Department of Bioprocess Development, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technological Applications, Alexandria, Egypt.
Department of Protein Research, Genetic Engineering and Biotechnology Research Institute, City of Scientific Research &Technological Applications, Alexandria, Egypt.
Sci Rep. 2016 Sep 8;6:32926. doi: 10.1038/srep32926.
L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml(-1)min(-1), respectively. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS-PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82.
L-天冬酰胺酶是一种重要的酶，作为治疗剂与其他药物联合用于治疗急性淋巴细胞白血病。一株新分离的放线菌菌株，链霉菌 sp. NEAE-82，具有潜在的产生细胞外 L-天冬酰胺酶的能力，被鉴定为弗雷氏链霉菌 NEAE-82，测序产物在 GenBank 数据库中登记，登录号为 KJ467538。L-天冬酰胺酶从粗酶中通过硫酸铵沉淀、透析和离子交换层析（使用 DEAE Sepharose CL-6B）进行纯化。进一步进行了纯化酶的动力学研究。发现最大 L-天冬酰胺酶活性的最适 pH、温度和孵育时间分别为 8.5、40°C 和 30 分钟。最佳底物浓度被发现为 0.06M。酶的 Km 和 Vmax 分别为 0.01007M 和 95.08Uml(-1)min(-1)。半衰期（T1/2）在 50°C 时为 184.91 分钟，而在 60°C 时为 179.53 分钟。SDS-PAGE 分析表明，L-天冬酰胺酶亚基的分子量约为 53kDa。纯化的 L-天冬酰胺酶的最终比活为 30.636U/mg 蛋白，纯化倍数为 3.338 倍。本研究首次报道了更多关于新分离的弗雷氏链霉菌 NEAE-82 产生的 L-天冬酰胺酶的生产、纯化和特性的信息。
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