Partial purification and characterization of proteases from Norway lobster (Nephrops norvegicus) and their role in the phenolase activation process
Partial purification and characterization of proteases from Norway lobster (Nephrops norvegicus) and their role in the phenolase activation process
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DOI：
10.1016/0308-8146(95)00158-1
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发表时间：
1996-05
期刊：
影响因子：
8.8
通讯作者：
A. Zotos;K. Taylor
中科院分区：
文献类型：
--
作者：
A. Zotos;K. Taylor
研究了挪威龙虾（Nephrops norvegicus）的蛋白水解活性。通过丙酮沉淀和DEAE-Sepharose CL-6B柱层析，从挪威龙虾头中分离和部分纯化了三种蛋白酶（指定为蛋白酶I， II和III）。蛋白酶I、II和III在pH 8.2时的纯化率分别为63倍、25倍和217倍，在pH 6.4时的纯化率分别为40倍、25倍和160倍。以酪蛋白为底物的蛋白酶III在pH 8.2时活性最强，而蛋白酶I和蛋白酶II在pH范围内均表现出较强的活性。蛋白酶III被PMSF、大豆胰蛋白酶抑制剂、Co2+、Mn2+和1-10菲罗啉强烈抑制，为碱性锌丝氨酸蛋白酶。蛋白酶I被对苯醌、碘乙酰胺、重金属（Ag+、Cu2+）和1-10菲罗啉强烈抑制，因此被表征为锌硫醇蛋白酶。蛋白酶II也被与蛋白酶I相同的抑制剂抑制（但程度较轻），并被表征为硫醇蛋白酶。蛋白酶I、II和III的分子量分别为22.5、45和42.5 kDa（活性为18 kDa）。发现蛋白酶III在pH值8.2时激活酚酶，而蛋白酶II和蛋白酶I在pH值6.7和8.2时都能激活酚酶。
Proteolytic activity in Norway lobster (Nephrops norvegicus) was studied. An improved separation and partial purification of the three proteases (designated as proteases I, II and III) was achieved from Norway lobster heads by a combination of acetone precipitation and DEAE-Sepharose CL-6B column chromatography. The purification achieved was 63-, 25- and 217-fold at pH 8.2, and 40-, 25- and 160-fold at pH 6.4 for protease I, II and III, respectively. With casein as substrate, protease III was most active at pH 8.2, whilst proteases I and II showed activity over a wide range of pH. Protease III was characterized as an alkaline Zn-serine protease as it was strongly inhibited by PMSF, soybean trypsin inhibitor, Co2+, Mn2+and 1–10 phenanthroline. Protease I was strongly inhibited by p-benzoquinone, iodo-acetamide, heavy metals (Ag+, Cu2+) and 1–10 phenanthroline and was thus characterized as a Zn-thiol protease. Protease II was also inhibited by the same inhibitors as protease I (but to a lesser extent) and was characterized as a thiol protease. The molecular weights were determined to be 22.5, 45 and 42.5 kDa (with activity at 18 kDa) for proteases I, II and III, respectively. It was found that protease III activates phenolase at pH 8.2, whilst proteases II and I can activate phenolase at both pH 6.7 and 8.2.