Li Xian, Kushad Mosbah M
Department of Natural Resources and Environmental Sciences, University of Illinois, 279 ERML, 1201 W. Gregory Drive, Urbana, IL 61801, USA.
Plant Physiol Biochem. 2005 Jun;43(6):503-11. doi: 10.1016/j.plaphy.2005.03.015.
Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.
通过硫酸铵分级沉淀、Q-琼脂糖凝胶柱层析和伴刀豆球蛋白A琼脂糖亲和层析，从辣根（Armoracia rusticana）根中纯化得到了纯度达到均一的黑芥子酶（β-硫代葡萄糖苷葡萄糖水解酶；EC 3.2.3.147）。在SDS-聚丙烯酰胺凝胶电泳中，纯化后的蛋白质迁移为一条单一的条带，质量约为65 kDa。使用液相色谱-串联质谱法（LC-MS/MS），这条带被鉴定为黑芥子酶。利用抗黑芥子酶单克隆抗体3D7进行的蛋白质免疫印迹分析表明，辣根粗提物和纯化后的黑芥子酶均显示出一条约65 kDa的单一条带。使用凝胶过滤法估算，纯化后的黑芥子酶的天然分子量约为130 kDa。基于这些数据，辣根根中的黑芥子酶似乎由两个分子量相似、约为65 kDa的亚基组成。该酶在较宽的pH范围（pH 5.0 - 8.0）和温度（37和45摄氏度）下均表现出高活性。纯化后的酶在4摄氏度下可保持稳定超过1年。以黑芥子硫苷为底物，纯化后的酶的米氏常数（Km）和最大反应速度（Vmax）值分别估计为0.128 mM和0.624 μmol min⁻¹。该酶被0.5 mM抗坏血酸强烈激活，并且能够分解西兰花粗提物中的完整硫代葡萄糖苷。
