Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China; College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China.
College of Chemical Engineering, Nanjing Forestry University, 159 Long Pan Road, Nanjing 210037, China.
Enzyme Microb Technol. 2022 Aug;158:110040. doi: 10.1016/j.enzmictec.2022.110040. Epub 2022 Apr 6.
The α-L-rhamnosidase BtRha from Bacteroides thetaiotao VPI-5482 is a specific enzyme that selectively hydrolyzes the α-1,2 glycosidic bond between rhamnose and rhamnose, allowing the bioconversion of epimedin C to icariin. In this study, BtRha was molecularly modified using B-factor-saturation mutagenesis strategy and the introduction of disulfide bonds, resulting in a mutant with significantly improved catalytic efficiency, S592C, and two thermally stable mutants, E39W and E39W-S592C. The results showed that the half-lives of E39W and E39W-S592C at 55 °C were 10.4 and 9.4-fold higher, respectively, than that of the original enzyme, The mutant S592C showed a 63.3% reduction in K value and a 163.6% increase in catalytic efficiency (k/K value), which improved the ability to hydrolyze epimedin C to icariin effectively. In addition, high-level expression of α-L-rhamnosidase mutant S592C was established. With 0.1 mM IPTG as an inducer, induction temperature of 32 °C, induction pH of 7.0 and induction OD of 50, the maximum activity of mutant S592C reached 182.0 U/mL in terrific broth medium after 22 h. This is the highest enzyme activity of α-L-rhamnosidase which can convert epimedin C to icariin to date. All the results provide a specific and cost-effective α-L-rhamnosidase mutant, which will raise its potential interest for the food and pharmaceutical applications.
来自拟杆菌属的 α-L-鼠李糖苷酶 BtRha 是一种特异性酶，它选择性地水解鼠李糖和鼠李糖之间的 α-1,2 糖苷键，从而使淫羊藿素 C 转化为淫羊藿苷。在这项研究中，采用 B 因子饱和诱变策略和引入二硫键对 BtRha 进行了分子修饰，得到了一个催化效率显著提高的突变体 S592C，以及两个热稳定性提高的突变体 E39W 和 E39W-S592C。结果表明，E39W 和 E39W-S592C 在 55°C 时的半衰期分别比原始酶高 10.4 倍和 9.4 倍，突变体 S592C 的 K 值降低了 63.3%，催化效率（k/K 值）提高了 163.6%，有效提高了水解淫羊藿素 C 生成淫羊藿苷的能力。此外，还建立了α-L-鼠李糖苷酶突变体 S592C 的高效表达。以 0.1mM IPTG 为诱导剂，诱导温度 32°C，诱导 pH 值 7.0，诱导 OD 值 50，在 Terrific 肉汤培养基中，突变体 S592C 的最大酶活在 22h 时达到 182.0U/mL。这是迄今为止可以将淫羊藿素 C 转化为淫羊藿苷的α-L-鼠李糖苷酶的最高酶活。所有结果都提供了一种特异性和成本效益高的α-L-鼠李糖苷酶突变体，这将提高其在食品和制药应用方面的潜在兴趣。
