Department of Biology, Chemistry and Geography, Université du Québec à Rimouski, Canada.
Appl Biochem Biotechnol. 2011 Mar;163(6):765-79. doi: 10.1007/s12010-010-9081-2. Epub 2010 Sep 20.
Sequential acidic precipitation followed by a single chromatographic step (gel filtration) allowed the recovery of a collagenolytic fraction containing several proteases from by-products of snow crab (Chionoecetes opilio). The partial purification was particularly efficient to recover tryptic (purification fold = 1,352.5; yield = 110%) but also chymotryptic, elastolytic, and collagenolytic activities. A temperature of 40 °C and pH 8.0-8.5 were optimal for enzyme activity, which was stable for 2 h under these conditions. Calcium was not required for stability and thus activity. The isoelectric points of the protein components ranged from 3.7 to 4.6. Zymography revealed 29 and 48 kDa major components and others from 22 to 56 kDa. Enzymes were inhibited by PMSF and TLCK but were insensitive to TPCK. In view of these properties, the proteases likely belong to the serine collagenase group. Inhibition by EDTA could be due to a mechanism other than Ca(2+) chelation. Using a food system (ground fish), the fraction was more proteolytic than a commercial bacterial protease, suggesting potential applications in enzymatic hydrolysis processes.
顺序酸性沉淀后进行单一的色谱步骤（凝胶过滤），从雪蟹（Chionoecetes opilio）副产物中回收了含有几种蛋白酶的胶原水解物。部分纯化特别有效地回收了胰蛋白酶（纯化倍数= 1,352.5；产率= 110%），但也回收了糜蛋白酶、弹性蛋白酶和胶原酶活性。40°C 和 pH 8.0-8.5 的温度最适合酶活性，在这些条件下稳定 2 小时。钙对稳定性和活性不是必需的。蛋白质成分的等电点范围为 3.7 至 4.6。同工酶显示 29 和 48 kDa 的主要成分以及其他 22 至 56 kDa 的成分。蛋白酶被 PMSF 和 TLCK 抑制，但对 TPCK 不敏感。鉴于这些特性，这些蛋白酶可能属于丝氨酸胶原酶组。EDTA 的抑制作用可能不是由于 Ca(2+)螯合。在食品体系（粉碎鱼）中，该部分比商业细菌蛋白酶更具蛋白水解活性，表明其在酶水解过程中具有潜在应用。
