Abstract

The axenic culture of Aspergillus candidus (Asp-C) produced an anti-leukemic L-asparaginase while Aspergillus sydowii (Asp-S) produced the acrylamide-reduction type. Upon mutagenesis by atmospheric and room-temperature plasma (ARTP), their individual L-asparaginase activities improved 2.3-folds in each of Ile^-Thr^-Asp-C-180-K and Val^-Asp-S-180-E stable mutants. Protoplast fusion of selected stable mutants generated fusant-09 with improved anti-leukemic activity, acrylamide reduction, higher temperature optimum and superior kinetic parameters. Submerged (SmF) and solid-state fermentation (SSF) types were compared; likewise batch, fed-batch and continuous fermentation modes; and fed-batch submerged fermentation was selected on the basis of impressive techno-economics. Fusant L-asparaginase was purified by PEG/Na⁺ citrate aqueous two-phase system and molecular exclusion chromatography to 69.96 and 146.21-fold, respectively, and characterized by molecular weight, specificity, activity and stability to chemical and physical agents. Michaelis–Menten kinetics, evaluated under optimum conditions gave K_m, V_max, K_cat, and K_cat/K_m as 1.667 × 10⁻³ M, 1666.67 µmol min⁻¹ mg⁻¹ protein, 645.99 s⁻¹ and 3.88 × 10⁵ M⁻¹ s⁻¹ respectively. In-vitro cytotoxicity of HL-60 cell lines by fusant-09 L-asparaginase improved 3.00 and 18.71-folds from mutants Ile^-Thr^-Asp-C-180-K and Val^-Asp-S-180-E, and from 5.73 and 32.55 from respective original strains. Free-radical scavenging and acrylamide reduction improvements were intermediate. Fusant-09 L-asparaginase is strongly recommended for sustainable economic anti-leukemic and food industry applications.

摘要

白色曲霉(Asp-C)的无菌培养物产生抗白血病 L-天冬酰胺酶，而sydowii 曲霉(Asp-S) 则产生丙烯酰胺还原酶。^{通过大气和室温等离子体 (ARTP) 诱变后，Ile -} Thr ^- Asp-C-180-K 和 Val ^- Asp-S-180-E 稳定突变体各自的 L-天冬酰胺酶活性提高了 2.3 倍。所选稳定突变体的原生质体融合产生了具有改进的抗白血病活性、丙烯酰胺减少、更高的最适温度和优异的动力学参数的fusant-09。比较了深层发酵 (SmF) 和固态发酵 (SSF) 类型；同样是分批、补料分批和连续发酵模式；基于令人印象深刻的技术经济性，选择了分批补料深层发酵。^{Fusant L-天冬酰胺酶经PEG/Na +}柠檬酸盐水两相系统和分子排阻色谱法分别纯化至69.96和146.21倍，并对其分子量、特异性、活性以及对化学和物理试剂的稳定性进行了表征。在最佳条件下评估的米氏动力学得出K _m、V _max、K _cat和K _cat / K _m为 1.667 × 10 ^-3 M、1666.67 µmol min ^-1 mg ^-1蛋白质、645.99 s ^-1和 3.88 ×分别为10 ⁵ M ^-1 s ^-1。^{Fusant-09 L-天冬酰胺酶对 HL-60 细胞系的体外细胞毒性较突变体 Ile -} Thr ^- Asp-C-180-K 和 Val ^- Asp-S-180-E分别提高了 3.00 和 18.71 倍，以及 5.73 倍和 32.55 来自各自的原始菌株。自由基清除和丙烯酰胺减少的改善程度中等。Fusant-09 L-天冬酰胺酶强烈推荐用于可持续经济的抗白血病和食品工业应用。