Efficient Bioconversion of Stevioside and Rebaudioside A to Glucosylated Steviol Glycosides Using an Alkalihalobacillus oshimesis-Derived Cyclodextrin Glucanotransferase

Abstract

The enzymatic transglycosylation of steviol glycosides can improve the edulcorant quality of steviol glycosides. Cyclodextrin glucanotransferase (CGTase) is one of the most popular glucanotransferases applied in this reaction. Herein, the CGTase-producing strain Alkalihalobacillus oshimensis CGMCC 23164 was isolated from Stevia planting soil. Using mass spectrometry-based secretome profiling, a high-efficiency CGTase that converted steviol glycosides to glucosylated steviol glycosides was identified and termed CGTase-13. CGTase-13 demonstrated optimal transglycosylation activity with 10 g/L steviol glycoside and 50 g/L soluble starch as substrates at <40 °C. Under the above conditions, the conversion rate of stevioside and rebaudioside A, two main components of steviol glycosides, reached 86.1% and 90.8%, respectively. To the best of our knowledge, this is the highest conversion rate reported to date. Compared with Toruzyme® 3.0 L, the commonly used commercial enzyme blends, glucosylated steviol glycosides produced using CGTase-13 exhibited weaker astringency and unpleasant taste, faster sweetness onset, and stronger sweetness intensity. Thus, CGTase provides a novel option for producing high-quality glucosylated steviol glycoside products and has great potential for industrial applications.

Engineering of cyclodextrin glycosyltransferase improves the conversion efficiency of rebaudioside A to glucosylated steviol glycosides and increases the content of short-chain glycosylated steviol glycoside

“…Among them, 5 CGTases exhibited relatively good transglycosylation activity. The best of the five was CGTase-13, followed by CGTase-8, CGTase-15, CGTase14, and CGTase-7 (data can be found in [ 44 ]).…”

“…CGTase-13 is a CGTase discovered by our laboratory with great potential in the production of glycosylated steviol glycosides. The glucosylated steviol glycoside product produced by CGTase-13 exhibited excellent taste [ 44 ]. Through mutation, its potential in the production of glucosylated steviol glycoside was further improved.…”

“…Sensory analysis was conducted by Zhucheng Haotian Pharmaceutical Co., Ltd. For details of the method, please refer to the paper by Zhang et al [ 44 ].…”