Frosco M, Chase T, Macmillan J D
Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08903.
Infect Immun. 1992 Mar;60(3):728-34. doi: 10.1128/iai.60.3.728-734.1992.
Elastase, a potential virulence factor from the opportunistic pathogen Aspergillus fumigatus, was purified 220-fold from culture broth by fast-performance liquid chromatography employing anion exchange (Q Sepharose fast flow), cation exchange (S Sepharose fast flow), and gel filtration (Superose 12). Purified to near homogeneity, the elastase had an apparent molecular mass of 32 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver stain) but a mass of about 19.1 kDa as determined by gel filtration on Superdex 75. The elastase is not glycosylated and is positively charged at neutral pH, having a pI of 8.75. Inhibition by 0.2 mM phenylmethylsulfonyl fluoride (100%) and 0.21 mM leupeptin (60%) implies that the elastase is a serine protease. However, the enzyme is also inhibited by 5 mM EDTA (100%) and 10 mM 1,10-orthophenanthroline (30%), suggesting a requirement for divalent cations. The enzyme acts optimally at pH 7.4 and 45 degrees C in 50 mM sodium borate buffer, but in Tris HCl, the pH optimum shifts to 8.8.
弹性蛋白酶是机会致病菌烟曲霉的一种潜在毒力因子，通过使用阴离子交换（Q Sepharose快速流动）、阳离子交换（S Sepharose快速流动）和凝胶过滤（Superose 12）的快速液相色谱法从培养液中纯化了220倍。纯化至接近同质，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（银染）测定，弹性蛋白酶的表观分子量为32 kDa，但通过Superdex 75凝胶过滤测定，其分子量约为19.1 kDa。该弹性蛋白酶无糖基化，在中性pH下带正电荷，其pI为8.75。0.2 mM苯甲基磺酰氟（100%）和0.21 mM亮抑酶肽（60%）的抑制作用表明该弹性蛋白酶是一种丝氨酸蛋白酶。然而，该酶也受到5 mM乙二胺四乙酸（100%）和10 mM 1,10-邻菲罗啉（30%）的抑制，表明需要二价阳离子。该酶在50 mM硼酸钠缓冲液中，pH 7.4和45℃时作用最佳，但在Tris HCl中，最适pH值移至8.8。
