Characterization of important new industrial enzymes CGTase and their application in the transglycosylation of stevia glycosides

Abstract

Background: Stevia rebaudiana is a key source of natural sweeteners, but its glycosides, such as stevioside, have limitations in terms of sweetness and taste. Cyclodextrin glycosyltransferase (CGTase), an enzyme that catalyzes cyclodextrin formation, has potential to enhance stevia glycosides  via transglycosylation. Alkalophilic Bacilli, which thrive in high-pH environments, have emerged as a promising source of CGTase. The present study focuses on the following objectives: firstly, the isolation and identification of CGTase-producing alkalophilic Bacilli; secondly, the characterization of the enzyme; and thirdly, the investigation of its application in improving stevia glycoside production, with a view to potentially leading to more efficient and sustainable sweeteners.

Objective: is to isolate and identify CGTases-producing new strains and to characterize the enzymes in terms of molecular weight, temperature, and pH optimization, heat stability, metal tolerance, and hydrolytic and cyclizing activities. 

Methods: The “Harikoshi” medium was used for the isolation and cultivation of CGTase-producing strains. To identify the strains under discussion, a molecular taxonomic investigation was undertaken. The purified enzymes were characterized using SDS-PAGE, electrophores method. The biochemical characteristics of the enzymes were determined using standard accepted methods.

Results: Based on 16S rRNA analysis, strains A-12 and A-19 isolated from saline soils were identified as Halalkalibacter akibai and Salipaludibacillus agaradhaerens, respectively. The maximum CGTase activity for strain A-12 was observed at 60°C, while for A-19 it was at 65°C. The pH optima for A-12 and A-19 CGTases were determined to be 8.5 and 9, respectively. In optimal conditions, the enzyme activities were 115U/mL (A-12) and 140U/mL (A-19). Subsequently, optimal conditions were developed for the transglycosylation of Stevia glycosides and the decolorization of the obtained products.                   

Conclusion: The purification and characterization of a novel CGTase from alkalophilic Bacilli has the potential to significantly impact the development of more efficient and sustainable stevia-based sweeteners. This research contributes to the potential industrial application of CGTases for improving the quality of natural sweeteners, with implications for the food and pharmaceutical industries. The study demonstrated that CGTases produced by alkalophilic cultures can function as effective biocatalysts in the enzymatic transglycosylation of stevia glycosides using starch as a donor. 

Keywords: Alkalophilic Bacilli, Cyclodextrin glycosyltransferase (CGTase), stevia glycosides, transglycosylation, stevioside modification, sustainable sweeteners