Serge Ngangoum Eric, Laurette Blandine Mezajoug Kenfack, Kumar Sanjit, Clergé Tchiégang, Vijayalakshmi Mookambeswaran
a Bioprocess Laboratory, Institute University of Technology , University of Ngaoundere , Ngaoundere , Cameroon.
b Centre for Bio-separation Technology , VIT University , Vellore , Tamil Nadu , India.
Prep Biochem Biotechnol. 2017 Nov 26;47(10):1016-1024. doi: 10.1080/10826068.2017.1373289. Epub 2017 Aug 31.
A protease from fresh leaves of Abrus precatorius was purified using two classical chromatography techniques: ion-exchange (DEAE-Sepharose) and Gel filtration (Sephadex G-75). The purified protease showed a molecular weight of ∼ 28 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature for the purified protease was 8 and 40°C, respectively. The purified protease was stable throughout a wide temperature range from 10 to 80°C and pH from 2 to 12. Protease activity was inhibited in the presence of Co, Ni, Hg, and Zn while its activity has increased in the presence of Ca and Mg. The protease was highly specific to casein when compared to its specificity for gelatin, bovine serum albumin, hemoglobin, and defatted flour of Ricinodendron heudelotii. Its V and K determined using casein as a substrate were 94.34 U/mL and 349.07 µg/mL respectively. Inhibition studies showed that this purified protease was inhibited by both phenylmethane sulfonyl fluoride and aprotinin which are recognized as competitive inhibitors of serine proteases.
采用两种经典色谱技术（离子交换色谱（DEAE-琼脂糖）和凝胶过滤色谱（葡聚糖凝胶G-75））对相思子鲜叶中的一种蛋白酶进行了纯化。在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上，纯化后的蛋白酶分子量约为28 kDa。纯化后的蛋白酶的最适pH值和温度分别为8和40℃。纯化后的蛋白酶在10至80℃的宽温度范围以及2至12的pH范围内均保持稳定。在存在Co、Ni、Hg和Zn时，蛋白酶活性受到抑制，而在存在Ca和Mg时，其活性增强。与明胶、牛血清白蛋白、血红蛋白和海红豆脱脂面粉相比，该蛋白酶对酪蛋白具有高度特异性。以酪蛋白为底物测定的其V和K分别为94.34 U/mL和349.07 μg/mL。抑制研究表明，这种纯化后的蛋白酶受到苯甲基磺酰氟和抑肽酶的抑制，这两种物质被认为是丝氨酸蛋白酶的竞争性抑制剂。
