Mass-production of thermostable tryptophan indole-lyase and enzymatic production of opticallypure L-tryptophan

Abstract

In this study, we developed the practical processes for the production of L- tryptophan, which is widely used in food and pharmaceutical industry from indole, pyruvate and ammonia using thermostable tryptophanase from Symbiobacterium toebii. A gene encoding thermostable tryptophanase of Symbiobacterium toebii has previously cloned. Firstly, we employed the constitutive expression system to mass produce the thermostable tryptophanase. We established the optimal cultivation conditions for the constitutive expression system in E. coli. Obviously for a lower growth rate (μ < 0.1), intracellular conditions can be provided which are particularly favorable for expressing the heterologous protein in constitutive expression system. In this condition (μ=0.1), the final cell density reached at 35g DCW. We obtained 7.35g/L of thermostable tryptophanase, which is 42.0 % of total protein in soluble form. Secondly, we optimized the process for the production of L-tryptophan from indole, pyruvate and ammonia using thermostable tryptophanase which previously massproduced. In order to reduce the inhibition of tryptophanase by indole, we used the whole E. coli cells highly expressing thermostable tryptophanase as a biocatalyst. Optimal temperature and pH were determined to be 37℃ and 8.5, respectively, by taking into account the reaction rate and conversion yield. Enzymatic production of L-tryptophan was carried out in bench-scale reactor. Under optimal conditions, we obtained 183g/L of L-tryptophan for 37h reaction. This value corresponds to a 97% of conversion yield for indole and an 87% yield for sodium pyruvate.