School of Biological and Food Engineering, Suzhou University, Suzhou, 234000, China.
Appl Biochem Biotechnol. 2021 Oct;193(10):3407-3417. doi: 10.1007/s12010-021-03589-7. Epub 2021 Jun 7.
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 k/K of the mutant enzyme (V231A/K382G) is 4.36 mM∙s. 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.
L-5-羟色氨酸是一种重要的氨基酸，广泛应用于食品和医药领域。本研究采用改良色氨酸合酶合成 L-5-羟色氨酸。应用易错 PCR 对大肠杆菌色氨酸合酶进行定向进化改造，以提高 L-5-羟色氨酸的合成效率。采用高通量筛选方法获得高活性突变酶（V231A/K382G）。突变酶（V231A/K382G）的酶活比其亲本提高了 3.79 倍，k/K 值提高到 4.36mM·s。突变酶（V231A/K382G）催化 L-5-羟色氨酸合成的最佳反应条件为：L-丝氨酸 100mmol/L，pH8.5，35℃反应 15h，L-5-羟色氨酸的产率可达 86.7%。定向进化是提高色氨酸合酶酶活的有效策略。
