Production and partial characterization of a therapeutic enzyme, L-asparaginase, from marine filamentous fungal strains
Project no. 22265 · Status: Kapanmış · 2020
Summary
L-asparaginase, which has a high therapeutic effect in various cancer diseases, hydrolyzes L-asparagine to aspartate and ammonia. While healthy cells can synthesize asparagine, tumor cells cannot perform this process, so they die due to nutrient deficiency. L-asparaginase, which is used in clinical applications, has various side effects, especially anaphylaxis. In addition, the presence of glutaminase and urease activities of the enzyme also causes side effects. Glutaminase breaks down glutamine, which is necessary for healthy cells, while urease causes hydrolysis of urea, thus increasing ammonia. In this project, it is aimed to evaluate the glutaminase and urease negative L-asparaginase production potential of marine filamentous fungi and to partial purification and characterization of the enzyme of the selected strain. In this context, 94 marine filamentous fungi were screened by rapid plate screening method to determine L-asparaginase activity. Then, 89 fungi determined to be L-asparaginase producers were screened for glutaminase and urease activity and 4 candidate producers (Penicillum crustosum MF5, Aspergillus flavus MF10, Penicillum canescens MF20, Cladosporium cladosporioides MF7) were selected. In a small scale fermentation study, the highest asparaginase activity was obtained with A. flavus MF10. Extracellular L-asparaginase produced by A. flavus MF10 was concentrated by ultrafiltration followed by acetone precipitation. It was purified 4,8 times by partial purification, and its molecular weight was approximately 100 kDa. While urease and glutaminase activities were not determined in the fermentation broth, low glutaminase activity (1.27 U/mL) was determined in the concentrated preparation, but urease activity was not detected. With partial characterization studies, the optimum temperature of the enzyme was determined as 40°C and the optimum pH was determined as 8.0. It was determined that the enzyme could maintain its activity 70% at 40°C after one hour and maintains its activity at pH 7 and 8 by pH stability study. It was determined that partially purified L-asparaginase decreased the viability of SH-SY5Y, LNCap cell lines after 24 hours, did not show cytotoxic effects in healthy cell line L929 and showed low cytotoxicity in HaCaT cells.