Filamentous Fungi Producing l-Asparaginase with Low Glutaminase Activity Isolated from Brazilian Savanna Soil.

Marcela Freitas,Danilo Batista Pinho,Pérola O Magalhães,Paula Souza,Letícia Santos Abrunhosa,Samuel Cardoso,Kellen Cruvinel,Adalberto Pessoa,Edivaldo X Ferreira Filho,João Inácio

doi:10.3390/pharmaceutics13081268

Marcela Freitas, Danilo Batista Pinho + Show 8 more

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https://doi.org/10.3390/pharmaceutics13081268

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Abstract

l-asparaginase is an enzyme used as treatment for acute lymphoblastic leukemia (ALL) due to its ability to hydrolyze l-asparagine, an essential amino acid synthesized by normal cells unlike neoplastic cells. The adverse effects of l-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of l-asparaginase-producing eukaryotic microorganisms with low glutaminase activity. This work evaluated the biotechnological potential of filamentous fungi isolated from Brazilian Savanna soil and plants for l-asparaginase production. Thirty-nine isolates were screened for enzyme production using the plate assay, followed by measuring enzymatic activity in cells after submerged fermentation. The variables influencing l-asparaginase production were evaluated using Plackett–Burman design. Cell disruption methods were evaluated for l-asparaginase release. Penicillium sizovae 2DSST1 and Fusarium proliferatum DCFS10 showed the highest l-asparaginase activity levels and the lowest glutaminase activity levels. Penicillium sizovae l-asparaginase was repressed by carbon sources, whereas higher carbon concentrations enhanced l-asparaginase by F. proliferatum. Maximum enzyme productivity, specific enzyme yield and the biomass conversion factor in the enzyme increased after Plackett–Burman design. Freeze-grinding released 5-fold more l-asparaginase from cells than sonication. This study shows two species, which have not yet been reported, as sources of l-asparaginase with possible reduced immunogenicity for ALL therapy.

Highlights

IntroductionThe leading cause of death from disease among children is cancer, with leukemias being the most common type diagnosed in children up to 14 years of age, mainly acute lymphoblastic leukemia (ALL)
Thirty-nine fungal isolates from the Brazilian Savanna were evaluated for L-asparaginase production, out of which 21 were isolated from the soil and 18 from plants
This study evaluated the biotechnological potential of filamentous fungal isolates from different samples of Brazilian Savanna for the production of L-asparaginase of industrial and pharmaceutical interest

Summary

Introduction

The leading cause of death from disease among children is cancer, with leukemias being the most common type diagnosed in children up to 14 years of age, mainly acute lymphoblastic leukemia (ALL). L-asparaginase (EC 3.5.1.1; L-asparagine amidohydrolase), listed by the World Health Organization as an essential medicine, is an important enzyme in the pharmaceutical field that has been used for ALL treatment in children since 1970 [1,2]. Its anti-leukemic effect relies on the hydrolysis of L-asparagine, an amino acid required for the biosynthesis of proteins that neoplastic cells cannot synthesize due to lack of Lasparagine synthetase, unlike normal cells. L-asparaginase and asparagine synthetase can use glutamine as substrate: L-asparaginase cleaves its amino group off at a slower

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