Zhang Yuwei, Li Jie, Yuan Yong, Gu Jijuan, Chen Peng
College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China.
Sheng Wu Gong Cheng Xue Bao. 2017 May 25;33(5):796-807. doi: 10.13345/j.cjb.160414.
Rutin-degrading enzymes (RDE) can degrade rutin into poorly water soluble compound, quercetin, and cause the bitter taste in tartary buckwheat. In the present study RDE from Yu 6-21 tartary buckwheat seeds was purified by ammonium sulphate precipitation, followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, ion exchange chromatography on CM-Cellulose and gel filtration chromatography on Sephadex G-150. Purified RDE showed single band with molecular weight of 66 kDa on SDS-PAGE. The optimum pH and temperature of RDE were 5.0 and 50 ℃ respectively. The Km was 0.27 mmol/L, and the Vmax was 39.68 U/mg. The RDE activity could be inhibited by Cu²⁺, Zn²⁺, Mn²⁺ and EDTA, and showed tolerance to 50% methanol (V/V). The N terminal sequence (TVSRSSFPDGFLFGL) was obtained by Edman degradation method and 15 internal peptide sequences were determined by MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry). These results established the foundations for identification of the candidate gene of RDE via transcriptome data and further studying RDE biological function.
芦丁降解酶（RDE）可将芦丁降解为水溶性较差的化合物槲皮素，并导致苦荞产生苦味。在本研究中，通过硫酸铵沉淀法从榆6-21苦荞种子中纯化RDE，随后依次进行苯基琼脂糖凝胶CL-4B疏水作用色谱、CM-纤维素离子交换色谱和葡聚糖凝胶G-150凝胶过滤色谱。纯化后的RDE在SDS-PAGE上显示出一条分子量为66 kDa的单带。RDE的最适pH值和温度分别为5.0和50℃。Km为0.27 mmol/L，Vmax为39.68 U/mg。RDE活性可被Cu²⁺、Zn²⁺n²⁺、Mn²⁺和EDTA抑制，并对50%甲醇（V/V）表现出耐受性。通过Edman降解法获得了N端序列（TVSRSSFPDGFLFGL），并通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）测定了15个内部肽段序列。这些结果为通过转录组数据鉴定RDE候选基因以及进一步研究RDE生物学功能奠定了基础。
