关键词: 大肠杆菌, 色氨酸酶, 酶学性质, 反应动力学

Abstract:

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.

Key words: Escherichia coli, tryptophanase, enzymatic properties, kinetics