High efficiency transformation of stevioside into a single mono-glycosylated product using a cyclodextrin glucanotransferase from Paenibacillus sp. CGMCC 5316

Abstract

Stevioside is a non-caloric, natural, high-intensity sweetener. However, the bitter aftertaste of stevioside restricts its utilization for human consumption and limits its application in the food industry. In this study, a high efficiency enzymatic modification system was investigated to improve stevioside taste quality. A cyclodextrin glucanotransferase (CGTase) producing strain Paenibacillus sp. CGMCC 5316 was isolated from Stevia planting soil. With starch as glycosyl donor, this CGTase can transform stevioside into a single specific product which is an isomer of rebaudioside A and identified as mono-glycosylated stevioside. The taste of stevioside is improved noticeably by generating mono-glycosylated stevioside, which possesses a sucrose-like taste and has sweetness increased significantly by 35.4%. Next, the parameters influencing CGTase production were optimized. Compared to initial conditions, CGTase activity increased by 214.7% under optimum conditions of 3.9 g/L starch, 17.9 g/L tryptone, and 67.6 h of culture time, and the transglycosylation rate of stevioside was remarkably increased by 284.8%, reaching 85.6%. This CGTase modification system provides a promising solution for improving the sweetness and taste quality of stevioside. The efficiency of CGTase transformation can be greatly increased by optimizing the culture conditions of Paenibacillus sp. CGMCC 5316.

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

“…Therefore, significant research effort has been focused on the addition of glycosyl moieties in steviol glycosides, aiming at the improvement of their organoleptic properties. Cyclodextrin glucanotransferases (CGTases, 1,4-α-d-glucan: 1,4-α-d-glucopyranosyltransferase, cyclizing, EC 2.4.1.19) are the most common enzymes used for steviol glycosides transglycosylation, introducing α-1,4 glycosidic bonds with very high yields, exceeding 70% ( Li et al, 2013 ; Yu et al, 2015 ; Zhang et al, 2015 ). However, the hydrolysis of α- 1,4 bonds by human amylases can increase the caloric value of the modified steviosides, resulting in a final product with low appeal to target consumer groups, e.g., diabetics ( Gerwig et al, 2016 ).…”

Preparation of Crosslinked Enzyme Aggregates of a Thermostable Cyclodextrin Glucosyltransferase from Thermoanaerobacter sp. Critical Effect of the Crosslinking Agent

“…In addition to the cyclization reaction (CD production), this enzyme also catalyzes other two intermolecular transglycosylation reactions (coupling and disproportionation) and the hydrolysis reaction of the starch [3,5]. Although it is mainly used at industrial scale for the production of cyclodextrins, recent studies performed by Yu et al [6] have demonstrated that CGTase from Paenibacillus sp. CGMCC 5316 can be used to eliminate the bitter taste of a natural sweetener known as stevioside by a reaction of transglycosylation using starch as a glucose donor.…”

RQ3, A Natural Rebaudioside D Isomer, Was Obtained from Glucosylation of Rebaudioside A Catalyzed by the CGTase Toruzyme 3.0 L

“…For example, rebaudioside D (Figure 1s) and rebaudioside M, which carry two and three C-19 linked glycosyl units, respectively, both demonstrate improved edulcorant quality over that of stevioside or rebaudioside A. Hence, enzymatic glucosylation of stevioside and rebaudioside A have been extensively studied − to improve the edulcorant quality; ,− the resultant products are so-called glucosyl steviol glycosides (GSGs).…”

“…In industry, the most popular glucosyltransferases, such as CGTase and other α-amylase, have been extensively applied to catalyze the transglucosylation of stevioside or rebaudioside A with dextrins as glucosyl donors. ,,− ,,, The CGTase-intermediated reaction has multiple pathways that could yield many products via hydrolysis, coupling, and disproportionation . The obtained GSGs that possess a high percentage of glucosyl units are used as sweet flavoring agents in both perfumes and drug formulas .…”