Laboratório de Bioquímica e Microbiologia Aplicada, Departamento de Ciência de Alimentos, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9500, 91501-970 Porto Alegre, Brazil.
Appl Biochem Biotechnol. 2010 Sep;162(2):548-60. doi: 10.1007/s12010-009-8835-1. Epub 2009 Nov 21.
A novel keratinase from Chryseobacterium sp. strain kr6 was purified to homogeneity by (NH(4))(2)SO(4) precipitation, gel permeation on Sephadex G-100, and Q-Sepharose Fast Flow anion-exchange chromatography. The molecular weight of the purified enzyme was around 20 kDa. Kinetic and thermodynamic parameters for thermal inactivation were determined. The influence of Ca(2+) and Mg(2+) ions and purification degree on the enzyme stability was evaluated in the range of 50 to 60 degrees C. The results showed that first-order kinetics explained well the thermal denaturation of the keratinase in this temperature interval. The presence of Ca(2+) increases significantly the enzyme stability. Compared with the controls, the half-life of the purified enzyme after two purification steps in the presence of Ca(2+) increased 7.3, 20.2, and 9.8 fold at 50, 55, and 60 degrees C, respectively. Thermodynamics parameters for thermal inactivation were also determined.
一株 Chryseobacterium sp. 菌株 kr6 来源的新型角蛋白酶经（NH4）2SO4 沉淀、Sephadex G-100 凝胶过滤和 Q-Sepharose Fast Flow 阴离子交换层析纯化至均一性。纯化酶的分子量约为 20 kDa。测定了热失活动力学和热力学参数。在 50 至 60°C 的范围内，评估了 Ca2+和 Mg2+离子以及纯化程度对角蛋白酶稳定性的影响。结果表明，在该温度范围内，一级动力学很好地解释了角蛋白酶的热变性。Ca2+的存在显著提高了酶的稳定性。与对照相比，在 Ca2+存在下经过两步纯化后，纯化酶在 50、55 和 60°C 时的半衰期分别延长了 7.3、20.2 和 9.8 倍。还确定了热失活动力学参数。
