Taguchi H, Hamaoki M, Matsuzawa H, Ohta T
J Biochem. 1983 Jan;93(1):7-13. doi: 10.1093/oxfordjournals.jbchem.a134179.
Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T. caldophilus sp. n. strain GK24. The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth. Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography. The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S. The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2). The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C. The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents. The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA). From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin. The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).
在一种新分离出的属于嗜热栖热菌属的嗜热细菌的培养上清液中检测到了蛋白水解活性，该细菌暂命名为嗜热栖热菌新种GK24菌株。细胞进入生长稳定期后，酶活性至少持续增加三天。尝试通过硫酸铵分级沉淀、凝胶过滤和离子交换色谱法对蛋白水解酶进行纯化。由此获得的最纯酶组分在色谱分析中似乎是均一的，但在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳上仍有七条蛋白带。用变性剂或有机溶剂处理蛋白酶不会改变色谱图谱，纯化的酶样品显示出约11S的大沉降系数。该酶对酪蛋白的水解活性最佳pH值约为7.8，对N-苄氧羰基-L-亮氨酰-L-酪氨酸酰胺（Z-Leu-Tyr-NH2）的最佳pH值为7.2。该酶在pH值5至11的范围内，于4℃可稳定保存1天，或在70℃可稳定保存1小时。酶样品在90℃时显示出最大活性，并且对热和变性剂处理具有极高的稳定性。酶样品几乎完全被二异丙基氟磷酸（DFP）灭活，但不被乙二胺四乙酸（EDTA）或乙二醇双（β-氨基乙基醚）N,N'-四乙酸（EGTA）灭活。从这些结果来看，该酶似乎是一种丝氨酸蛋白酶，而不是诸如嗜热菌蛋白酶之类的金属酶。该酶对酯化合物N-苯甲酰-L-酪氨酸乙酯（BTEE）也具有水解活性，但对N-苯甲酰-L-精氨酸乙酯（BAEE）没有水解活性。
