Title : Highly efficient biotransformation of Astragaloside IV to Cycloastragenol by sugar-stimulated β-glucosidase and β-xylosidase from Dictyoglomusthermophilum

β-Glucosidases and β-xylosidases are two categories of enzymes that could 1 cleave out non-reducing terminal β-D-glucosyl and β-D-xylosyl residues with release 2 of D-glucose and D-xylose, respectively. In this paper, two functional β-glucosidase 3 Dth3 and β-xylosidase Xln-DT from Dictyoglomus thermophilum were heterologously 4 expressed in E.coli BL21 (DE3). Dth3 and Xln-DT were relatively stable at 75C and 5 were tolerant or even stimulated by glucose and xylose. Dth3 was highly tolerant to 6 glucose with a Ki value approximately 3 M. Meanwhile, it was not affected by xylose 7 in high concentration. The activity of Xln-DT was remarkably stimulated 2.13-fold by 8 1 M glucose and 1.29-fold by 0.3 M xylose, respectively. Furthermore, the β9 glucosidase Dth3 and β-xylosidase Xln-DT showed excellent selectivity to cleave the 10 outer C-6 and C-3 sugar moieties of ASI, which established an effective and green 11 method to produce the more pharmacological active CAG, an exclusive telomerase 12 activator. We measured temperature, pH and dosage of enzyme using a single-factor 13 experiment in ASI biotransformation. After optimization, the optimal reaction 14 conditions were as follow: 75C, pH 5.5, 1 U of Dth3 and 0.2 U of Xln-DT, respectively. 15 Under the optimized conditions, 1 g/L ASI was transformed into 0.63 g/L CAG with a 16 corresponding molar conversion of 94.5% within 3 h. This is the first report to use the 17 purified thermostable and sugar-tolerant enzymes from Dictyoglomus thermophilum to 18 hydrolysis ASI synergistically, which provides a specific, environment-friendly and 19 cost-effective way to produce CAG. 20