The thermostable GH3 family β-glucosidase DthBgl3 and thermostable GH78 family α-l-rhamnosidase DthRha from Dictyoglomus thermophilum DSM3960 were successfully immobilized by industrial amino resin 1000NH. The optimal reaction temperature and pH of 1000NH-DthBgl3 was 85 °C and pH 5.0. Over 65% residual enzyme activity maintained for 1000NH-DthBgl3 after a 3-h incubation under 85 °C, while about 20% residual enzyme activity maintained for free DthBgl3. The optimal reaction temperature and pH of 1000NH-DthRha was 95 °C and pH 6.5. Over 90% residual enzyme activity maintained for 1000NH-DthRha after a 3-h incubation under 90 °C, while about 22% residual enzyme activity maintained for free DthRha. Meanwhile, immobilized 1000NH-DthBgl3 and 1000NH-DthRha were successfully and could completely transform all major ingredient of 10 g/L total flavonoids extract from Epimedium (TFEE) into icaritin. After 15 cycles (45 h) of repeated use at 85 °C, 3 U 1000NH-DthBgl3 showed a molar conversion rate of 73.12%, initial activity of 30.09% and productivity of 124 mg/L/h, while 15 U 1000NH-DthRha showed a molar conversion rate of 88.50%, initial activity of 85.31% and productivity of 75 mg/L/h after ten cycles (60 h) of repeated use at 85 °C. The cooperation of two immobilized enzymes showed a molar conversion rate of 87.21% and productivity of 141 mg/L/h after 15 cycles of repeated use at 85 °C. This is the first report on immobilization of two thermostable glycosidases from thermophilic bacteria and their cooperated biocatalysis of all major ingredients of TFEE into icaritin with high productivity under a high substrate concentration.

Graphical Abstract

中文翻译：

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嗜热链球菌DSM3960的热稳定β-葡糖苷酶和α-1-鼠李糖苷酶的固定化及其淫羊med中总黄酮提取物协同生物转化为叶黄素的作用

嗜热双歧杆菌的热稳定GH3家族β-葡萄糖苷酶DthBgl3和热稳定GH78家族α- 1-鼠李糖苷酶DthRhaDSM3960成功地被工业氨基树脂1000NH固定。1000NH-DthBgl3的最佳反应温度和pH为85°C，pH为5.0。在85°C下孵育3小时后，对于1000NH-DthBgl3保留了超过65％的残留酶活性，而对于游离DthBgl3则保持了约20％的残留酶活性。1000NH-DthRha的最佳反应温度和pH为95°C和pH 6.5。在90°C下孵育3小时后，对于1000NH-DthRha保留了90％以上的残留酶活性，而对于游离DthRha则保留了约22％的残留酶活性。同时，成功地固定了1000NH-DthBgl3和1000NH-DthRha，并且可以将淫羊Epi总黄酮提取物（TFEE）中的10 g / L总黄酮的所有主要成分完全转化为伊卡立汀。在85°C下重复使用15个周期（45小时）后，3 U 1000NH-DthBgl3的摩尔转化率为73.12％，初始活性为30.09％，产率为124 mg / L / h，而15 U 1000NH-DthBgl3的摩尔转化率为88.50％，初始活性为85.31％在85°C下重复使用十个周期（60 h）后，生产率为75 mg / L / h。在85°C下重复使用15个循环后，两种固定化酶的协同作用显示摩尔转化率为87.21％，生产率为141 mg / L / h。这是关于将嗜热细菌中的两种热稳定糖苷酶固定化，以及它们在高底物浓度下以高生产率协同作用将TFEE的所有主要成分生物催化合成二十碳叉蛋白的报道。在85°C重复使用十个周期（60 h）后，初始活性为85.31％，生产率为75 mg / L / h。在85°C下重复使用15个循环后，两种固定化酶的协同作用显示摩尔转化率为87.21％，生产率为141 mg / L / h。这是关于将嗜热细菌中的两种热稳定糖苷酶固定化，以及它们在高底物浓度下以高生产率协同作用将TFEE的所有主要成分生物催化合成二十碳叉蛋白的报道。在85°C重复使用十个周期（60 h）后，初始活性为85.31％，生产率为75 mg / L / h。在85°C下重复使用15个循环后，两种固定化酶的协同作用显示摩尔转化率为87.21％，生产率为141 mg / L / h。这是关于将嗜热细菌中的两种热稳定糖苷酶固定化，以及它们在高底物浓度下以高生产率协同作用将TFEE的所有主要成分合成为二十碳五烯酸的生物催化的首次报道。

图形概要