Purification, characterization and immunogenicity assessment of glutaminase free L-asparaginase from Streptomyces brollosae NEAE-115

Abstract

Abstract Background L-asparaginase is a potential therapeutic enzyme widely used in the chemotherapy protocols of pediatric and adult patients with acute lymphoblastic leukemia. However, its use has been limited by a high rate of hypersensitivity in the long-term used. Hence, there is a continuing need to search for other L-asparaginase sources capable of producing an enzyme with less adverse effects. Methods Production of extracellular L-asparaginase by Streptomyces brollosae NEAE-115 was carried out using submerged fermentation. L-asparaginase was purified by ammonium sulphate precipitation and pure enzyme was reached using ion-exchange chromatography, followed by enzyme characterization. Anticancer activity towards Ehrlich Ascites Carcinoma (EAC) cells was investigated in female Swiss albino mice by determination of tumor size and the degree of tumor growth inhibition. The levels of anti-L-asparaginase IgG antibodies in mice sera were measured using ELISA method. Results The purified L-asparaginase showed a total activity of 795.152 with specific activity of 76.671 U/mg protein and 7.835 − purification fold. The enzyme purity was confirmed by using SDS–PAGE separation which revealed only one distinctive band with a molecular weight of 67 KDa. The enzyme showed maximum activity at pH 8.5, optimum temperature of 37 °C, incubation time of 50 min and optimum substrate concentration of 7 mM. A Michaelis-Menten constant analysis showed a Km value of 2.139 × 10− 3 M with L-asparagine as substrate and Vmax of 152.6 UmL− 1 min− 1. The half-life time (T1/2) was 65.02 min at 50°С, while being 62.65 min at 60°С. Furthermore, mice treated with Streptomyces brollosae NEAE-115 L-asparaginase showed higher cytotoxic effect (79% tumor growth inhibition) when compared to commercial L-asparaginase group (67% tumor growth inhibition). Conclusions The study reveals the excellent property of this enzyme which makes it highly valuable for development of chemotherapeutic drug.

Keywords

• Streptomyces brollosae NEAE-115
• L-asparaginase
• Purification
• DEAE Sepharose CL-6B
• Characterization
• Immunogenicity assessment