Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
Int J Biol Macromol. 2024 Jan;255:128258. doi: 10.1016/j.ijbiomac.2023.128258. Epub 2023 Nov 19.
The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior.
SUMO 蛋白酶（Ulps）是一组半胱氨酸蛋白酶，以其有效切割感兴趣蛋白质上的 SUMO 标签并生成具有真实 N 末端的生物治疗药物的能力而闻名。然而，Ulps 的稳定性仍然是在大肠杆菌中经济生产难以生产的蛋白质的挑战。因此，本研究旨在建立使用不同酶固定化策略开发稳定的 S. pombe Ulp1 制剂的方法。使用毕赤酵母 Pir1 锚蛋白开发的全细胞生物催化剂在反应孵育 24 小时内切割 SUMO 标签。使用商业环氧和氨基甲基丙烯酸酯珠的化学固定化显著提高了 SpUlp1 的操作可重复使用性，达到 24 个循环。硅胶珠进一步将固定化酶的重复使用提高到 65 个循环。SpUlp1 在实验室开发的壳聚糖涂覆的氧化铁纳米粒子上的固定化表现出对不同底物的 SUMO 标签的切割效率超过 90%，即使在 100 次连续反应后也是如此。此外，在孵育 10 分钟内观察到有效的 SUMO 标签去除。固定化酶的操作稳定性在 pH 值为 5 到 13 的范围内得到了确认。FESEM 和 TEM 结果证实了纳米粒子的球形性质。FT-IR 证实了壳聚糖的成功涂层和随后用戊二醛的活化。此外，HRTEM、SAED 和 XRD 证明了纳米粒子的结晶性质，而 VSM 则证实了超顺磁性行为。
