Ekpenyong Maurice, Asitok Atim, Antigha Richard, Ogarekpe Nkpa, Ekong Ubong, Asuquo Marcus, Essien Joseph, Antai Sylvester
Environmental Microbiology and Biotechnology Unit, Department of Microbiology, University of Calabar, Calabar, Nigeria.
Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, University of Calabar, Calabar, Nigeria.
Int J Pept Res Ther. 2021;27(2):1501-1527. doi: 10.1007/s10989-021-10188-x. Epub 2021 Mar 9.
Sequential optimization of bioprocess nutritional conditions for production of glutaminase-near-free L-asparaginase by UCCM 00117 was conducted under shake flask laboratory conditions. Catalytic and anti-cancer activities of the poly-peptide were evaluated using standard in vitro biochemical methods. Medium nutrients were selected by one-factor-at-a-time (OFAT) approach while Plackett-Burman design (PBD) screened potential factors for optimization. Path of steepest ascent (PSA) and response surface methodology (RSM) of a Min-Run-Res V fractional factorial of a central composite rotatable design (CCRD) were employed to optimize factor levels towards improved enzyme activity. A multi-objective approach using desirability function generated through predictor importance and weighted coefficient methodology was adopted for optimization. The approach set optimum bioprocess conditions as 49.55 g/L molasses, 64.98% corn steep liquor, 44.23 g/L asparagine, 1.73 g/L potassium, 0.055 g/L manganese and 0.043 g/L chromium (III) ions, at a composite desirability of 0.943 and an L-asparaginase activity of 5216.95U. The Sephadex-200 partially-purified polypeptide had a specific activity of 476.84 U/mg; 0.087U glutaminase activity, 36.46% yield and 20-fold protein purification. Anti-cancer activity potentials of the catalytic poly-peptide were dose-dependent with IC (µg/mL): 4.063 (HL-60), 13.75 (HCT-116), 15.83 (HeLa), 11.68 (MCF-7), 7.61 (HepG-2). The therapeutic enzyme exhibited 15-fold more cytotoxicity to myeloid leukemia cell line than to normal (HEK 238 T) cell. Optimum temperature and pH for activity were within physiological range. However, significant interactions between exposure time and levels of each of temperature and pH made interpretations of residual enzyme activities difficult. The manganese-dependent L-asparaginase from UCCM 00117 is recommended for further anticancer drug investigations.
在摇瓶实验室条件下，对UCCM 00117生产谷氨酰胺酶含量近乎为零的L-天冬酰胺酶的生物过程营养条件进行了顺序优化。使用标准体外生化方法评估了该多肽的催化活性和抗癌活性。通过一次一因素（OFAT）方法选择培养基营养成分，同时采用Plackett-Burman设计（PBD）筛选潜在的优化因素。采用最速上升路径（PSA）和中心复合旋转设计（CCRD）的最小运行分辨率V分数析因的响应面方法（RSM）来优化因素水平以提高酶活性。采用通过预测器重要性和加权系数方法生成的合意函数的多目标方法进行优化。该方法将最佳生物过程条件设定为49.55 g/L糖蜜、64.98%玉米浆、44.23 g/L天冬酰胺、1.73 g/L钾、0.055 g/L锰和0.043 g/L铬（III）离子，综合合意度为0.943，L-天冬酰胺酶活性为5216.95 U。经Sephadex-200部分纯化的多肽比活性为476.84 U/mg；谷氨酰胺酶活性为0.087 U，产率为36.46%，蛋白质纯化了20倍。催化多肽的抗癌活性潜力呈剂量依赖性，IC（μg/mL）分别为：4.063（HL-60）、13.75（HCT-116）、15.83（HeLa）、11.68（MCF-7）、7.61（HepG-2）。该治疗性酶对髓系白血病细胞系的细胞毒性比对正常（HEK 238 T）细胞高15倍。活性的最佳温度和pH在生理范围内。然而，暴露时间与温度和pH各自水平之间的显著相互作用使得对残余酶活性的解释变得困难。推荐来自UCCM 00117的锰依赖性L-天冬酰胺酶用于进一步的抗癌药物研究。
