Abstract

L-asparaginase is an enzyme commonly used to treat acute lymphoblastic leukemia. Commercialized bacterial L-asparaginase has been reported to cause several life-threatening complications during treatment, hence the need to seek alternative sources of L-asparaginase. In this study, the novelty of upstream and downstream bioprocessing of L-asparaginase from a fungal endophyte, Colletotrichum gloeosporioides, and the cytotoxicity evaluation was demonstrated. Six variables (carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate) known to influence L-asparaginase production were studied using One-Factor-At-A-Time (OFAT) approach, with four significant variables further optimized using Response Surface Methodology (RSM). The crude extract produced using optimized condition was purified, characterized and examined for its anticancer effect. Purification of fungal L-asparaginase was performed via ultrafiltration and size exclusion chromatography, which are less common techniques. The protein profile and monomeric weight of L-asparaginase were determined using SDS-PAGE and Western blot. Cytotoxicity of purified L-asparaginase on leukemic Jurkat E6 and oral carcinoma cells were studied using MTS assay for 24 h and 48 h. OFAT results from optimization showed that glucose and L-asparagine concentrations, incubation period and temperature, were significant factors affecting L-asparaginase production by C. gloeosporioides. RSM analysis further evidence the significant interaction between glucose and L-asparagine concentrations in inducing L-asparaginase production. Purified L-asparaginase was profiled with specific activity of 255.02 IU/mg protein, purification fold of 6.12, and 34.63% of enzyme recovery. SDS and Western blot revealed that the purified L-asparaginase might be a tetramer with monomeric units of 25 kDa. Purified L-asparaginase was discovered to be more efficient against Jurkat leukemic cells than against H103 oral carcinoma cells, as lower IC₅₀ value was observed for Jurkat cell lines (46 .36 ± 1.52 µg/mL for Jurkat and 125.56 ± 7.28 µg/mL for H103). In short, purified L-asparaginase derived from endophytic C. gloeosporioides showed high purity and significant anticancer effect toward cancer cells. This study therefore demonstrated the potential of fungal L-asparaginase as alternative chemotherapy drug in the future.

摘要

L-天冬酰胺酶是一种常用于治疗急性淋巴细胞白血病的酶。据报道，商业化细菌 L-天冬酰胺酶在治疗过程中会引起多种危及生命的并发症，因此需要寻找 L-天冬酰胺酶的替代来源。在这项研究中，来自内生真菌胶孢炭疽菌 L-天冬酰胺酶的上游和下游生物加工的新颖性，并进行了细胞毒性评价。使用一次一因子 (OFAT) 方法研究了已知影响 L-天冬酰胺酶生产的六个变量（碳源和浓度、氮源和浓度、培养期、温度、pH 和搅拌速率），其中四个显着使用响应面方法（RSM）进一步优化变量。使用优化条件产生的粗提物经过纯化、表征并检查其抗癌作用。真菌 L-天冬酰胺酶的纯化是通过超滤和尺寸排阻色谱法进行的，这是不太常见的技术。使用 SDS-PAGE 和蛋白质印迹测定 L-天冬酰胺酶的蛋白质谱和单体重量。使用 MTS 测定法研究纯化的 L-天冬酰胺酶对白血病 Jurkat E6 和口腔癌细胞的细胞毒性 24 小时和 48 小时。OFAT 优化结果表明，葡萄糖和 L-天冬酰胺浓度、培养时间和温度是影响 L-天冬酰胺酶产量的重要因素C. 胶孢子虫。RSM 分析进一步证明葡萄糖和 L-天冬酰胺浓度在诱导 L-天冬酰胺酶产生方面存在显着的相互作用。纯化的 L-天冬酰胺酶的比活性为 255.02 IU/mg 蛋白质，纯化倍数为 6.12，酶回收率为 34.63%。SDS和Western blot显示纯化的L-天冬酰胺酶可能是单体单位为25 kDa的四聚体。纯化的 L-天冬酰胺酶被发现对 Jurkat 白血病细胞比对 H103 口腔癌细胞更有效，因为 Jurkat 细胞系的IC ₅₀值较低（Jurkat 为 46 .36 ± 1.52 µg/mL，Jurkat 为 125.56 ± 7.28 µg/mL）对于 H103）。简而言之，纯化的 L-天冬酰胺酶源自内生C. gloeosporioides纯度高，对癌细胞具有显着的抗癌作用。因此，这项研究证明了真菌L-天冬酰胺酶作为未来替代化疗药物的潜力。