大肠杆菌色氨酸酶的制备及酶学性质

以硫酸铵沉淀法分离提取大肠杆菌色氨酸酶，进行色氨酸酶学性质检测及动力学初步探讨。结果表明：使用50℃水浴法进行细胞壁的破碎，提取粗TPase 酶液效果较好；(NH₄)₂SO₄ 粗提色氨酸酶有较好效果；色氨酸酶的最佳反应pH 值为8.0，在pH5.5～7.5 较稳定；最高耐受温度65℃，最佳反应温度45℃；K⁺、NH⁴⁺ 能够明显地提高色氨酸酶活性，而Na+ 则明显地抑制酶活性。对色氨酸酶的动力学研究表明，在相同反应条件下，针对底物L- 丝氨酸的米式常数Km 值远远大于吲哚的Km 值。

Preparation and Enzymatic Properties of Tryptophanase from Escherichia coli

The suspension of cultured Escherichia coli was subjected to cell disruption and enzyme separation/extraction to prepare tryptophanase. The preparation process was investigated and the crude enzyme solution obtained was characterized for its enzymatic properties and reaction kinetic characteristics. Among four cell disruption methods, 50 ℃water bath heating and adding TritonX-100 were considered to be two better methods than adding SDS and microwave treatment due to ease of operation, mild reaction, good preservation of enzyme activity, and so on, and 50 ℃water bath heating method was selected in this study. Fractional salting-out with ammonium sulphate was the best method for the separation of tryptophanase among three methods. The enzyme revealed an optimal reaction temperature of 45 ℃ and was stable in a pH range of 5.5 to 7.5. Its maximum tolerant and optimal reaction temperatures were 65 ℃ and 45 ℃, respectively. K+ and NH4+ could effectively elevate the activity of the enzyme, whereas the enzyme was remarkably inactivated by Na+. Kinetic studies demonstrated that the kinetic parameter Km of the enzyme towards the substrate L-serine was much larger than that towards indole.