Abstract:β-glucosidase may stimulate hydrolysis of stevioside to produce rubusoside. However,there are three β-1,2-glucose residues in stevioside molecule, which may produce rubusoside, steviolbioside, steviol glucosyl ester, steviolmonoside and steviol during the hydrolysis. To date, the structures of by-products remain unclear, and the analysis of reaction route is even more limited. The reported production throughput is not high enough for industry production. β-glucosidase from Aspergillus niger was applied to catalyze the hydrolysis of stevioside for rubusoside production in high-throughput. This reaction route was analyzed through substrate specificity test, structural characterization of products and by-products, and analysis of real-time composition. The results indicate that the enzyme is specific to stevioside. The main hydrolysis product is rubusoside, and it is demonstrated for the first time that the by-product, steviol glucosyl ester, is directly produced by hydrolyzing sophorosyl group of stevioside instead of the hydrolysate of rubusoside.
