桔青霉产核酸酶P1酶分离纯化及其酶学性质

李明; 余华顺; 喻晨; 吴尧; 龚大春

doi:10.13386/j.issn1002-0306.2020050212

桔青霉产核酸酶P1酶分离纯化及其酶学性质

Isolation, Purification and Enzymatic Properties of Nuclease P1 Fermented by Penicillium citrinum

摘要

摘要: 桔青霉（Penicillium citrinum）产核酸酶P1浓缩液采用活性炭脱色、硫酸铵分级沉淀、脱盐和凝胶层析等分离技术，得到核酸酶P1纯组分，并研究了该酶的酶学性质。该酶纯化后比酶活达到33967 U/mg，纯化倍数为8.48倍；该酶的米氏常数K_m、最大反应速度V_m和催化常数K_cat分别为2.50 mmol/L、0.0864 mmol/（mL·min）和252.43 s⁻¹。该酶最适温度为75 ℃，热稳定范围60～75 ℃；最适pH为5.5，pH稳定范围为4.0～6.0；Zn²⁺在1 mmol/L条件下对核酸酶P1有很好的激活作用，Cu²⁺和Co²⁺对该酶的抑制作用明显，而Ni²⁺、Fe²⁺、Mn²⁺等离子具有不同程度的抑制作用。本研究对于该酶的广泛应用奠定了科学基础。

Abstract: The nuclease P1 was purified to obtain pure component by activated carbon decolorization, (NH₄)₂SO₄ precipitation, desalination and gel chromatography and its enzymatic properties was investigated. This purified enzyme had a specific activity of 33967 U/mg protein after 8.48-fold purification. The Michaelis constant (K_m), the maximum reaction rates (V_m) and the catalytic constant (K_ca_t) of the purified enzyme were 2.50 mmol/L, 0.0864 mmol/(mL·min) and 252.43 s⁻¹, respectively. The optimization pH and temperature for the nuclease P1 were at pH5.5 and 75 ℃. The enzyme was stable in the temperature range from 60 to 75 ℃ and in the pH range from 4.0 to 6.0. Zn²⁺ had a positive effect on the enzyme activity, while Cu²⁺ was a strong inhibitor of nuclease P1, Ni²⁺、Fe²⁺、Mn²⁺ had the different inhibition. This research laid a scientific foundation for the extensive application of the enzyme.