Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing, China; College of Chemical Engineering, Nanjing Forestry University, Nanjing, China; Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing, China.
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing, China; College of Chemical Engineering, Nanjing Forestry University, Nanjing, China; Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing, China.
Bioorg Chem. 2021 Jul;112:104926. doi: 10.1016/j.bioorg.2021.104926. Epub 2021 Apr 20.
Orientin and vitexin, important components of bamboo-leaf extracts, are C-glycosylflavones which exhibit a number of interesting biological properties. In this work, we developed an efficient biocatalytic cascade for orientin and vitexin production consisting of Trollius chinensis C-glycosyltransferase (TcCGT) and Glycine max sucrose synthase (GmSUS). In order to relieve the bottleneck of the biocatalytic cascade, the biocatalytic efficiency, reaction condition compatibilities and the ratio of the enzymes were determined. We found that the specific activity of TcCGT was significantly influenced by enzyme dose and Triton X-100 or Tween 20 (0.2%). Co-culture of BL21-TcCGT-Co and BL21-GmSUS-Co affected the catalytic efficiency of TcCGT and GmSUS, and the maximum orientin production rate reached 47 μM/min at the inoculation ratio of 9:1. The optimal pH and temperature for the biocatalytic cascade were pH 7.5 and 30 °C, respectively. Moreover, the high dose of the enzymes can improve the tolerance of biocatalytic cascade to substrate inhibition in the one-pot reaction. By using a fed-batch strategy, maximal titers of orientin and vitexin reached 7090 mg/L with a corresponding molar conversion of 98.7% and 5050 mg/L with a corresponding molar conversion of 97.3%, respectively, which is the highest titer reported to date. Therefore, the method described herein for efficient production of orientin and vitexin by modulating catalytic efficiencies of enzymes can be widely used for the C-glycosylation of flavonoids.
竹叶提取物中的重要成分——Orientin 和 Vitexin 是 C-糖苷黄酮，具有多种有趣的生物学特性。在这项工作中，我们开发了一种有效的生物催化级联反应，用于 Orientin 和 Vitexin 的生产，该反应由 Trollius chinensis C-糖基转移酶 (TcCGT) 和 Glycine max 蔗糖合酶 (GmSUS) 组成。为了缓解生物催化级联反应的瓶颈问题，我们确定了生物催化效率、反应条件兼容性和酶的比例。我们发现 TcCGT 的比活性受到酶剂量和 Triton X-100 或 Tween 20（0.2%）的显著影响。BL21-TcCGT-Co 和 BL21-GmSUS-Co 的共培养影响了 TcCGT 和 GmSUS 的催化效率，接种比例为 9:1 时，Orientin 的最大产率达到 47 μM/min。生物催化级联的最佳 pH 和温度分别为 pH 7.5 和 30°C。此外，高剂量的酶可以提高一锅反应中生物催化级联对底物抑制的耐受性。通过分批补料策略，最大 Orientin 和 Vitexin 的浓度分别达到 7090 mg/L，相应的摩尔转化率为 98.7%和 5050 mg/L，相应的摩尔转化率为 97.3%，这是迄今为止报道的最高浓度。因此，本文所述的通过调节酶催化效率来高效生产 Orientin 和 Vitexin 的方法可广泛应用于黄酮类化合物的 C-糖基化。
