Co-innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China.
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China.
Bioorg Chem. 2018 Dec;81:461-467. doi: 10.1016/j.bioorg.2018.08.004. Epub 2018 Sep 6.
In this study, a α-l-rhamnosidase gene from Bacteroides thetaiotaomicron VPI-5482 was cloned and expressed in Escherichia coli. The specific activity of rhamnosidase was 0.57 U/mg in LB medium with 0.1 mM Isopropyl β-d-Thiogalactoside (IPTG) induction at 28 °C for 8 h. The protein was purified by Ni-NTA affinity, which molecular weight approximately 83.3 kDa. The characterization of BtRha was determined. The optimal activity was at 55 °C and pH 6.5. The enzyme was stable in the pH range 5.0-8.0 for 4 h over 60%, and had a 1-h half-life at 50 °C. The Kcat and Km for p-nitrophenyl-α-l-rhamnopyranoside (pNPR) were 1743.29 s and 2.87 mM, respectively. The α-l-rhamnosidase exhibited high selectivity to cleave the α-1,2 and α-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively, which could hydrolyze rutin, hesperidin, epimedin C and 2″-O-rhamnosyl icariside II. Under the optimal conditions, BtRha transformed epimedin C (1 g/L) to icariin by 90.5% in 4 h. This study provides the first demonstration that the α-l-rhamnosidase could hydrolyze α-1,2 glycosidic bond between rhamnoside and rhamnoside.
在这项研究中，从拟杆菌属 VPI-5482 中克隆并在大肠杆菌中表达了 α-l-鼠李糖苷酶基因。在含有 0.1mM异丙基-β-d-硫代半乳糖苷（IPTG）的 LB 培养基中，28°C 诱导 8 小时后，鼠李糖苷酶的比活为 0.57 U/mg。该蛋白通过 Ni-NTA 亲和层析进行纯化，分子量约为 83.3kDa。确定了 BtRha 的特性。最佳活性为 55°C 和 pH6.5。该酶在 pH5.0-8.0 范围内 4 小时内稳定，在 50°C 下半衰期为 1 小时。对 p-硝基苯基-α-l-鼠李吡喃糖苷（pNPR）的 Kcat 和 Km 分别为 1743.29s 和 2.87mM。α-l-鼠李糖苷酶对裂解鼠李糖苷和鼠李糖苷之间的α-1,2 和α-1,6 糖苷键、鼠李糖苷和糖苷之间的α-1,2 和α-1,6 糖苷键具有很高的选择性，分别可以水解芦丁、橙皮苷、淫羊藿苷 C 和 2″-O-鼠李糖苷淫羊藿苷 II。在最佳条件下，BtRha 在 4 小时内将淫羊藿苷 C（1g/L）转化为 90.5%的淫羊藿苷。本研究首次证明 α-l-鼠李糖苷酶可以水解鼠李糖苷和鼠李糖苷之间的α-1,2 糖苷键。
