Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China; College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China.
Jiangsu Kanion Pharmaceutical Co., Ltd., 58 Haichang South Road, Lianyungang, Jiangsu 222001, China.
Enzyme Microb Technol. 2023 Jan;162:110131. doi: 10.1016/j.enzmictec.2022.110131. Epub 2022 Sep 15.
Icariin, the most effective bioactive component in Epimedium, is also the index component of Epimedium quality control in Pharmacopoeia. It was a very attractive approach for bioconversion from epimedin C to icariin. However, its potential was impeded by poor stability and non-recyclable properties of free enzymes. In this study, we have fused the linker (4LP) to α-L-rhamnosidase BtRha and successfully prepared the immobilized enzyme (incubated 4LP-BtRha@Na-Y) to produce icariin from epimedin C. The activity recovery of 4LP-BtRha@Na-Y was 79.6 %, and enzyme activity was 209.8 U/g, which was 1.75-fold and 1.6-fold higher than that of immobilized BtRha (BtRha@Na-Y), respectively. The optimal reaction temperature and pH of 4LP-BtRha@Na-Y was 55 °C and 6.5, respectively. The thermal stability of immobilized enzyme was significantly improved by incubation in phosphate buffer containing 20 % glycerol and 10 % fructose. The k/K value of incubated 4LP-BtRha@Na-Y was 7.98 × 10 sM, which increased by 8 % compared with free BtRha. Finally, under suitable conditions, 1 g/L epimedin C was transformed into icariin with icariin yield 75.1 %, and the relative conversion rate retained 74.9 % after reused 13 cycles. This experiment provides a new idea for one-step purification and immobilization of α-L-rhamnosidase for direct biotransformation from epimedin C to icariin, which will have great prospects in food and pharmaceutical production.
朝藿定 C 是淫羊藿中最有效的生物活性成分，也是《中国药典》中淫羊藿质量控制的指标成分。将朝藿定 C 转化为淫羊藿苷是一种非常有吸引力的方法。然而，由于游离酶的稳定性差和不可回收性，其潜力受到了阻碍。在这项研究中，我们将接头（4LP）与 α-L-鼠李糖苷酶 BtRha 融合，并成功制备了固定化酶（孵育 4LP-BtRha@Na-Y），用于从朝藿定 C 生产淫羊藿苷。4LP-BtRha@Na-Y 的酶活回收率为 79.6%，酶活为 209.8 U/g，分别比固定化 BtRha（BtRha@Na-Y）提高了 1.75 倍和 1.6 倍。4LP-BtRha@Na-Y 的最佳反应温度和 pH 值分别为 55°C 和 6.5。在含有 20%甘油和 10%果糖的磷酸盐缓冲液中孵育可显著提高固定化酶的热稳定性。孵育的 4LP-BtRha@Na-Y 的 k/K 值为 7.98×10 sM，比游离 BtRha 提高了 8%。最后，在合适的条件下，1 g/L 的朝藿定 C 转化为淫羊藿苷，淫羊藿苷的产率为 75.1%，重复使用 13 次后相对转化率保留 74.9%。该实验为α-L-鼠李糖苷酶的一步纯化和固定化提供了新思路，可直接将朝藿定 C 转化为淫羊藿苷，在食品和医药生产中具有广阔的前景。
