Directed evolution  improves the enzymatic synthesis of L-5-hydroxytryptophan by an  engineered tryptophan synthase

Abstract

L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) was 4.36 mM− 1∙s− 1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

Residual Life Prediction and Design Correction Method of Corroded Circular Steel Tubes Based on Time-Varying Reliability

“…A large number of engineering applications and measurements have indicated that there is significant randomness and variability in both load and resistance. The uncertain statistical parameters of loads according to the provisions of "Unified Standard for Reliability Design of Building structures" GB50068-2018 [4], "Code for load of Building structures" GB50009-2012 [25], and "Technical Specification for Spatial Grid structures" JGJ7-2010 [33] are shown in Table 1.…”

“…As the service life of a large number of steel structures progresses, durability issues [1][2][3] related to corrosion problems come to the fore [4,5]. However, there are no operational assessment and design correction methods that take into consideration the development and effects of corrosion under different environments.…”