School of Life Sciences, Anhui University, 230601 Hefei, Anhui, China; Anhui Key Laboratory of Biocatalysis and Modern Biomanufacturing, 230601 Hefei, Anhui, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, 230601 Hefei, Anhui, China.
School of Life Sciences, Anhui University, 230601 Hefei, Anhui, China; Anhui Key Laboratory of Biocatalysis and Modern Biomanufacturing, 230601 Hefei, Anhui, China; Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, 230601 Hefei, Anhui, China.
Int J Biol Macromol. 2024 Oct;277(Pt 4):134534. doi: 10.1016/j.ijbiomac.2024.134534. Epub 2024 Aug 5.
A fungal laccase-mediator system capable of high effectively oxidizing tetracyclines under a wide pH range will benefit environmental protection. This study reported a directed evolution of a laccase PIE5 to improve its performance on tetracyclines oxidization at alkaline conditions. Two mutants, namely MutA (D229N/A244V) and MutB (N123A/D229N/A244V) were obtained. Although they shared a similar optimum pH and temperature as PIE5, the two mutants displayed approximately 2- and 5-fold higher specific activity toward the mediators ABTS and guaiacol at pHs 4.0 to 6.5, respectively. Simultaneously, their catalytic efficiency increased by 8.0- and 6.4-fold compared to PIE5. At a pH range of 5-8 and 28 °C, MutA or MutB at a final concentration of 2.5 U·mL degraded 77 % and 81 % of 100 mg·L tetracycline within 10 min, higher than PIE5 (45 %). Furthermore, 0.1 U·mL MutA or MutB completely degraded 100 mg·L chlortetracycline within 6 min in the presence of 0.1 mM ABTS. At pH 8.0, MutA degraded tetracycline and chlortetracycline following the routine pathways were reported previously based on LC-MS analysis.
一种能够在较宽 pH 范围内高效氧化四环素的真菌漆酶-介体系统将有益于环境保护。本研究报道了漆酶 PIE5 的定向进化，以提高其在碱性条件下氧化四环素的性能。得到了两个突变体，即 MutA（D229N/A244V）和 MutB（N123A/D229N/A244V）。尽管它们与 PIE5 具有相似的最适 pH 和温度，但这两个突变体在 pH 4.0 至 6.5 下对 ABTS 和愈创木酚的介体的比活性分别提高了约 2 倍和 5 倍。同时，与 PIE5 相比，它们的催化效率分别提高了 8.0 倍和 6.4 倍。在 pH 5-8 和 28°C 的条件下，终浓度为 2.5 U·mL 的 MutA 或 MutB 在 10 分钟内可降解 100 mg·L 的四环素 77%和 81%，高于 PIE5（45%）。此外，在 0.1 mM ABTS 的存在下，0.1 U·mL 的 MutA 或 MutB 在 6 分钟内可完全降解 100 mg·L 的金霉素。在 pH 8.0 下，通过 LC-MS 分析，MutA 降解四环素和金霉素的途径与先前报道的常规途径一致。
