Fraction B (retention time 31.325 min).

[0225] The retention time of fraction A was consistent with reb D, indicating unreacted starting material from the biotransformation reaction.

[0226] The retention time of purified fraction B (Figure 6) was consistent with reb X, indicating successful biotransformation from reb D. The identity of the material recovered in fraction B as reb X was confirmed by co-injection of purified fraction B with a reb X standard (available from Pure Circle; the HPLC trace of the reb X standard is shown in Figure 7). Both fraction B and the reb X standard were found to elute at the same retention time (Figure 8), indicating that fraction B was reb X.

[0227] The identity of fraction B as reb X was independently confirmed by NMR and HRMS. For sampling, fraction B was concentrated on a rotovapor, lyophilized, and dried for 40 hours at 40°C.

[0228] NMR samples were dissolved in deuterated pyridine (CDN) and spectra were acquired on a Varian Unity Plus 600 MHz instrument using standard pulse sequences. The NMR spectrum of Fraction B was compared to that of reb X. An overlay of the two spectra (FIG. 9) showed a match between the peaks of Fraction B and reb X. A table of NMR assignments for reb X is shown below:

[0229] [Table 4-1]

[0230] [Table 4-2]

[0231] [Table 4-3]

[0232] HRMS (Figure 10) was generated on a Waters Premier Quadropole Time-of-Flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source operated in positive ion mode. Samples were dissolved in methanol, eluted with 2:2:1 methanol:acetonitrile:water, and introduced by infusion using an on-board syringe pump. The presence of reb X was confirmed by the [M+Na]+ adduct at m / z 1313.5265, which corresponds to the molecular formula C56H90O33:

[0233] [ka]

Claims

1. 1. A method for adding at least one glucose unit to a steviol glycoside substrate to provide a target steviol glycoside, the method comprising contacting the steviol glycoside substrate with a biocatalytic protein enzyme comprising UGT76G1, wherein the steviol glycoside substrate is rebaudioside D and the target steviol glycoside is rebaudioside X.

2. 10. The method of claim 1, further comprising purifying rebaudioside X to a purity of greater than 80% by weight.

3. 10. The method of claim 1, further comprising purifying rebaudioside X to a purity of greater than 90% by weight.

4. 10. The method of claim 1, further comprising purifying rebaudioside X to a purity of greater than 95% by weight.

5. The method of claim 1, wherein the UDP-glucosyltransferase is expressed in a host microorganism.

6. 6. The method of claim 5, wherein the host microorganism is selected from the group consisting of E. coli, Saccharomyces sp., Aspergillus sp., and Pichia sp.

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