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

Abstract

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(β1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 β-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties.

Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate

“…18,21,31 Finally, the importance of residue Q1140 was shown in a study where Gtf180-ΔN was used to transglycosylate steviol glycosides. 15…”

“…1,3,10,11 These products are attracting strong interest for industrial applications as food or feed ingredients and in pharmacy and medicine. 12−15…”

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

“…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., ).…”

“…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. To elucidate the structure‐sweetness relationship, the stevioside or rebaudioside A derivatives produced by cyclomaltodextrin glucanotransferase, glucansucrase from L. citreum (Ko et al., ), β‐fructofuranosidase, or glucansucrase from Lactobacillus returi by transglucosylation (Te Poele et al., ). However, the structure‐sweetness relationship of various steviol derivatives remains to be elucidated.…”

Enzymatic Synthesis and Characterization of a Novel α-1→6-Glucosyl Rebaudioside C Derivative Sweetener

“…This α-1→6 glycosylated reb A compound was reported to have a superior taste profile, with significantly less bitterness than the parent reb A. In fact, the author claimed that it displayed almost no bitterness at all [15]. Reb A was also modified by using the method previously described for reb C, and the product is highly likely to contain the same α-1→6 glycosylated reb A, although it has not been fully characterized (Figure 3).…”