Department of R&D of Zhejiang zhengshuo Biological Co., Ltd, Huzhou 313000, Zhejiang, China; College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
College of life science, Fujian normal university, Fujian 350000, China.
Enzyme Microb Technol. 2020 Jan;132:109393. doi: 10.1016/j.enzmictec.2019.109393. Epub 2019 Aug 8.
In our previous study, we produced α-keto acids by using an L-amino acid deaminase PmiLAAD (wide-type) from Proteus mirabilis, however, the catalytic efficiency was low due to its low substrate affinity. In this study, protein engineering of PmiLAAD was performed to improve the α-keto acid production. PmiLAAD was engineered by iterative CASTing to improve its catalytic performance. The four mutant PmiLAAD-SAVS (PmiLAAD-Phe93Ser-Pro186Ala- Met394Val-Phe184Ser) with 6.6 -fold higher specific activity compared with that of wild-type PmiLAAD has been obtained by high-throughput screening. Comparative kinetics analysis showed that the four mutant PmiLAAD-SAVS had a higher substrate-binding affinity and catalytic efficiency than that of PmiLAAD wild-type. The K, k, and k/K values of the PmiLAAD(SAVS) variant was better (-42.7%, 75.11%, and 85.79%, respectively) than the corresponding values of PmiLAAD wild type. Finally, the whole cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS) has been applied to α-keto acids production. The conversion rate of L-phenylalanine reached 99% by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS). The conversion of (D/L)-4-phenylalanine was reached 49.5% after 7 h by whole-cell biocatalyst E. coli-pETDuet-1-PmiLAAD(SAVS), while the conversion of E. coli-pETDuet-1-PmiLAAD (wild type) was only 18% after an extension of the reaction time (24 h). This study has developed a robust whole-cell E. coli biocatalyst for α-keto acids production by protein engineering, and this strategy may be useful for the construction of other biotransformation biocatalysts.
在我们之前的研究中，我们使用来自奇异变形杆菌的 L-氨基酸脱氨酶 PmiLAAD（野生型）生产了α-酮酸，但是由于其底物亲和力低，催化效率很低。在这项研究中，通过迭代 CASTing 对 PmiLAAD 进行了蛋白质工程改造，以提高 α-酮酸的产量。通过高通量筛选获得了与野生型 PmiLAAD 相比，比活提高了 6.6 倍的四个突变体 PmiLAAD-SAVS（PmiLAAD-Phe93Ser-Pro186Ala-Met394Val-Phe184Ser）。比较动力学分析表明，与野生型 PmiLAAD 相比，四个突变体 PmiLAAD-SAVS 具有更高的底物结合亲和力和催化效率。PmiLAAD(SAVS)变体的 K、k 和 k/K 值分别提高了(-42.7%、75.11%和 85.79%)，优于 PmiLAAD 野生型的相应值。最后，应用全细胞生物催化剂 E. coli-pETDuet-1-PmiLAAD(SAVS)生产α-酮酸。全细胞生物催化剂 E. coli-pETDuet-1-PmiLAAD(SAVS)的 L-苯丙氨酸转化率达到 99%。通过全细胞生物催化剂 E. coli-pETDuet-1-PmiLAAD(SAVS)，(D/L)-4-苯丙氨酸的转化率在 7 小时后达到 49.5%，而全细胞生物催化剂 E. coli-pETDuet-1-PmiLAAD（野生型）在延长反应时间（24 小时）后仅达到 18%。本研究通过蛋白质工程开发了一种用于α-酮酸生产的稳健全细胞大肠杆菌生物催化剂，该策略可能对其他生物转化生物催化剂的构建有用。
