Abstract：
BACKGROUND: Microbial L-asparaginase (L-ASNase, EC 3.5.1.1) is a pivotal biopharmaceutical drug-protein that catalyzes the hydrolysis of the non-essential amino acid L-asparagine (L-Asn) into L-aspartic acid (L-Asp) and ammonia , resulting in deplenishing the cellular L-Asn pool, which leads to the ultimate death of the L-asparagine synthetase (L-ASNS) deficient cancerous cells.
OBJECTIVE: This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction.
METHODS: The recombinant P. aeruginosa L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein\'s N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized.
RESULTS: The Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 μmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 μmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced LASNase thermostability at 37, 45, 50, and 55 °C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37 °C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an in vivo study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form\'s activity vanished entirely from the rats\' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme.
CONCLUSIONS: For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged in vivo half-life duration, which could be considered an alternative to mitigate the high molecular weight of PEGylation\'s drawbacks.
OBJECTIVE: This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction.
METHODS: The recombinant P. aeruginosa L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein\'s N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized.
RESULTS: The Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 μmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 μmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced LASNase thermostability at 37, 45, 50, and 55 °C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37 °C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an in vivo study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form\'s activity vanished entirely from the rats\' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme.
CONCLUSIONS: For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged in vivo half-life duration, which could be considered an alternative to mitigate the high molecular weight of PEGylation\'s drawbacks.
摘要：
背景：微生物L-天冬酰胺酶（L-ASNase，EC3.5.1.1）是一种关键的生物制药药物蛋白，可催化非必需氨基酸L-天冬酰胺（L-Asn）水解为L-天冬氨酸（L-Asp）和氨，导致细胞L-Asn池耗尽，这导致L-天冬酰胺合成酶（L-ASNS）缺陷的癌细胞最终死亡。
目的：本研究旨在通过药代动力学特性研究低分子量聚乙二醇对重组铜绿假单胞菌L-ASNase的影响，对底物的亲和力，以及反应前后的酶稳定性。
方法：将重组铜绿假单胞菌L-ASNase亲和纯化，然后通过将聚乙二醇（MW=330Da）位点特异性地连接到蛋白质的N末端进行PEG化。之后,通过SDS-PAGE(15%)检查聚乙二醇化的L-ASNase,FTIR,和UV/Vis分光光度法，随后进行生化表征。
结果：确定游离铜绿假单胞菌rL-ASNase的Km和Vmax值为0.318±1.76mM和2915μmolmin-1，它们分别为0.396±1.736mM和3193μmolmin-1。聚乙二醇（330Da）在37、45、50和55°C时具有显着增强的LASNase热稳定性，与游离酶相反，在37°C孵育1小时后保留了19.5%。发现聚乙二醇化的L-ASNase在与人血清孵育28小时后是稳定的，与孵育4小时后游离rL-ASNase的残留生物活性急剧下降相反。因此，体内研究用于验证，证明聚乙二醇化rL-ASNase表现出更长的生物活性24小时，而游离形式的活性在8小时后从大鼠血清中完全消失。分子动力学模拟表明，PEG（330Da）影响了L-ASNase的流体动力学体积并增加了其结构稳定性。对接分析探索了PEG相对于结合位点的位置，并预测了与游离酶相似的结合亲和力。
结论：第一次，通过共价连接PEG(330Da)修饰重组L-ASNase。所得的新提出的PEG化rL-ASNase具有显着增加的稳定性和延长的体内半衰期，这可以被认为是减轻高分子量聚乙二醇化缺点的替代方法。
目的：本研究旨在通过药代动力学特性研究低分子量聚乙二醇对重组铜绿假单胞菌L-ASNase的影响，对底物的亲和力，以及反应前后的酶稳定性。
方法：将重组铜绿假单胞菌L-ASNase亲和纯化，然后通过将聚乙二醇（MW=330Da）位点特异性地连接到蛋白质的N末端进行PEG化。之后,通过SDS-PAGE(15%)检查聚乙二醇化的L-ASNase,FTIR,和UV/Vis分光光度法，随后进行生化表征。
结果：确定游离铜绿假单胞菌rL-ASNase的Km和Vmax值为0.318±1.76mM和2915μmolmin-1，它们分别为0.396±1.736mM和3193μmolmin-1。聚乙二醇（330Da）在37、45、50和55°C时具有显着增强的LASNase热稳定性，与游离酶相反，在37°C孵育1小时后保留了19.5%。发现聚乙二醇化的L-ASNase在与人血清孵育28小时后是稳定的，与孵育4小时后游离rL-ASNase的残留生物活性急剧下降相反。因此，体内研究用于验证，证明聚乙二醇化rL-ASNase表现出更长的生物活性24小时，而游离形式的活性在8小时后从大鼠血清中完全消失。分子动力学模拟表明，PEG（330Da）影响了L-ASNase的流体动力学体积并增加了其结构稳定性。对接分析探索了PEG相对于结合位点的位置，并预测了与游离酶相似的结合亲和力。
结论：第一次，通过共价连接PEG(330Da)修饰重组L-ASNase。所得的新提出的PEG化rL-ASNase具有显着增加的稳定性和延长的体内半衰期，这可以被认为是减轻高分子量聚乙二醇化缺点的替代方法。
