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) is 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.
Highlights
L-5-Hydroxytryptophan is a special amino acid found in both plants and animals
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-5hydroxytryptophan synthesis
Summary
L-5-Hydroxytryptophan is a special amino acid found in both plants and animals. L-5-Hydroxytryptophan increases brain serum concentrations and brain melatonin levels, improves symptoms of spinal cord atrophy and promotes sleep [1]. L-5-Hydroxytryptophan was prepared by plant extraction and biosynthesis. L-5-Hydroxytryptophan was prepared by tryptophan hydroxylase, which promoted the synthesis of L-5-hydroxytryptophan by metabolic engineering [7, 8]. The yield of L-5-hydroxytryptophan prepared by the plant extraction method was lower compared with the yield prepared by the biosynthesis method, and the enzymatic reaction was specific with fewer by-products. Enzymatic synthesis is an effective strategy to synthesize short-chain flavor esters, phytosteryl lipoate, Lo-galloylglycerol [9,10,11]. In this study, directed evolution of tryptophan synthase was performed by error-prone PCR. L-5-Hydroxytryptophan was synthesized by L-serine and 5hydroxyindole using tryptophan synthase as the catalyst. This provides a new strategy for the enzymatic synthesis of L-5-hydroxytryptophan
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