Roy P, Colas B, Durand P
Laboratoire Biochimie et Molécules Marines, VP/BM, IFREMER, Nantes, France.
Comp Biochem Physiol B Biochem Mol Biol. 1996 Sep;115(1):87-95. doi: 10.1016/0305-0491(96)00090-9.
A serine collagenolytic protease was purified from a water soluble fraction of greenshore crab digestive gland by acidic precipitation, gel filtration on a Sephadex G-50 column, ion-exchange chromatography on a Fractogel TSK DEAE column, immobilized metal ion affinity chromatography (IMAC) on IDA (Cu2+) Sepharose 6B and ion-exchange chromatography on Hyper D column. The molecular mass of the monomeric Carcinus serine collagenase (CSC) was estimated to be 23,000 by SDS PAGE and its isoelectric point was found to be 4.0. The CSC is optimally active at pH 7 and 30 degrees C and is stable over a month at room temperature. The CSC activity is strongly inhibited by PMSF, 3,4-DCI, soybean trypsin inhibitor, alpha 1 proteinase inhibitor and elastatinal. The CSC hydrolyzes native collagen (Type I and III). CSC N-terminal sequence is similar to shrimp chymotrypsin-like protease and crab collagenolytic protease sequences. Kinetic parameters of the CSC were determined using some peptidyl-p-nitroanilides. The catalytic efficiency (kcat/Km) is Leu > Phe > Ala.
通过酸性沉淀、在Sephadex G - 50柱上进行凝胶过滤、在Fractogel TSK DEAE柱上进行离子交换色谱、在IDA（Cu2+）Sepharose 6B上进行固定化金属离子亲和色谱（IMAC）以及在Hyper D柱上进行离子交换色谱，从绿岸蟹消化腺的水溶性部分中纯化出一种丝氨酸胶原分解蛋白酶。通过SDS - PAGE估计单体的滨蟹丝氨酸胶原酶（CSC）的分子量为23,000，其等电点为4.0。CSC在pH 7和30℃时活性最佳，在室温下一个月内稳定。CSC的活性受到苯甲基磺酰氟（PMSF）、3,4 - 二氯异苯丙氨酸（3,4 - DCI）、大豆胰蛋白酶抑制剂、α1蛋白酶抑制剂和弹性蛋白酶抑制剂的强烈抑制。CSC可水解天然胶原蛋白（I型和III型）。CSC的N端序列与虾类胰凝乳蛋白酶样蛋白酶和蟹类胶原分解蛋白酶序列相似。使用一些肽基 - p - 硝基苯胺测定了CSC的动力学参数。催化效率（kcat/Km）为亮氨酸＞苯丙氨酸＞丙氨酸。
