School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan.
Department of Microbiology, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei Province, China.
Int J Biol Macromol. 2024 Nov;281(Pt 3):136442. doi: 10.1016/j.ijbiomac.2024.136442. Epub 2024 Oct 9.
l-Asparaginases catalyze the hydrolysis of l-asparagine to l-aspartic acid and ammonia. These enzymes have potential applications in therapeutics and food industry. Tk1656, a highly active and thermostable l-asparaginase from Thermococcus kodakarensis, has been proved effective in selective killing of acute lymphocytic leukemia cells and in reducing acrylamide formation in baked and fried foods. However, it displayed <5 % activity under physiological conditions compared to the optimal activity at 85 °C and pH 9.5. We have attempted engineering of this valuable enzyme to improve the characteristics required for therapeutic and industrial applications. Based on the literature and crystal structure of Tk1656, nine specific mutant variants were designed, produced in Escherichia coli, and the purified mutant enzymes were compared with the wild-type. One of the mutants, K299L, displayed >20 % increase in activity at 85 °C. H158S substitution resulted in >5 °C increase in the optimal temperature. Similarly, a mesophilic-like mutation L56D, resulted in >5-fold increase in activity at pH 7.0 and 37 °C compared to that of the wild-type enzyme. The substrate specificity of the mutant variants remained unchanged. These results demonstrate that L56D and K299L variants of Tk1656 are the potent enzymes for therapeutics and acrylamide mitigation applications, respectively.
l-天冬酰胺酶催化 l-天冬酰胺水解为 l-天冬氨酸和氨。这些酶在治疗学和食品工业中有潜在的应用。来自 Thermococcus kodakarensis 的高活性和热稳定的 l-天冬酰胺酶 Tk1656 已被证明可有效选择性杀死急性淋巴细胞白血病细胞，并减少烘焙和油炸食品中丙烯酰胺的形成。然而，与在 85°C 和 pH 9.5 下的最佳活性相比，它在生理条件下的活性<5%。我们已经尝试对这种有价值的酶进行工程改造，以改善治疗和工业应用所需的特性。基于 Tk1656 的文献和晶体结构，设计了九个特定的突变变体，在大肠杆菌中生产，并比较了纯化的突变酶与野生型。突变体之一 K299L 在 85°C 时的活性增加了>20%。H158S 取代导致最适温度升高了>5°C。同样，类似于嗜温菌的突变 L56D 在 pH 7.0 和 37°C 时的活性比野生型酶增加了>5 倍。突变变体的底物特异性保持不变。这些结果表明，Tk1656 的 L56D 和 K299L 变体分别是治疗学和丙烯酰胺缓解应用的有效酶。
