Purification and Characterization of Cathepsin B from the Gut of the Sea Cucumber (Stichopus japonicas)
Purification and Characterization of Cathepsin B from the Gut of the Sea Cucumber (Stichopus japonicas)
复制标题
海参 (Stichopus japonicas) 肠道组织蛋白酶 B 的纯化和表征
DOI：
10.1007/s10068-011-0127-1
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发表时间：
2011-08-01
影响因子：
2.9
通讯作者：
Murata, Yoshiyuki
中科院分区：
文献类型：
--
作者：
Sun, Li-Ming;Zhu, Bei-Wei;Murata, Yoshiyuki
通过硫酸铵分级沉淀和DEAE SepharoseCL-6B、Sephadex G-75、TSK-Gel 3000 SWxl层析，从海参肠道中分离纯化组织蛋白酶B，纯化倍数为81倍，回收率为3%。纯化蛋白在Native-PAGE上显示为单一条带，而在SDS-PAGE上显示为23和26 kDa两条带。该酶的最适pH为5.5，最适温度为45℃。该酶在pH4.5-6.0范围内稳定，热稳定性高达50℃。E-、碘乙酸和止痛剂对该酶有较强的抑制作用，表明该酶是一种含有巯基的半胱氨酸水解酶。Cu2+、Ni2+、Zn2+对酶有较强的抑制作用。用质谱仪测定了该酶的氨基酸序列，与已报道的组织蛋白没有同源性，提示它可能是一个新的成员。
Cathepsin B from the gut of sea cucumber (Stichopus japonicas) was purified 81-fold with a 3% recovery by ammonium sulfate fractionation and a series chromatography on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-Gel 3000 SWxl. The purified protein appeared as a single band on Native-PAGE but showed 2 bands of 23 and 26 kDa on SDS-PAGE. The optimum activity was found at pH 5.5 and 45 degrees C. The enzyme was stable at pH 4.5-6.0 and the thermal stability was up to 50 degrees C. The enzyme was strongly inhibited by E-64, iodoacetic acid, and antipain, demonstrating it is a cysteine protease containing sulfhydryl groups. Cu2+, Ni2+, and Zn2+ could strongly inhibit the enzyme activity. The amino acid sequences of the purified enzyme were acquired by mass spectrometer, which did not show any homology with previously described cathepsins, suggesting it may be a novel member.