Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation

Abstract

Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation Gerwig, Gerrit; te Poele, Evelien; Dijkhuizen, Lubbert; Kamerling, Johannis P a b s t r a c tThe wild-type Gtf180-DN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the a-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically a-D-glucosylated at the steviol C-19 b-D-glucosyl moiety (55% conversion). The main product is a mono-(a1 / 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (a1 / 3)-and (a1 / 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties. IntroductionThe leaves of the Stevia rebaudiana BERTONI plant contain a high variety of sweet substances, called steviol glycosides [1,2], and so far more than 40 different structures have been elucidated (see review Ref. [3]). Stevioside (~5e20% w/w of dried leaves) and rebaudioside A (~2e5% w/w of dried leaves) are the most abundant components (Fig. 1), tasting about 200e300 times sweeter than sucrose (0.4% aqueous solution). Structurally, steviol glycosides have ent-13-hydroxykaur-16-en-19-oic acid as aglycone, called steviol, but are differing in carbohydrate composition at the C-13-tert-hydroxyl and C-19-carboxyl functions.Due to the growing awareness and concerns for human health related to excessive consumption of sugar (sucrose), the application of steviol glycosides as non-caloric bio-alternatives for sucrose and as substitutes for artificial (synthetic) sweeteners is strongly promoted nowadays [4e8]. Since a couple of years, steviol glycosides have been permitted for use as food additive and sweetener in the USA [9] and in Europe (E960) [10,11]. However, despite their intense sweetness and diverse beneficial pharmacological properties [12e15], the main drawback for successful commercialization of Stevia sweeteners is their slight bitterness and unpleasant (metallic) aftertaste, experienced by more than half of the human population.For natural steviol glycosides with b-D-glucopyranosyl units as constituents, it has been reported that the ratio of the number of glucose units at the C-13 site to that at the C-19 site of the steviol core has a relationship with the sweetness as well as with the quality of taste of the steviol glycosides [16,17]. To improve the Abbreviations: FID, free induction decay; FWHM, full width at half maximum; GLC-EI-MS, gas-liquid chromatography electron impact mass spectrometry; HPAEC, high-pH anion-exchange chromatography; HSQC, 1 H-detected hetero-nucl...

Glucansucrase (mutant) enzymes from Lactobacillus reuteri 180 efficiently transglucosylate Stevia component rebaudioside A, resulting in a superior taste

“…Recently, we have shown that the wild-type Gtf180-ΔN (N-terminally truncated) glucansucrase enzyme of L . reuteri 180 was able to glucosylate the steviol glycoside RebA, using sucrose as glucosyl donor substrate 23 . About 55% of RebA was glucosylated with up to eight α-D-glucosyl units attached (RebA-G).…”

Enzymatic Synthesis of Glucosyl Rebaudioside A and its Characterization as a Sweetener

“…The glucosylation of rebaudioside A by dextransucrase has many advantages. First, dextransucrase showed the highest conversion yield (approximately 86%) of rebaudioside A compared to another enzymatic synthesis (55% yield, Gerwig et al, 2017). Second, compared to other applied glucosyl transferases (Gerwig et al, 2017), dextransucrase yielded a single product with different regio-selective glucosyl derivative at the 19-carboxyl group of rebaudioside A.…”

“…First, dextransucrase showed the highest conversion yield (approximately 86%) of rebaudioside A compared to another enzymatic synthesis (55% yield, Gerwig et al., ). Second, compared to other applied glucosyl transferases (Gerwig et al., ), dextransucrase yielded a single product with different regio‐selective glucosyl derivative at the 19‐carboxyl group of rebaudioside A. Third, the transglucosylation product showed a lower precipitation than rebaudioside A in aqueous solutions after 15 days of incubation at room temperature.…”

“…Moreover, it has shown that plant‐derived glucosyltransferases such as UGT76G1 (Wang et al., ) can insert and produce rebaudioside A from stevioside in Saccharomyces cerevisiae (Olsson et al., ; Li et al., ) and Escherichia coli system (Wang et al., ). Production of rebaudioside I, rebaudioside D, and glucosyl rebaudioside A from rebaudioside A by enzymatic biotransformation has also been reported (Gerwig, Te Poele, Dijkhuizen & Kamerling, ; Te Poele et al., ; Chen et al., ). Lactobacillus reuteri 180 glucansucrase can transferred several glucosyl residues from one moiety to nine moieties into rebaudioside A from sucrose (Gerwig et al., ).…”

“…Production of rebaudioside I, rebaudioside D, and glucosyl rebaudioside A from rebaudioside A by enzymatic biotransformation has also been reported (Gerwig, Te Poele, Dijkhuizen & Kamerling, ; Te Poele et al., ; Chen et al., ). Lactobacillus reuteri 180 glucansucrase can transferred several glucosyl residues from one moiety to nine moieties into rebaudioside A from sucrose (Gerwig et al., ). Although there have been many biotransformation trials for stevioside or rebaudioside A, cyclomaltodextrin glucanotransferase is the most well‐known enzyme for stevioside derivatives and the sole one used in industrial production of enzymatically modified stevioside.…”