苦荞籽粒芦丁降解酶的纯化、酶学性质及部分一级结构分析

Purification, characterization and partial primary structure analysis of rutin-degrading enzyme in tartary buckwheat seeds

摘要:

芦丁降解酶催化芦丁降解为水溶性降低的槲皮素，是形成苦荞苦味的主要因素。研究以榆6-21苦荞籽粒粉为材料，通过硫酸铵分级沉淀、疏水层析、阳离子交换层析、凝胶过滤层析分离纯化芦丁降解酶，SDS-PAGE显示纯化的芦丁降解酶为分子量66 kDa的单一条带。芦丁降解酶的最适pH值为5.0，最适温度为50 ℃。对其催化特性研究表明，该酶的Km值为0.27 mmol/L，Vmax值为39.68 U/mg。Cu2＋、Zn2＋、Mn2＋和EDTA对其有不同程度的抑制作用。纯化的芦丁降解酶可耐受50% (V/V) 的甲醇。Edman降解法测定其N端序列为TVSRSSFPDGFLFGL。MALDI-TOF-MS (基质辅助激光解吸电离飞行时间质谱) 二级质谱获得了其15个内肽序列。该研究为从转录组数据中确定芦丁降解酶的候选基因及深入研究芦丁降解酶的生物学功能奠定了基础。

Abstract:

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.