Laboratório de Enzimologia, Departamento de Bioquímica and Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco, Brazil.
J Agric Food Chem. 2010 May 26;58(10):6433-9. doi: 10.1021/jf100111e.
A trypsin from the viscera of the lane snapper (Lutjanus synagris) was purified by heat treatment, fractionation with ammonium sulfate and affinity chromatography. The molecular weight of the enzyme was estimated to be 28.4 kDa (SDS-PAGE). The purified enzyme was capable of hydrolyzing the specific substrate for trypsin benzoyl-arginine-p-nitroanilide (BApNA) and was inhibited by benzamidine and tosyl lysine chloromethyl ketone (TLCK), synthetic trypsin inhibitors and phenylmethylsulfonyl fluoride (PMSF), which is a serine-protease inhibitor. The enzyme exhibited maximal activity at pH 9.0 and 45 degrees C and retained 100% of the activity after incubation at the optimal temperature for 30 min. At a concentration of 10 mM, activity was slightly activated by Ca(2+) and inhibited by the following ions in decreasing order: Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+). The effects of Ba(2+), K(1+) and Li(1+) proved to be less intensive. Using 1% (w/v) azocasein as substrate, the enzyme revealed high resistance (60% residual activity) when incubated with 10% H(2)O(2) for 75 min. The enzyme retained more than 80% activity after 60 min in the presence of different surfactants (Tween 20, Tween 80 and sodium choleate). The alkaline protease demonstrated compatibility with commercial detergents (7 mg/mL), such as Bem-te-vi, Surf and Ala, retaining more than 50% of initial activity after 60 min at 25 degrees C and 30 min at 40 degrees C. The thermostability and compatibility of this enzyme with commercial detergents suggest a good potentiality for application in the detergent industry.
从笛鲷（Lutjanus synagris）内脏中提取的胰蛋白酶经热处理、硫酸铵分级和亲和层析纯化。该酶的分子量估计为 28.4 kDa（SDS-PAGE）。纯化的酶能够水解胰蛋白酶特异性底物苯甲酰-精氨酸-对硝基苯胺（BApNA），并被苯甲脒和甲苯磺酰赖氨酰氯甲基酮（TLCK）、合成胰蛋白酶抑制剂和苯甲基磺酰氟（PMSF）抑制，苯甲基磺酰氟是一种丝氨酸蛋白酶抑制剂。该酶在 pH 9.0 和 45°C 时表现出最大活性，在最佳温度下孵育 30 分钟后保留 100%的活性。在 10mM 浓度下，活性被 Ca(2+) 轻微激活，被以下离子按降序抑制：Cd(2+) > Hg(2+) > Cu(2+) > Zn(2+) > Al(3+)。Ba(2+)、K(1+) 和 Li(1+) 的影响证明较弱。以 1%（w/v）的偶氮酪蛋白为底物，该酶在 10%H(2)O(2)存在下孵育 75 分钟时表现出高抗性（残留活性 60%）。在存在不同表面活性剂（吐温 20、吐温 80 和胆酸钠）的情况下，该酶在 60 分钟后保留超过 80%的活性。碱性蛋白酶与商业洗涤剂（Bem-te-vi、Surf 和 Ala，7mg/mL）兼容，在 25°C 下 60 分钟和 40°C 下 30 分钟后保留超过 50%的初始活性。该酶的热稳定性和与商业洗涤剂的兼容性表明其在洗涤剂工业中有很好的应用潜力。
