Barata Ricardo Andrade, Andrade Milton Hercules Guerra, Rodrigues Roberta Dias, Castro Ieso Miranda
Laboratório de Bioquímica e Fisiologia de Microorganismos, Núcleo de Pesquisa em Ciências Biológicas, Universidade Federal de Ouro Preto, CEP 35.400.000, Ouro Preto, MG, Brazil.
J Biosci Bioeng. 2002;94(4):304-8. doi: 10.1263/jbb.94.304.
An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.
尖孢镰刀菌亚麻专化型在以明胶作为唯一氮源和碳源的条件下生长时，会分泌一种能够水解Nα-苯甲酰-DL-精氨酸对硝基苯胺的碱性丝氨酸蛋白酶。该蛋白酶通过三步纯化程序从培养上清液中纯化了65倍，达到电泳纯，这三步程序包括Q Sepharose层析、亲和层析以及在MonoQ柱上进行快速蛋白质液相色谱（FPLC）。对纯化后的蛋白质进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，其估计分子量为41 kDa。该蛋白酶在45℃的反应温度下具有最佳活性，在48℃时活性迅速下降。最佳pH值约为8.0。对该蛋白酶的特性进行表征发现，Ca2+和Mg2+阳离子可提高其活性，且其活性不受乙二胺四乙酸（EDTA）或1,10-菲啰啉的抑制。4-（2-氨乙基）苯磺酰氟盐酸盐、对氨基苯甲脒二盐酸盐、抑肽酶、3,4-二氯异香豆素和N-对甲苯磺酰-L-赖氨酸氯甲基酮可抑制该酶对Nα-苯甲酰-DL-精氨酸对硝基苯胺的活性。底物浓度高于2.5×10-4M时也会抑制该酶的活性。在非抑制性底物浓度下进行测定时，该蛋白酶的米氏常数（Michaelis-Menten constant）为0.16 mM，最大反应速度（V(max)）为0.60 μmol产物释放·分钟-1·毫克-1酶。对氨基苯甲脒二盐酸盐竞争性抑制该酶对Nα-苯甲酰-DL-精氨酸对硝基苯胺的活性。获得的抑制常数（K(i)）值为0.04 mM。
