Protease D from the sarcocarp of honeydew melon fruit.

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.