大肠杆菌色氨酸酶的制备及酶学性质

Preparation and Enzymatic Properties of Tryptophanase from Escherichia coli

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。

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.