Uchikoba T, Niidome T, Sata I, Kaneda M
Department of Chemistry, Faculty of Science, Kagoshima University, Japan.
Phytochemistry. 1993 Jul;33(5):1005-8. doi: 10.1016/0031-9422(93)85012-g.
A serine protease was purified from commercially available honeydew melon fruit (Cucumis melo L. var. inodorus Naud) by five steps including CM-Sepharose CL-6B column chromatography. At the first CM-Sepharose CL-6B chromatography four active fractions appeared. No difference in enzymatic properties and M(r) in each fraction was found. Finally, honeydew melon protease D was isolated from the major active fraction D. A M(r) of 50,000 was determined for protease D on SDS-PAGE and gel filtration. Protease D was a glycoprotein. The protease exhibited a remarkable stability, even in 8 M urea or 2 M guanidine hydrochloride for 24 hr. The optimum pH of the enzyme was 11 at 35 degrees using casein as substrate. The enzyme was strongly inhibited by diisopropyl fluorophosphate and was not inhibited by metal-chelating reagents or cysteine protease inhibitors. The enzymatic properties of honeydew melon protease D were similar to those of cucumisin [EC 3.4.21.25], except for its stability at high temperature, wide pH range and against detergents.
通过包括CM-琼脂糖CL-6B柱色谱在内的五步从市售甜瓜（甜瓜变种Cucumis melo L. var. inodorus Naud）中纯化出一种丝氨酸蛋白酶。在第一次CM-琼脂糖CL-6B色谱分析中出现了四个活性组分。各组分的酶学性质和相对分子质量（Mr）未发现差异。最后，从主要活性组分D中分离出甜瓜蛋白酶D。在SDS-PAGE和凝胶过滤中测定蛋白酶D的相对分子质量为50,000。蛋白酶D是一种糖蛋白。该蛋白酶表现出显著的稳定性，即使在8M尿素或2M盐酸胍中处理24小时也是如此。以酪蛋白为底物时，该酶在35℃下的最适pH为11。该酶受到二异丙基氟磷酸的强烈抑制，不受金属螯合剂或半胱氨酸蛋白酶抑制剂的抑制。甜瓜蛋白酶D的酶学性质与黄瓜蛋白酶[EC 3.4.21.25]相似，但其在高温、宽pH范围和抗洗涤剂方面的稳定性除外。
