Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysuru, Karnataka, 570 006, India.
Department of Studies in Biochemistry, University of Mysore, Manasagangotri, Mysuru, Karnataka, 570 005, India.
World J Microbiol Biotechnol. 2023 Jul 14;39(9):252. doi: 10.1007/s11274-023-03684-3.
L-asparaginase is used as one of the prime chemotherapeutic agents to treat acute lymphoblastic leukemia. L-asparaginase obtained from bacteria exhibits hypersensitive reactions including various side effects. The present work aimed to optimize growth parameters for maximum production of L-asparaginase by Fusarium foetens through response surface methodology, its purification, and characterization. The optimization of L-asparaginase production by Fusarium foetens was initially done through a one-factor-at-a-time method. L-asparaginase production was further optimized using a central composite design based response surface methodology. The maximum L-asparaginase activity of 12.83 IU/ml was obtained under the following growth conditions; temperature-27.5 °C, pH-8, inoculum concentration-1.5 × 10 spores/ml, and incubation period-7 days. In comparison with the unoptimized growth conditions (4.58 IU/ml), the optimization led to a 2.65-fold increase in the L-asparaginase activity. The L-asparaginase from Fusarium foetens was purified 15.60-fold, with a yield of 39.89% using DEAE-cellulose column chromatography. After purification, the L-asparaginase activity was determined to be 127.26 IU/ml and the specific activity was found to be 231.38 IU/mg. The molecular mass was estimated to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme showed optimum activity at pH 5, and a temperature of 40 °C. The enzyme showed 100% specificity towards L-asparagine and no activity towards L-glutamine. Its activity was enhanced by Mn, Fe, and Mg, while it was inhibited by β-mercaptoethanol and EDTA. The Km and Vmax of the purified L-asparaginase were found to be 23.82 mM and 210.3 IU/ml respectively. The results suggest that Fusarium foetens could be a potent candidate for the bioprocessing of L-asparaginase at a large scale.
L-天冬酰胺酶被用作治疗急性淋巴细胞白血病的主要化疗药物之一。从细菌中获得的 L-天冬酰胺酶会引起过敏反应，包括各种副作用。本工作旨在通过响应面法优化产朊假丝酵母的生长参数，以最大限度地生产 L-天冬酰胺酶，并对其进行纯化和表征。首先通过单因素法优化产朊假丝酵母的 L-天冬酰胺酶生产。进一步通过基于响应面法的中心复合设计优化 L-天冬酰胺酶的生产。在以下生长条件下获得最大的 L-天冬酰胺酶活性 12.83 IU/ml；温度为 27.5°C，pH 值为 8，接种浓度为 1.5×10 个孢子/ml，培养时间为 7 天。与未优化的生长条件（4.58 IU/ml）相比，优化使 L-天冬酰胺酶活性提高了 2.65 倍。产朊假丝酵母的 L-天冬酰胺酶通过 DEAE-纤维素柱层析纯化 15.60 倍，收率为 39.89%。纯化后，L-天冬酰胺酶的活性为 127.26 IU/ml，比活为 231.38 IU/mg。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计分子量约为 37 kDa。纯化酶在 pH5 和 40°C 时表现出最佳活性。该酶对 L-天冬酰胺具有 100%的特异性，对 L-谷氨酰胺没有活性。其活性受 Mn、Fe 和 Mg 增强，而受β-巯基乙醇和 EDTA 抑制。纯化的 L-天冬酰胺酶的 Km 和 Vmax 分别为 23.82 mM 和 210.3 IU/ml。结果表明，产朊假丝酵母可能是大规模生物加工 L-天冬酰胺酶的有力候选物。
