β-Glucosidase and β-Galactosidase-Mediated Transglycosylation of Steviol Glycosides Utilizing Industrial Byproducts

Abstract

Stevia rebaudiana Bertoni is a plant cultivated worldwide due to its use as a sweetener. The sweet taste of stevia is attributed to its numerous steviol glycosides, however, their use is still limited, due to their bitter aftertaste. The transglycosylation of steviol glycosides, aiming at the improvement of their taste, has been reported for many enzymes, however, glycosyl hydrolases are not extensively studied in this respect. In the present study, a β-glucosidase, MtBgl3a, and a β-galactosidase, TtbGal1, have been applied in the transglycosylation of two steviol glycosides, stevioside and rebaudioside A. The maximum conversion yields were 34.6 and 33.1% for stevioside, while 25.6 and 37.6% were obtained for rebaudioside A conversion by MtBgl3a and TtbGal1, respectively. Low-cost industrial byproducts were employed as sugar donors, such as cellulose hydrolyzate and acid whey for TtbGal1- and MtBgl3a- mediated bioconversion, respectively. LC-HRMS analysis identified the formation of mono- and di- glycosylated products from stevioside and rebaudioside A. Overall, the results of the present work indicate that both biocatalysts can be exploited for the design of a cost-effective process for the modification of steviol glycosides.

Transglycosylation of Stevioside by a Commercial β-Glucanase with Fungal Extracted β-Glucans as Donors

“…The obtained conversion yields were similar to those obtained from other glucosidases from our previous work [ 16 , 33 ]. Generally, the transglycosylation yields obtained from glucosidases are low, from 15 to 30%, and modern approaches include protein engineering and directed evolution in order to obtain mutant enzymes with minimal hydrolytic activity [ 38 , 39 ].…”

“…Specifically, for stevioside transglycosylation, the addition of β-linked glucosyl moieties to the molecule, could result to next-generation sweeteners with improved properties, and, most importantly, with minimal caloric value. β-linked glycosidic bonds are not hydrolysed in the human body (with an exception of β-linked galactose in the lactose molecule, catalysed by human β-galactosidase in the majority of adults), and therefore, the insertion of β-linked glucose to the stevioside molecule retains the caloric value of the product, and concomitantly improves the sensory characteristics, as described in previous studies [ 2 , 7 , 16 , 17 ]. Therefore, it is of high importance to uncover the potential of already commercially available biocatalysts in inserting β-bonds to the desired products.…”

“…Up to 24 h, the reduction in stevioside concentration is increasing, reaching 12.9 ± 0.8% stevioside conversion. This is rather unusual for glycosyl hydrolases, since most of them hydrolyze their transglycosylation products in prolonged incubation times [ 16 , 33 ]. In Fig.…”

“…Previous efforts towards the production of unconventional steviol glucosides include the use of glycoside hydrolases [ 16 , 17 ] for the introduction of alternative sugars in the stevioside backbone, as candidates for new bioactive compounds. Glycoside hydrolases are represented in microbial genomes from genes coding for a variety of enzymes with different specificities including enzymes for the degradation of β-1,3, β-1,6, β-1,3/1,4 β-1,3/1,6 glycosidic bonds in polymeric substrates.…”