3,4-Dihydroxyphenyl-L-alanine (L-DOPA) is the most important antiparkinsonian drug, and tyrosine phenol-lyase (TPL)-based enzyme catalysis process is one of the most adopted methods on industrial scale production. TPL activity and stability represent the rate-limiting step in L-DOPA synthesis. Here, 25 TPL mutants were predicted, and two were confirmed as exhibiting the highest L-DOPA production and named E313W and E313M. The L-DOPA production from E313W and E313M was 47.5 g/L and 62.1 g/L, which was 110.2 % and 174.8 % higher, respectively, than that observed from wild-type (WT) TPL. The Km of E313W and E313M showed no apparent decrease, whereas the kcat of E313W and E313M improved by 45.5 % and 36.4 %, respectively, relative to WT TPL. Additionally, E313W and E313M displayed improved thermostability, a higher melting temperature, and enhanced affinity between for pyridoxal-5'-phosphate. Structural analysis of the mutants suggested increased stability of the N-terminal region via enhanced interactions between the mutated residues and H317. Application of these mutants in a substrate fed-batch strategy as whole-cell biocatalysts allows realization of a cost-efficient short fermentation period resulting in high L-DOPA yield.

中文翻译：

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定点诱变可提高酪氨酸酚裂解酶的热稳定性。

3,4-二羟基苯基-L-丙氨酸（L-DOPA）是最重要的抗帕金森病药物，基于酪氨酸酚裂解酶（TPL）的酶催化工艺是工业规模生产中最常用的方法之一。TPL活性和稳定性代表了L-DOPA合成中的限速步骤。在这里，预测了25个TPL突变体，其中两个被证实表现出最高的L-DOPA产量，并命名为E313W和E313M。E313W和E313M的L-DOPA产量分别为47.5 g / L和62.1 g / L，分别比野生型（WT）TPL产量高110.2％和174.8％。相对于WT TPL，E313W和E313M的Km没有明显降低，而E313W和E313M的kcat分别提高了45.5％和36.4％。此外，E313W和E313M表现出更高的热稳定性，更高的熔化温度，和对5'-磷酸吡-醛的亲和力增强。突变体的结构分析表明，通过增强突变残基与H317之间的相互作用，可以增强N末端区域的稳定性。这些突变体作为全细胞生物催化剂在底物补料分批策略中的应用允许实现具有成本效益的短发酵期，从而导致高L-DOPA产量。