Park Pyo-Jam, Lee Sang-Hoon, Byun Hee-Guk, Kim Soo-Hyun, Kim Se-Kwon
Department of Chemistry, Pukyong National University, Busan 608-737, Korea.
J Biochem Mol Biol. 2002 Nov 30;35(6):576-82. doi: 10.5483/bmbrep.2002.35.6.576.
Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.
利用丙酮沉淀、在DEAE - Sephadex A - 50上进行离子交换色谱、在Sephadex G - 100上进行凝胶过滤色谱、在DEAE - Sephacel上进行离子交换色谱以及在Sephadex G - 75柱上进行凝胶过滤色谱，对鲭鱼内脏中的胶原酶进行了纯化。通过凝胶过滤和SDS - PAGE估计纯化酶的分子量为14.8 kDa。与起始粗提物相比，纯化倍数和产率分别为39.5倍和0.1%。该酶活性的最适pH和温度分别约为pH 7.5和55℃。该酶对I型胶原的K(m)和V(max)分别约为1.1mM和2343 U。纯化酶受到Hg2 +、Zn2 +、PMSF、TLCK和大豆胰蛋白酶抑制剂的强烈抑制。
