Methods for the Enzymatic Modification of Steviol Glycosides, Modified Steviol Glycosides Obtainable Thereby, and the Use Thereof as Sweeteners

Abstract

The present invention relates generally to the production of steviol glycosides. Provided is a method for enzymatically providing a modified steviol glycoside, comprising incubating a steviol glycoside substrate in the presence of sucrose and the glucansucrase GTF180 of Lactobacillus reuteri strain 180, or a mutant thereof having the desired transglycosylation activity. Also provided are modified steviol glycosides obtainable by a method of the invention, and the use thereof as low-glycemic sweetener.

Claims

1-12. (canceled)

13. A modified steviol glycoside obtainable by incubating a steviol glycoside substrate in the presence of sucrose as a glucose donor and the glucansucrase GTF180 of Lactobacillus reuteri strain 180, or a mutant thereof having the desired transglycosylation activity.

14. A modified steviol glycoside selected from the group consisting of (i) 13-({-D-glucopyranosyl-(1.fwdarw.2)-[-D-glucopyranosyl-(1.fwdarw.3)-]-D-glucopyranosyl}oxy)ent-kaur-16-en-19-oic acid -D-glucopyranosyl-(1.fwdarw.6)--D-glucopyranosyl ester; (ii) 13-({-D-glucopyranosyl-(1.fwdarw.2)-[-D-glucopyranosyl-(1.fwdarw.3)-]-D-glucopyranosyl}oxy)ent-kaur-16-en-19-oic acid -D-glucopyranosyl-(1.fwdarw.6)--D-glucopyranosyl-(1.fwdarw.6)--D-glucopyranosyl ester; and (iii) 13-({-D-glucopyranosyl-(1.fwdarw.2)-[-D-glucopyranosyl-(1.fwdarw.3)-]-D-glucopyranosyl}oxy)ent-kaur-16-en-19-oic acid -D-glucopyranosyl-(1.fwdarw.3)--D-glucopyranosyl-(1.fwdarw.6)--D-glucopyranosyl ester.

15. (canceled)

16. A sweetening composition comprising at least one modified steviol glycoside according to claim 13.

17. A consumable comprising at least one modified steviol glycoside according to claim 13, optionally combined with other sweetener and/or sweetness enhancer.

18. The consumable according to claim 17, selected from the group consisting of beverages, foodstuff, an oral care product, a tobacco product, a pharmaceutical products and nutraceutical products.

19.-22. (canceled)

23. A sweetening composition comprising at least one modified steviol glycoside according to claim 14.

24. A consumable comprising at least one modified steviol glycoside according to claim 14, optionally combined with other sweetener and/or sweetness enhancer.

Description

LEGENDS TO THE FIGURES

[0065] FIG. 1. Chemical structures of Stevioside, Rebaudioside A and B, and Steviolbioside. [Glc1], etc. denotation of glucose residues for NMR assignment.

[0066] FIG. 2A-C. Effect of Rebaudioside A and sucrose concentrations on the transglucosylation (solid bars) and hydrolysis (hatched bars) activity using 0.12 mg/mL purified enzyme: (A) GTF180-N, (B) GTF180-N mutant 51137Y and (C) GTF180-N mutant Q1140E.

[0067] FIG. 3. NP-HPLC product profiles of a 4 hour incubation of 10 U/mL GTF180-N(WT), mutant S1137Y and mutant Q1140E with 50 mM Rebaudioside A(*) and 1 M sucrose (**=RebAG1).

[0068] FIG. 4A-C. Time course of Rebaudioside A utilization (dashed line) and RebAG1 formation by -glucosylation (solid line) using 10 U/mL enzyme: (A) GTF180-N, (B) GTF180-N mutant S1137Y and (C) GTF180-N mutant Q1140E, showing 55%, 73% and 96% conversion, respectively.

[0069] FIG. 5A-D. Structures of modified Rebaudioside A glucosides produced by GTF180-N and mutants GTF180-N S1137Y and GTF180-N Q1140E: (A) RebAG1=13-({-D-glucopyranosyl-(1.fwdarw.2)-[-D-glucopyranosyl-(1.fwdarw.3)-]-D-glucopyranosyl}oxy)ent-kaur-16-en-19-oic acid -D-glucopyranosyl-(1.fwdarw.6)--D-glucopyranosyl ester (B) 13-({-D-glucopyranosyl-(1.fwdarw.2)-[-D-glucopyranosyl-(1.fwdarw.3)-]-D-glucopyranosyl}oxy)ent-kaur-16-en-19-oic acid -D-glucopyranosyl-(1-6)--D-glucopyranosyl-(1.fwdarw.6)--D-glucopyranosyl ester (C) 13-({f-D-glucopyranosyl-(1.fwdarw.2)-[-D-glucopyranosyl-(1.fwdarw.3)-]-D-glucopyranosyl}oxy)ent-kaur-16-en-19-oic acid -D-glucopyranosyl-(1.fwdarw.3)--D-glucopyranosyl-(1.fwdarw.6)--D-glucopyranosyl ester (D) The 500-MHz .sup.1H NMR spectra of RebAG1 product of GTF180-N (a) and mutants Q1140E (b) and S1137Y (c) recorded in D.sub.2O at 334K.

[0070] FIG. 6. TLC analysis of products obtained in a 1 hour incubation of 50 mM Stevioside and 50 mM Rebaudioside A with 100 mM sucrose with and without 20 U/ml Q1140E, before and after alkaline saponification with 1 M NaOH.

[0071] FIG. 7. Sensory evaluation (n=12) of several sweeteners: Stevioside (250 mg/L), Rebaudioside A (300 mg/L), RebAG1 (350 mg/mL), and sucrose (60 g/L). Evaluated taste attributes were sweetness (hatched bars) and bitterness (solid bars). Score 0=not sweet/not bitter, score 5=very sweet/very bitter.

[0072] FIG. 8. TLC analysis of products obtained in a 2 hour incubation of 50 mM Rebaudioside A and 200 mM sucrose with GTF180-NV (Q), GTF180-NV Q1140 amino acid substitution mutants Q1140 G/S/I/V/W/D/M/C/E/L/K/T/P/R/F/N/Y, or GTF180-N Q1140E (E*).

[0073] FIG. 9A-C. Amino acid sequence of glucansucrase GTF180 from Lactobacillus reuteri 180. Panel (A) full length protein; panel (B)N-terminally truncated mutant GTF180-N; panel (C)N-terminally truncated and domain V truncated mutant GTF180-NV.

EXPERIMENTAL SECTION

[0074] The section below exemplifies the advantageous use of glucansucrase GTF180-N of Lactobacillus reuteri strain 180 and its derived single amino acid substitution mutants to -glucosylate Rebaudioside A. GTF180-N and derived mutant enzymes glucosylate Rebaudioside A specifically at the C-19 site, introducing (1.fwdarw.6) and (1.fwdarw.3) glycosidic linkages, which are resistant to hydrolysis by the amylolytic enzymes present in saliva. Several GTF180-N mutants displayed a much higher transglucosylating activity towards Rebaudioside A than GTF180-N. Interestingly, one mutant, Q1140E, showed nearly 100% conversion of Rebaudioside A and attached mostly a single (1.fwdarw.6)-glucose at the C-19 site of Rebaudioside A.

[0075] The produced novel Rebaudioside A glucosides are very interesting, carrying one and two (1.fwdarw.6)-linked glucose units specifically at the C-19 -linked glucose. The mono--glucosylated Rebaudioside A product, RebAG1, has an increased and more natural sweetness and reduced bitterness compared to Rebaudioside A. These improved novel steviol glycosides of the invention are of great interest as functional food ingredients.

Materials and Methods

Steviol Glycoside Substrates

[0076] Rebaudioside A (2) and Stevioside (1) were purchased from Sigma Aldrich.

Glucansucrase Enzymes

[0077] All glucansucrase and fructansucrase enzymes were produced as described by Meng et al (2014) and purified as described by Kralj et al (2004b). GTF180-N is the 117 kDa N-terminally truncated (741 residues) fragment of the GTF180 full-length protein (Kralj et al. 2004a). The construction of truncation mutant GTF180-NV, consisting of amino acids 794-1636 of the GTF180 enzyme is described in Meng et al. (2015a). GTF180-N mutant enzymes were created by van Leeuwen et al. (2009), Meng et al. (2015a), and Meng et al. (2015b). Amino acid substitutions in truncation mutant GTF180-NV were created as described by Meng et al. (2015b)

Enzyme Activity Assays

[0078] Enzyme activity assays were performed at 100 mM and 1000 mM sucrose, with and without 50 mM Rebaudioside A in 25 mM sodium acetate (pH 4.7); 1 mM CaCl.sub.2); and 0.12 mg/mL purified GTF180-N enzyme or GTF180-N mutant enzyme at 37 C. Samples of 100 L were taken every 30 sec for 4 min and the reaction was immediately stopped by incubating with 20 L 1000 mM NaOH for 30 min. The inactivated samples were diluted two times in deionized water and from 10 L of the diluted sample the glucose and fructose concentrations were determined enzymatically by monitoring the reduction of NADP with the hexokinase and glucose-6-phosphate dehydrogenase/phosphoglucose isomerase assay (Roche) as described previously (Mayer 1987). Quantitative determination of the release of glucose and fructose from sucrose allowed estimation of the activities of the glucansucrase enzymes (van Geel-Schutten et al. 1999). Fructose release corresponds to the total enzyme activity and glucose release to the hydrolytic activity. The transglycosylation activity can be obtained by subtracting the hydrolytic activity from the total activity. One unit (U) of enzyme is defined as the amount of enzyme required for producing 1 mol monosaccharide per min in a reaction mixture containing 25 mM sodium acetate (pH 4.7); 1 mM CaCl.sub.2; and 1000 mM sucrose at 37 C.

Enzymatic Glycosylation of Steviol Glycosides

[0079] Incubation reactions were performed in 25 mM sodium acetate (pH 4.7), 1 mM CaCl.sub.2), 50 to 1,000 mM sucrose, 50-100 mM steviol glycoside, and 2-30 U/mL purified GTF180-N enzyme or GTF180-N mutant enzyme at 37 C. for 15 min to 24 hours. Reactions were stopped by heat inactivation (100 C. for 15 min). From the inactivated samples 250 uL was mixed with 1000 ul of 10 mM catechol (internal standard) and subsequently purified by solid phase extraction using Strata-X 33u Polymeric Reversed Phase columns (Phenomenex). For HPLC analysis 20 L of the purified sample was injected on a Luna 10 m NH2 chromatography column (250 mm4.6 mm; Phenomenex). Reaction components were separated at a flow-rate of 1 mL/min under gradient elution conditions, starting with a 2 min isocratic step of 70% solvent A followed by a linear gradient from 70 to 55% solvent A over 9 min (solvent A=acetonitrile; solvent B=0.025% acetic acid). Rebaudioside A and the mono--glucosylated Rebaudioside A product concentrations were determined with NP-HPLC, using their corresponding calibration curves ranging from 1.56 to 50 mM. All data were normalized with catechol as internal standard. The standard deviation of the response was less than 5%. All NP-HPLC analyses were performed on an UltiMate 3000 chromatography system (ThermoFischer Scientific, Amsterdam, The Netherlands), equipped with an Endurance autosampler (Spark Holland, The Netherlands).

Quantitative Synthesis of -Glucosylated Rebaudioside A Products

[0080] For quantitative synthesis of -glucosylated Rebaudioside A products using GTF180-N and its derived mutants, incubations were performed in 5 mL 25 mM sodium acetate (pH 4.7), 1 mM CaCl.sub.2), 50 mM steviol glycoside with two batches of 1,000 mM equivalent of sucrose donor (t=0 and 3 h) to a total of 2,000 mM sucrose, using 10 U/mL enzyme at 37 C. for 24 hours. Products were purified from the incubation mixture by solid phase extraction using Strata-X 33u Polymeric Reversed Phase columns (Phenomenex). Products were separated on a Luna 10 m NH2 semi-preparative chromatography column (250 mm10 mm, Phenomenex) and were manually collected at a flow-rate of 4.6 mL/min, starting with a 2 min isocratic step of 80% solvent A followed by a linear gradient of 80 to 50% solvent A over 38 min (solvent A=acetonitrile; solvent B=0.025% acetic acid). The solvent of the collected fractions was evaporated under a stream of nitrogen gas and the dried materials were dissolved in deionized water.

Thin-Layer Chromatography

[0081] Samples were spotted on TLC sheets (Merck Kieselgel 60 F254, 2020 cm), which were developed with n-butanol:acetic acid:water=2:1:1. Spots were visualized by orcinol/sulfuric acid staining and compared with a simultaneous run of standard compounds.

Alkaline Saponification

[0082] To release the 19-O-linked glycosyl moiety, 4 mg of each steviol glycoside product was dissolved in 1 M NaOH (1 mL) and heated at 80 C. for 2.5 h.

Mass Spectrometry

[0083] Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) experiments were performed using an Axima mass spectrometer (Shimadzu Kratos Inc., Manchester, UK) equipped with a Nitrogen laser (337 nm, 3 ns pulse width). Positive-ion mode spectra were recorded using the reflector mode at a resolution of 5000 FWHM and delayed extraction (450 ns). Accelerating voltage was 19 kV with a grid voltage of 75.2%. The mirror voltage ratio was 1.12 and the acquisition mass range was 200-6000 Da. Samples were prepared by mixing 1 L sample solutions with 1 L 10% 2,5-dihydroxybenzoic acid in 70% ACN as matrix solution.

NMR Spectroscopy

[0084] Resolution-enhanced 1D/2D 500-MHz .sup.1H NMR spectra were recorded in D.sub.2O on a Bruker DRX-500 spectrometer (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University) at a probe temperature of 334K. Data acquisition and processing were done with Bruker Topspin 2.1. Prior to analysis, samples were exchanged twice in D.sub.2O (99.9 atom % D, Cambridge Isotope Laboratories, Inc., Andover, Mass.) with intermediate lyophilization, and then dissolved in 0.6 mL D.sub.2O. Suppression of the deuterated water signal (HOD at 4.40 ppm) was achieved by applying a WEFT (water eliminated Fourier transform) pulse sequence for 1D NMR experiments and by a pre-saturation of 1 s during the relaxation delay in 2D experiments. The 2D TOCSY spectra were recorded using an MLEV-17 (composite pulse devised by Levitt et al (1982)) mixing sequence with spin-lock times of 40-200 ms. The 2D ROESY spectra were recorded using standard Bruker XWINNMR software with mixing time of 200 ms. The carrier frequency was set at the downfield edge of the spectrum in order to minimize TOCSY transfer during spin-locking. Natural abundance 2D .sup.13C-.sup.1H HSQC experiments (.sup.1H frequency 500.0821 MHz, .sup.13C frequency 125.7552 MHz) were recorded without decoupling during acquisition of the .sup.1H FID. Resolution enhancement of the spectra was performed by a Lorentzian-to-Gaussian transformation for 1D spectra or by multiplication with a squared-bell function phase shifted by /(2.3) for 2D spectra, and when necessary, a fifth order polynomial baseline correction was performed. Chemical shifts () are expressed in ppm by reference to internal acetone ( 2.225 for .sup.1H and 31.07 for .sup.13C)

Sensory Evaluation of Novel -Glucosylated Products of Rebaudioside A

[0085] Taste evaluations were performed in which novel -glucosylated products of Rebaudioside A (350 mg/L) were compared to sucrose (60 g/L), Rebaudioside A (300 mg/L), and stevioside (250 mg/L). In a blind test, twelve test persons that were able to perceive the bitter aftertaste of steviol glycosides were asked to rate sweetness and bitterness on a scale from 0 to 5, with 0 indicating not sweet/not bitter and 5 indicating very sweet/very bitter.

Results

Screening Glucan and Fructansucrase Enzymes for -Glucosylation of Rebaudioside A

[0086] Over a hundred glucan and fructansucrase wild type and mutant enzymes, mostly from Lactobacillus reuteri, were screened for Rebaudioside A -glucosylation. For this, enzymes were incubated in 50 mM Rebaudioside A (FIG. 1) and 1000 mM sucrose for 3 hours. HPLC and TLC analysis of the reaction mixtures showed that only GTF180-N enzyme and mutant enzymes of GTF180-N were able to glucosylate Rebaudioside A (Table 1). Interestingly, two single GTF180-N mutants S1137Y and Q1140E, which are single amino acid substitutions close to the transition state stabilizing residue D1136 (van Leeuwen et al. 2009) displayed much better transglucosylating activity towards Rebaudioside A than GTF180-N. Also GTF180-N mutants L981A and W1065L (Meng et al. 2015b) were able to a-glucosylate Rebaudioside A (Table 1), but showed almost no polymerization (i.e. oligosaccharide and glucan formation from sucrose) activity (data not shown). This is a clear advantage during downstream processing, the purification of the -glucosylated Rebaudioside A products from mono- and disaccharides, oligosaccharides and glucans. By eliminating -glucan synthesis, the most important side reaction, higher glycosylation yields were obtained for Rebaudioside A. At lower sucrose concentration (200 mM), these mutants had similar or even higher transglucosylating activity with Rebaudioside A than GTF180-N and mutants S1137Y and Q1140E (data not shown).

TABLE-US-00001 TABLE 1 Overview of the Rebaudioside A -glucosylation potential of glucansucrase and fructansucrase enzymes from various Lactobacillus strains. Activity Enzyme Mutation *Glp (1.fwdarw. .fwdarw.3)Glp (.fwdarw. .fwdarw.4)Glp (.fwdarw. .fwdarw.6)Glp (.fwdarw. .fwdarw.3,6)Glp (.fwdarw. on RebA GTF180-N.sup.a N-terminal truncated 12 24 52 12 + GTF180.sup.a (**AY697430); Met-Gly-742-1772-His.sub.6 GTF180-NV.sup.b domain V deletion mutant of 12 23 52 13 + GTF180-N; Met-794-1636-His.sub.6 GTF180-N-PNNS.sup.c triple amino acid mutant 18 10 12 42 18 + (V1027P: S1137N: A1139S) of GTF180-N GTF180-N-SNAE.sup.d single amino acid mutant 12 16 2 52 18 ++++++ (Q1140E) of GTF180-N GTF180-N-SNAA.sup.d single amino acid mutant 11 6 69 14 +/ (Q1140A) of GTF180-N GTF180-N-SNAH.sup.d single amino acid mutant 8 8 76 8 +/ (Q1140H) of GTF180-N GTF180-N-NNA.sup.d single amino acid mutant 12 26 3 47 12 ++ (S1137N) of GTF180-N GTF180-N-YDA.sup.d double amino acid mutant 19 23 7 31 20 ++++ (S1137Y: N1138D) of GTF180-N GTF180-N-YNA.sup.d single amino acid mutant 18 21 4 39 18 ++++ (S1137Y) of GTF180-N GTF180-N-SDA.sup.d single amino acid mutant 10 24 56 10 + (N1138D) of GTF180-N GTF180-N-XM1.sup.e single amino acid mutant ++ (L981A) of GTF180-N GTF180-N-XM2.sup.e single amino acid mutant ++ (W1065L) of GTF180-N GTFA-N.sup.f N-terminal truncated 9 46 34 12*** **** GTFA (AX306822).sup.g GTFA-N N1134S.sup.h single amino acid mutant 8 12 76 4*** (N1134S) of GTFA-N GTFA-N N1134E.sup.h single amino acid mutant 8 49 36 7*** (N1134E) of GTFA-N GTFA-N N1134A.sup.h single amino acid mutant 13 25 49 13*** (N1134A) of GTFA-N GTFA-N NEV.sup.h double amino acid mutant 10 49 29 12*** (N1135E: S1136V) of GTFA-N GTFB.sup.a wild type (AY697435) GTFMLI.sup.a N-terminal truncated 47 10 26*** GTFMLI (AY697431).sup.a GTFO.sup.i N-terminal truncated 67 13 15 GTFO (AY911856).sup.i InuJ.sup.j N-terminal truncated InuJ.sup.j fructansucrase InuGA-RM.sup.k wild type fructansucrase InuGB-R.sup.k wild type fructansucrase LevG-R.sup.k wild type fructansucrase .sup.aKralj et al (2004a); .sup.bMeng et al (2015a); .sup.cVan Leeuwen et al (2008); .sup.dVan Leeuwen et al (2009); .sup.eMeng et al. (2015b); .sup.fKralj et al (2004b); .sup.gKralj et al (2002); .sup.hKralj et al (2006); .sup.iKralj et al (2005); .sup.jAnwar et al (2008); .sup.kAnwar et al (2010); *linkage distribution; **Genbank accession number; ***.fwdarw.4,6)Glp (.fwdarw.; ****not active on Rebaudioside A (RebA)

[0087] To optimize the reaction conditions towards glucosylation of Rebaudioside A the effect of Rebaudioside A and sucrose on the transglucosylation activity of GTF180-N and mutants S1137Y and Q1140E was determined. For this, enzyme activity assays were performed at 100 mM and 1000 mM sucrose with and without 50 mM Rebaudioside A. All three enzymes were more hydrolytic at low sucrose concentrations (FIG. 2). Both mutant enzymes were almost completely hydrolytic at 100 mM sucrose. However, when 50 mM Rebaudioside A was added to the reaction or when the sucrose concentration was increased to 1000 mM, the transglucosylation activity of all three enzymes was noticeably increased, showing the highest overall activity and highest transglucosylation to hydrolysis ratio at 1 M sucrose and 50 mM Rebaudioside A. These reaction conditions were used to follow the -glucosylation of Rebaudioside A by GTF180-N and mutants S1137Y and Q1140E in more detail.

[0088] When 50 mM Rebaudioside A, 1000 mM sucrose, and 10 U/mL enzyme was used, mutants S1137Y and Q1140E glucosylated respectively 73% and 96% Rebaudioside A compared to 55% by GTF180-N (FIG. 4). To our surprise, mutant Q1140E mainly produced mono-glucosylated Rebaudioside A (RebAG1), yielding 35 mM RebAG1 from 50 mM Rebaudioside A (FIG. 3 and FIG. 4). Mutant S1137Y produced a similar amount of RebAG1 as the GTF180-N enzyme (13 mM) (FIG. 3), but synthesized a higher amount of multiple glycosides (FIG. 3).

[0089] From all the tested glucan- and fructansucrase enzymes, mutant GTF180-N Q1140E showed the highest Rebaudioside A glucosylation activity and displayed mainly mono-glucosylation of Rebaudioside A. Therefore, besides mutations Q1140 E/A/H also additional amino acid substitutions at position Q1140 were created in mutant GTF180-NV and tested for Rebaudioside A glucosylation (FIG. 8). For this, 1 mg/ml of the enzymes was incubated for 2 hours at 37 C. with 50 mM Rebaudioside A and 200 mM sucrose in buffer (25 mM sodium acetate (pH 4.7), 1 mM CaCl.sub.2). The incubation mixtures were analyzed by TLC (FIG. 8). Under these conditions, several Q1140 substitution mutants (for e.g. Q1140 F/N/Y) showed even a higher Rebaudioside A glucosylation than mutant Q1140E, although the latter one still had the highest mono-glucosylation yield. Some mutations (for e.g. Q1140D) had a slightly negative effect on Rebaudioside A glucosylation. Interestingly, mutant Q1140R showed almost no activity on sucrose, although Rebaudioside A glucosylation was hardly affected by the mutation. It appears that the sucrose was mainly used for glucosylation of Rebaudioside A and not for side reactions, such as oligosaccharide formation.

Isolation and Characterization of -Glucosylated Products of Rebaudioside A Glucosides Produced by GTF180-N and GTF180-N Mutants S1137Y and Q1140E

[0090] Looking at the molecular structure of Rebaudioside A (FIG. 1), there are four Glcp residues (Glc1, Glc2, Glc3 and Glc4) with a total of 14 free hydroxyl groups, which can act as acceptors for transglucosylation.

[0091] GTF180-N converts sucrose into oligo- and polysaccharides, catalyzing the transglucosylation of Glcp residues in (1.fwdarw.3)- and (1.fwdarw.6)-linkages (van Leeuwen et al. 2008), there are 3 potential (1.fwdarw.3) sites and 4 potential (1.fwdarw.6) sites present at Rebaudioside A for the first attachment of a Glc residue.

[0092] In order to isolate -glucosylated products of Rebaudioside A glucosides for structural characterization, incubations were done with 10 U/mL enzyme with 50 mM Rebaudioside A and 1000 mM sucrose. After 3 hours 1000 mM extra sucrose was added to the reaction mixtures and incubated for an additional 21 hours. Glucosides were isolated from the reaction mixtures using semi-preparative NP-HPLC. Interestingly, NMR structural analysis and methylation analysis of the mono--glucosylated product showed that GTF180-N and mutants S1137Y and Q1140E specifically and only glucosylated Rebaudioside A at the C-19-linked glucose, attaching an (1.fwdarw.6)-linked glucose (100%), yielding RebAG1 (see also FIG. 5A).

##STR00001##

[0093] The di-glucosylated Rebaudioside A products of GTF180-N and mutant S1137Y were both linear elongations of the structure of RebAG1 with an (1.fwdarw.3)-linked glucose (75%) (FIG. 5C) or another (1.fwdarw.6)-linked glucose (25%) (FIG. 5B). FIG. 5D panels (a), (b) and (c) show the 500-MHz 1H NMR spectra of RebAG1 produced by, respectively, GTF180-N, GTF180-N Q1140E and GTF180-N S1137Y.

[0094] To confirm that introduction of extra Glcp residues via transglucosylation by GTF180-N only occurred on the C-19 -glucosyl moiety of Rebaudioside A, the isolated fractions were subjected to an alkaline saponification to specifically hydrolyze the 19-carboxyl-glucosyl ester bound (FIG. 1 and FIG. 6). Identical products were obtained from Rebaudioside A and the -glucosylated products of Rebaudioside A, according to TLC, MALDI-TOF-MS (m/z 826 [M+H]+) and NMR spectroscopy, being Rebaudioside A missing the complete glucosyl moiety at C-19. The resultant structure is known as Rebaudioside B (3) (FIG. 1).

C-19-Site Specific -Glucosylation of Stevioside by GTF180-N Mutant Q1140E

[0095] For commercial purposes it may be desirable to improve sweetness and decrease bitterness of the whole steviol glycoside leave extract. Since Stevioside (5-10% w/w of dried leaves) is the most abundant and one of the most bitter tasting steviol glycosides, our aim was also to enhance the taste profile of Stevioside. Therefore, glucosylation reactions were also performed with GTF180-N and substitution mutants derived thereof with Stevioside as the acceptor molecule. All three enzymes were also able to -glucosylate stevioside. As observed with Rebaudioside A as acceptor molecule, mutant Q1140E converted Stevioside mainly into a single mono--glucosylated product (data not shown). In order to determine whether GTF180-N mutant Q1140E glucosylates Stevioside also specifically at the C-19 site, steviol glucosides produced by Q1140E were subjected to alkaline saponification of the 19-carboxyl-glucosyl ester linkage with 1 M NaOH, to specifically remove the C-19 moiety. If alkaline saponification of the Q1140E steviol glucosides yields Steviolbioside (4)(FIG. 1) (i.e. Stevioside minus the C-19 moiety), then glucosylation occurred specifically at the C-19 site of Stevioside. On TLC plates multiple steviol glucosides were visible in the 1 hour incubation of Stevioside with Q1140E (FIG. 6). However, only one spot was observed when the incubation mixture was treated with 1 M NaOH and it migrated at the same height as the product formed after saponification of the Stevioside positive control. With MALDI-TOF analysis a molecular mass of 665.59 was detected, corresponding with the sodium-adduct of Steviolbioside. These results show that GTF180-N Q1140E also specifically -glucosylates Stevioside at the C-19 -linked glucosyl moiety.

Taste Evaluation of the Newly Synthesized -Glucosylated Products of Rebaudioside A

[0096] To determine the effect of (1.fwdarw.6) glucosylation at the 19-O-glucosyl moiety of Rebaudioside A on sweetness and bitterness, a taste evaluation was performed in which one the novel -glucosylated products of Rebaudioside A, RebAG1, was compared to Rebaudioside A. For this, in a blind test, twelve test persons that were able to perceive the bitter aftertaste of steviol glycosides were asked to rate sweetness and bitterness on a scale from 0 to 5, with 0 indicating not sweet/not bitter and 5 indicating very sweet/very bitter. A clear trend was observed showing that the novel RebAG1 had an increased and a more natural sweetness and reduced bitterness compared to Rebaudioside A (FIG. 7).

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