Abstract

l-Tyrosine is a versatile compound used in the fine chemical, pharmaceutical, and functional food industries. Here, we report a bi-enzymatic cascade involving alanine racemase (ALR) and d-amino acid oxidase (DAAO) to produce pyruvate, as co-substrate for l-tyrosine production, from the cheap substrate l-alanine. The BpALR (ALR from Bacillus pseudofirmus) was used as a whole-cell biocatalyst, converting l-alanine to d, l-alanine. The FsDAAO (DAAO from Fusarium solani) was immobilized to oxidize the d-alanine generated in the first step to pyruvate. Both systems were combined as a continuous-flow reactor for maximized l-alanine-to-pyruvate conversion rates. The optimal parameters and appropriate conditions for FsDAAO immobilization were investigated. The pyruvate concentration of 86.6 g/L was achieved within 17 h. Subsequently, a whole-cell biocatalyst system for l-tyrosine production, catalyzed by the tyrosine phenol-lyase (TPL) from Erwinia herbicola (EhTPL), was developed, and a fed-batch approach was applied with phenol and the pyruvate produced with the ALR/DAAO system mentioned above. The concentration of phenol and pyruvate in the reactor should not exceed 7.5 g/L and 10 g/L, respectively. Significantly, the l-tyrosine concentration of 152.5 g/L was achieved within 10 h, demonstrating the great potential for high-efficiency production of l-tyrosine through the approach we established in this paper.

Graphical abstract

Key points

• A specific bioreactor system for pyruvate produced from l-alanine was developed

• The appropriate condition for immobilization of FsDAAO was investigated

• A fed-batch process was established to produce l-tyrosine with recombinant E. coli

• The bi-enzymatic cascade was successfully used for l-tyrosine production at low cost

中文翻译：

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一种双酶联反应级联反应，产生丙酮酸作为生产酪氨酸的辅助底物

摘要

升-酪氨酸处于精细化工，制药，和功能性食品工业中使用的一种通用化合物。在这里，我们报告了一种双酶级联反应，涉及丙氨酸消旋酶（ALR）和d-氨基酸氧化酶（DAAO），从廉价的底物l-丙氨酸生产丙酮酸，作为l-酪氨酸生产的共底物。BpALR（假芽孢杆菌的ALR ）用作全细胞生物催化剂，将1-丙氨酸转化为d，1-丙氨酸。将FsDAAO（Fusarium solani的DAAO ）固定化以氧化d在第一步将丙酮酸生成的-丙氨酸。将两个系统组合为连续流反应器，以实现1-丙氨酸至丙酮酸盐的最大转化率。研究了固定FsDAAO的最佳参数和合适条件。17小时内丙酮酸浓度达到86.6 g / L。随后，全细胞生物催化剂系统，用于升-酪氨酸生产，通过从酪氨酸酚裂合酶（TPL）催化的草生欧文氏（EhTPL），显影，和补料分批方法用苯酚和与所产生的丙酮酸施加上面提到的ALR / DAAO系统。反应器中苯酚和丙酮酸盐的浓度分别不应超过7.5 g / L和10 g / L。显著的升在10小时内达到了152.5 g / L的酪氨酸浓度，这表明通过我们在本文中建立的方法，高效生产1-酪氨酸的巨大潜力。

图形概要

关键点

•开发了一种用于由 1-丙氨酸生产丙酮酸的特定生物反应器系统

•研究了固定FsDAAO的合适条件

•建立了分批 补料工艺，用重组大肠杆菌生产1-酪氨酸

•在双酶级联被成功地用于 升-酪胺酸生产成本低