Tales A Costa-Silva, I M Costa, G S Agamez-Montalvo, A Pessoa and G Monteiro
Introduction: L-asparaginase (E.C.3.5.1.1) produced by bacteria is used in the treatment of acute lymphocytic leukemia (ALL). However, innumerable side effects were registered by the usage of bacterial L-ASNase during ALL treatment. Other drawbacks associated with prokaryotic L-asparaginase treatment are hypersensitivity reactions, low thermal stability, human proteases degradation and rapid clearance.
Objectives: Some techniques have been used to overcome these downsides such as bio prospecting eukaryotic sources or modification of commercial bacterial L-asparaginase by site-directed mutagenesis. In order to find eukaryotic sources of L-ASNase, 20 filamentous fungi were used in this study, which were isolated from the microbiome of the jellyfish Olindias sambaquiensis.
Results: Six fungi samples isolated from jellyfish tentacles (brown structures in jelly fish responsible to toxin production) showed L-asparaginase production by submerged fermentation process. The highest activity was shown by Strain OS02 with 2.7 U/g. This strain was selected for optimization of L-asparaginase production by central composite design of response surface methodology. For maximum enzyme production (11.45 U/g), the best condition was modified Czapek Dox medium supplemented with L-asparagine and adjusted to pH 7.4 at 32.5 �C and 190 rpm.
Conclusions: Regarding protein engineering of commercial bacterial L-asparaginase we used site-directed mutagenesis to obtain L-asparaginase protease-resistance: a new Escherichia coli L-asparaginase (EcAII) variant, triple mutant. The preliminary results showed that mutant enzyme was expressed in E. coli BL21 (DE3) and preserved original L-asparaginase activity. These L-asparaginase proteo forms may be alternative biopharmaceuticals with the potential of further improving outcome in ALL treatment.
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