Marín-Zamora María Elisa, Rojas-Melgarejo Francisco, García-Cánovas Francisco, García-Ruiz Pedro Antonio
Grupo de Química de Carbohidratos y Biotecnología de Alimentos (QCBA), Departamento de Química Orgánica, Facultad de Química, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
J Biotechnol. 2006 Nov 10;126(3):295-303. doi: 10.1016/j.jbiotec.2006.04.024. Epub 2006 May 30.
Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.
由于发生了强烈的疏水相互作用，蘑菇酪氨酸酶通过直接吸附从提取物固定到D - 山梨醇的完全肉桂酰化衍生物上。固定化pH值和冻干蘑菇的质量是影响固定化效率的重要参数，而固定化时间和固定化载体浓度在这方面并不重要。提取/固定化酶在pH 3.5以上能得到最佳测定效果，最佳测定温度为55℃。以4 - 叔丁基邻苯二酚为底物时，表观米氏常数为0.38±0.02 mM，低于Sigma公司的可溶性酶（1.41±0.20 mM）。固定化使提取的酶对热失活更稳定，并且在储存过程中使其活性损失更不易发生。其操作稳定性高于Sigma公司提供并固定在相同载体上的酪氨酸酶。结果表明，在酶提取和固定化过程中使用对硝基苯酚作为酶抑制底物，无需使用抗坏血酸，是提取和固定酪氨酸酶的合适方法，能提供良好的酶活性和稳定性。
