Chen F R, Liu P C, Lee K K
Department of Aquaculture, National Taiwan Ocean University, Republic of China.
Microbios. 1999;98(389):27-34.
Four chromogenic substrates for characterizing serine protease of Vibrio alginolyticus were evaluated. The protease activity of bacterial extracellular products, or the fractions of 33 kD protease purified by the AKTA purifier system with various columns, was completely inhibited by ethylenediamine tetra-acetic acid, ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), antipain and phenylmethylsulphonyl fluoride (PMSF) using water-soluble substrates (azoalbumin and azocasein). It was only completely inhibited by antipain and PMSF using water-insoluble substrates (azocoll and hide powder azure). The protease activity was not, or only partially, inhibited by 1,10-phenanthroline and sodium dodecyl sulphate (SDS) using all four substrates. Since chelating agents and 1,10-phenanthroline are commonly employed as inhibitors to identify metalloprotease, the two water-soluble substrates may not be appropriate for this purpose, except for using 1,10-phenanthroline as an inhibitor. Chelating agents may be still applicable as inhibitors using water-insoluble substrates and 1,10-phenanthroline is highly recommended in the characterization for metalloprotease to avoid confusion. In the present study, the 33 kD protease was further confirmed as an SDS-resistant serine protease and not a metalloprotease.
评估了四种用于表征溶藻弧菌丝氨酸蛋白酶的显色底物。使用水溶性底物（偶氮白蛋白和偶氮酪蛋白）时，细菌胞外产物或通过AKTA纯化系统用各种柱纯化的33 kD蛋白酶组分的蛋白酶活性被乙二胺四乙酸、乙二醇双（β-氨基乙醚）N,N,N',N'-四乙酸（EGTA）、抗蛋白酶和苯甲基磺酰氟（PMSF）完全抑制。使用水不溶性底物（偶氮胶原和皮粉天蓝）时，其蛋白酶活性仅被抗蛋白酶和PMSF完全抑制。使用所有四种底物时，蛋白酶活性均未被1,10-菲咯啉和十二烷基硫酸钠（SDS）抑制，或仅被部分抑制。由于螯合剂和1,10-菲咯啉通常用作鉴定金属蛋白酶的抑制剂，除了使用1,10-菲咯啉作为抑制剂外，这两种水溶性底物可能不适用于此目的。使用水不溶性底物时，螯合剂仍可作为抑制剂，并且在金属蛋白酶的表征中强烈推荐使用1,10-菲咯啉以避免混淆。在本研究中，33 kD蛋白酶进一步被确认为一种抗SDS的丝氨酸蛋白酶，而非金属蛋白酶。
