Abdelal A T, Kennedy E H, Ahearn D G
J Bacteriol. 1977 Jun;130(3):1125-9. doi: 10.1128/jb.130.3.1125-1129.1977.
Saccharomycopsis lipolytica 37-1 produced two inducible extracellular proteases, one under neutral or alkaline growth conditions and the second under acid conditions. Secretion of the neutral protease was repressed in the presence of glycerol or glucose, both of which supported rapid growth of the organism. Ammonium ions also repressed the secretion of the enzyme. The neutral protease activity copurified with esterase activity during ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose, and gel filtration on Sephadex G-150. The molecular weight of the enzyme was estimated to be 42,000 by sucrose density gradient centrifugation and 38,500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme had a pH optimum of 6.8. Phenylmethylsulfonylfluoride inhibited both protease and esterase activities, indicating the presence of a serine residue in the active center. Protease, but not esterase, activity was sensitive to ethylenediaminetetraacetate and was significantly activated by divalent ions. Dithiothreitol inhibited both protease and esterase activities, indicating the presence of a critical disulfide bridge. The enzyme hydrolyzed casein (K(m) = 25.6 muM) and hemoglobin as well as the nitrophenyl esters of tyrosine (K(m) = 2.4 mM), glycine, tryptophan, and phenylalanine.
解脂耶氏酵母37-1产生两种诱导型胞外蛋白酶，一种在中性或碱性生长条件下产生，另一种在酸性条件下产生。在甘油或葡萄糖存在的情况下，中性蛋白酶的分泌受到抑制，而这两种物质都能支持该生物体的快速生长。铵离子也会抑制该酶的分泌。在硫酸铵分级分离、二乙氨基乙基纤维素柱层析以及Sephadex G-150凝胶过滤过程中，中性蛋白酶活性与酯酶活性共同纯化。通过蔗糖密度梯度离心法估计该酶的分子量为42,000，在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳时估计为38,500。纯化后的酶最适pH值为6.8。苯甲基磺酰氟抑制蛋白酶和酯酶的活性，表明活性中心存在丝氨酸残基。蛋白酶活性对乙二胺四乙酸敏感，但酯酶活性不敏感，并且蛋白酶活性被二价离子显著激活。二硫苏糖醇抑制蛋白酶和酯酶的活性，表明存在关键的二硫键。该酶能水解酪蛋白（Km = 25.6 μM）、血红蛋白以及酪氨酸（Km = 2.4 mM）、甘氨酸、色氨酸和苯丙氨酸的硝基苯酯。
