Arredondo-Nuñez Annsy, Monteiro Gisele, Flores-Fernández Carol N, Antenucci Lina, Permi Perttu, Zavaleta Amparo Iris
Laboratorio de Biología Molecular, Facultad de Farmacia y Bioquímica, Universidad Nacional Mayor de San Marcos, Lima 01, Peru.
Department of Pharmaceutical and Biochemical Technology, School of Pharmaceutical Sciences, University of São Paulo, São Paulo 05508-000, Brazil.
Life (Basel). 2023 Oct 31;13(11):2145. doi: 10.3390/life13112145.
L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene of halotolerant CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl, MgCl, mercaptoethanol, and DL-dithiothreitol (-value < 0.01). Moreover, the and were 145.2 µmol mL min and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.
来自细菌源的L-天冬酰胺酶已用于抗肿瘤治疗和食品工业。从秘鲁奇尔卡盐沼分离的耐盐CH11的N端截短基因编码的II型L-天冬酰胺酶，利用异源系统在BL21(DE3)pLysS中表达。重组蛋白通过一步镍亲和层析纯化，活性为234.38 U mg，在pH 9.0和60℃时具有最大催化活性。通过凝胶过滤层析，该酶呈现同四聚体形式，估计分子量为155 kDa。该酶在60℃的半衰期为3小时48分钟，L-天冬酰胺酶在37℃下24小时保留其初始活性的50%。KCl、CaCl、MgCl、巯基乙醇和DL-二硫苏糖醇显著增强了其活性（P值<0.01）。此外，Km和Vmax分别为145.2 µmol mL min和4.75 mM。这些发现证明了一种有前景的新型II型L-天冬酰胺酶在未来工业应用中的潜力。
