School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian, Liaoning, 116023, China.
School of Life Science and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian, Liaoning, 116023, China.
Enzyme Microb Technol. 2019 Jan;120:36-42. doi: 10.1016/j.enzmictec.2018.09.004. Epub 2018 Sep 18.
A facile and economical method was established for the oriented immobilization of biotin ligase (BirA) on Co-NTA sepharose through HO oxidation of Co and His-tag. His-tag of the BirA were designed at both N-terminal (His-BirA) and C-terminal (BirA-His), respectively. Immobilization of the His-BirA was performed, realized to 92.85% using by 10 mM HO without compromising catalytic activity. Because amounts of ions on matrix were far more than that of the immobilized BirA, EDTA should be used to remove residual ions before catalyzing, while it should be limited to lower than 30 mM, and imidazole ranging from 50 to 250 mM could be added in the catalytic system. When 10 mM EDTA and 50 mM imidazole were used, over 90% of substrates were obtained from the matrix. Moreover, the His-BirA showed higher immobilization rate than the BirA-His, while both of them appeared high catalytic abilities at pH ranging from 6.5 to 9.0, indicating versatile options in the biotinylation of proteins with different pH stabilities. Under the best catalytic conditions, the both immobilized His-BirA and BirA-His exhibited the same activity as the free. When the enzyme was incubated at different pH (pH 3.0, 4.0, 5.0, 10.0 and 11.0) and temperature (40 °C, 50 °C and 60 °C), the immobilized His-BirA showed less pH-sensitive, overall preferable thermo-stability than the free, making it a more desirable option for storage and transportation. More importantly, the reusability of the immobilized His-BirA implied a promising value in industrialization.
建立了一种简便、经济的方法，通过 Co 和 His 标签的 HO 氧化，将生物素连接酶（BirA）定向固定在 Co-NTA 琼脂糖上。BirA 的 His 标签分别位于 N 端（His-BirA）和 C 端（BirA-His）。通过 10 mM 的 HO 进行固定化，His-BirA 的固定化率达到 92.85%，同时不影响其催化活性。由于基质上的离子数量远远超过固定化的 BirA，因此在催化之前，应该使用 EDTA 去除残留的离子，但 EDTA 的浓度应限制在 30 mM 以下，并且可以在催化体系中添加 50-250 mM 的咪唑。当使用 10 mM EDTA 和 50 mM 咪唑时，超过 90%的底物从基质中获得。此外，His-BirA 的固定化率高于 BirA-His，而它们在 pH 6.5-9.0 范围内都表现出较高的催化能力，这表明在不同 pH 稳定性的蛋白质生物素化方面有多种选择。在最佳催化条件下，固定化的 His-BirA 和 BirA-His 的活性与游离酶相同。当酶在不同的 pH（pH 3.0、4.0、5.0、10.0 和 11.0）和温度（40°C、50°C 和 60°C）下孵育时，固定化的 His-BirA 表现出较低的 pH 敏感性，整体热稳定性优于游离酶，使其成为储存和运输的更理想选择。更重要的是，固定化的 His-BirA 的可重复使用性暗示了其在工业化方面具有广阔的应用前景。
