北极海洋细菌BSw20353的胞外蛋白酶性质

CHARACTERIZATION OF EXTRACELLULAR PROTEASES PRODUCED BY ONE ARCTIC MARINE BACTERIUM BSw20353

从北极楚科奇海水中分离到一株产蛋白酶耐冷河豚毒素假交替单胞菌BSw20353。该菌可合成多种胞外蛋白酶,而培养温度影响蛋白酶的合成。分离纯化出其中一种在15℃、25℃培养时均有生成的酶:该酶最适作用温度55℃;最适pH8;金属离子Hg2+、Sr2+抑制酶活,Fe2+、Mn2+则具有激活作用;该酶属于金属蛋白酶,受螯合剂邻二氮杂菲、EDTA及EGTA抑制,基本不受丝氨酸蛋白酶抑制剂PMSF、TPCK及TLCK抑制,半胱氨酸蛋白酶抑制剂E-64对该酶无抑制作用,天冬氨酸蛋白酶抑制剂pepstatin则具有一定抑制作用;SDS、二硫苏糖醇、碘乙酸可抑制酶活;而在变性剂尿素、盐酸胍作用下,酶活反而有所增高。

A psychrotolerant protease-producing bacterial strain BSw20353 was isolated trom the Chuckchi Sea in the Arctic, and identified as Pseudoalteromonas tetraodon is based on 16S rDNA sequencing. It could grow at temperature no higher than 38℃. The use of zymogram gels revealed the presence of three or more proteolytic bands in the culture supernatant. These bands may represent individual isozymes, different proteases, or active subunits of a larger pro- teolytic complex. The change of cultivation temperature affected the production of bacterial proteases, resulting in a change in protease category and quantity. One of these proteolytic bands was purified to electrophoretic homogeneity from the culture supernatant. It could be produced when bacterium growing at 15℃ and 25℃. The optimum temperature and pH for enzyme activity were 55℃ and 8, respectively. On the basis of its biochemical characteristics, the enzyme can be included in the group of metalloproteases that are inhibited by chelators including 1,10- phenanthroline, EDTA and EGTA. It was resistant to PMSF, TPCK, TLCK, and E-64, indicating that the enzyme is neither a serine protease nor cysteine protease. The protease activity was inhibited by pepstatin, an aspartic acid protease inhibitor, Hg^2＋ and Sr^2＋ , but was stimulated by Fe^2＋ and Mn^2＋. SDS showed obvious inhibiting effect on the enzyme. In addition to iodoacetic acid, dithiothreitol produced a decrease of activity. However, protease activity was enhanced after treated by denaturant of urea or guanidine HCl.