School of Chemistry and Environmental Engineering, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai, 201418, China.
Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore, 117585, Singapore.
Bioprocess Biosyst Eng. 2022 Jun;45(6):1075-1088. doi: 10.1007/s00449-022-02729-5. Epub 2022 May 9.
A series of nickel-incorporated SBA-15 mesoporous molecular sieves (Ni-SBA-15) were prepared as support for the immobilization of his-tagged recombinant Microbacterium esterase. The Ni-SBA-15 could strongly and specific absorb the his-tagged esterase from cell disrupted supernatant. It was found that the nickel amount in Ni-SBA-15 has dramatic influence on the activity and thermo-stability of immobilized enzyme, while the kinds of nickel precursor had little effect on enzyme stability. The morphology, chemical composition and structure of the best support NiCl-SBA-15 (Ni-SBA-15 prepared from NiCl precursor) were characterized by various spectroscopy techniques. The immobilized esterase retained full activity of free esterase and showed high immobilized yield (> 90%) with higher thermo-stability, pH stability and organic solvent resistance compared with free enzyme. The optimum reaction temperature increased from 35 to 40 °C and the optimal reaction pH moved from 10.0 to 8.0 after enzyme immobilization. The immobilized esterase exhibited excellent storage stability and keeping 92% of the initial activity after 30 days' storage at 25 °C. In addition, the immobilized esterase had excellent reusability for the synthesis of key chiral intermediate of d-biotin and the substrate conversion could still keep 100% after 13 cycles continuously. Finally, optical pure (4S, 5R)-hemiester was obtained in 80.8% isolated yield and 99% purity in the gram preparative scale.
一系列镍掺杂的 SBA-15 介孔分子筛（Ni-SBA-15）被制备为固定化组氨酸标签重组微杆菌酯酶的载体。Ni-SBA-15 可以从细胞破碎的上清液中强烈且特异性地吸附组氨酸标签酯酶。结果表明，Ni-SBA-15 中的镍含量对固定化酶的活性和热稳定性有显著影响，而镍前体的种类对酶稳定性影响较小。通过各种光谱技术对最佳载体 NiCl-SBA-15（由 NiCl 前体制备的 Ni-SBA-15）的形貌、化学组成和结构进行了表征。固定化酯酶保留了游离酯酶的全部活性，与游离酶相比，具有更高的固定化产率（>90%）、更高的热稳定性、pH 稳定性和有机溶剂耐受性。固定化酶的最适反应温度从 35°C 升高到 40°C，最适反应 pH 从 10.0 移动到 8.0。固定化酯酶具有出色的储存稳定性，在 25°C 下储存 30 天后，仍保持初始活性的 92%。此外，固定化酯酶在合成关键手性中间体制备 d-生物素的反应中具有极好的可重复使用性，连续 13 次循环后，底物转化率仍保持 100%。最后，在克级规模的制备实验中，光学纯（4S,5R）-半酯以 80.8%的分离收率和 99%的纯度获得。
