Pan Min-Hsiung, Tsai Mei-Ling, Chen Wen-Ming, Hwang Ann, Sun Pan Bonnie, Hwang Yu-Ren, Kuo Jen-Min
Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung, Taiwan, R.O.C.
J Agric Food Chem. 2010 Dec 8;58(23):12541-6. doi: 10.1021/jf1034042. Epub 2010 Nov 3.
The objective of the present study is to purify and characterize the fish scale-degrading enzyme from Vogesella sp.7307-1, which was newly identified and isolated from fish scales. The enzyme from Vogesella sp.7307-1 was assayed with casein and confirmed as a protease. Crude protease was extracted, isolated, and purified 35.7-fold with 19.6% recovery using 20-80% saturation of ammonium sulfate fractionation, Q FF ion exchange chromatography, and Superdex 200 gelfiltration. The molecular weight of the purified enzyme was 119 kDa. The Km and Vmax were 0.067 mM and 425.5 U/mg-min, respectively using azo-casein as substrate. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 50 °C. The enzyme was stable at temperatures below 55 °C and pH range 7.5 to 9.0. The enzyme activity of the purified protease was completely inhibited by EDTA (ethylene diamine teraacetates), indicating the enzyme was a metalloprotease. Hydrolysates from fish scales treated with protease 7307-1 were found having low molecular weight peptides (<1 kDa). The protease 7307-1 is a promising enzyme for preparing smaller peptides from fish scales.
本研究的目的是纯化并表征从新鉴定和分离自鱼鳞的沃氏菌属7307-1中提取的鱼鳞降解酶。用酪蛋白对沃氏菌属7307-1中的酶进行了测定，并确认其为蛋白酶。使用20%-80%饱和度的硫酸铵分级分离、Q FF离子交换色谱和Superdex 200凝胶过滤法提取、分离并纯化粗蛋白酶，纯化倍数为35.7倍，回收率为19.6%。纯化后酶的分子量为119 kDa。以偶氮酪蛋白为底物时，Km和Vmax分别为0.067 mM和425.5 U/mg·min。纯化后酶的最适pH为7.5，最适温度为50℃。该酶在55℃以下的温度和pH值7.5至9.0的范围内稳定。纯化后的蛋白酶活性被乙二胺四乙酸（EDTA）完全抑制，表明该酶是一种金属蛋白酶。用蛋白酶7307-1处理鱼鳞后得到的水解产物含有低分子量肽（<1 kDa）。蛋白酶7307-1是一种从鱼鳞制备较小肽的有前景的酶。
