Vasu Prasanna, Savary Brett J, Cameron Randall G
Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA.
Arkansas Biosciences Institute, Arkansas State University, Jonesboro, AR, USA; College of Agriculture and Technology, Arkansas State University, Jonesboro, AR, USA.
Food Chem. 2012 Jul 15;133(2):366-72. doi: 10.1016/j.foodchem.2012.01.042. Epub 2012 Jan 24.
We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6-9) and moderate thermostability (optimum ca. 70°C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure.
我们从一种市售木瓜蛋白酶制剂中纯化出一种番木瓜果胶甲酯酶（CpL-PME；EC 3.1.1.11）。利用连续的疏水相互作用和阳离子交换色谱法将这种CpL-PME与大量的半胱氨酸内肽酶活性分离，然后使用固定在琼脂糖上的猕猴桃PME抑制蛋白通过亲和色谱法进行纯化，从而获得一种单一的电泳纯蛋白。该酶纯化了92倍，产率为38%，比活性为1200 U/mg。通过线性模式的基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）测定其分子量为35,135。胰蛋白酶消化后的MALDI-TOF-MS肽质量指纹图谱表明CpL-PME代表一种新型的番木瓜PME同工型。CpL-PME的活性需要盐，并且它表现出较宽的活性范围（pH 6 - 9）和适度的热稳定性（最适温度约70°C）。用CpL-PME处理使甲基化酸橙果胶的甲酯化程度降低6%，将底物转化为对高钙敏感，这表明其作用方式为连续作用模式。这些特性支持进一步研究将CpL-PME应用于定制果胶纳米结构。
