Mayerhofer H J, Marshall R T, White C H, Lu M
Appl Microbiol. 1973 Jan;25(1):44-8. doi: 10.1128/am.25.1.44-48.1973.
A heat-stable, extracellular proteolytic enzyme was isolated from Pseudomonas fluorescens P26. Brain heart infusion broth (Fisher Scientific Co.), pH 7.5, and incubation at 21 C provided the optimal conditions for bacterial growth for enzyme production. The organism had a D value of 2.6 min at 62.8 C (145 F). The enzyme, however, was quite heat-stable, requiring 15 hr at 62.8 C, 8 hr at 71.4 C, and 9 min at 121 C for complete inactivation. Milk, whey, and casein each had a protective effect on the enzyme against heat inactivation. Purification was accomplished by growth of organisms in broth, centrifugation, sterilization by filtration, ammonium sulfate precipitation (55% saturation), dialysis (against six changes of water), and protein separation by passage through a Sephadex G-100 column. Ultracentrifugation revealed a single band with a sedimentation coefficient of 1.51 which suggested a molecular weight of approximately 23,000. As little as 0.2 unit of the purified enzyme caused detectable flavor defects during 30 days of storage at 4 C, and the Hull test for liberation of tyrosine compared favorably in sensitivity with the sensory method in milk.
从荧光假单胞菌P26中分离出一种热稳定的细胞外蛋白水解酶。脑心浸液肉汤（Fisher科学公司），pH值7.5，在21℃下培养为细菌生长和酶产生提供了最佳条件。该微生物在62.8℃（145°F）下的D值为2.6分钟。然而，该酶相当耐热，在62.8℃下需要15小时、71.4℃下需要8小时、121℃下需要9分钟才能完全失活。牛奶、乳清和酪蛋白对该酶均有抗热失活的保护作用。通过在肉汤中培养微生物、离心、过滤灭菌、硫酸铵沉淀（55%饱和度）、透析（对六次换水）以及通过Sephadex G - 100柱进行蛋白质分离来完成纯化。超速离心显示有一条单一谱带，沉降系数为1.51，这表明分子量约为23,000。在4℃储存30天期间，仅0.2单位的纯化酶就会导致可检测到的风味缺陷，并且在牛奶中，酪氨酸释放的赫尔试验在灵敏度方面与感官方法相当。
