Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea

Abstract

Background L-asparaginase has been used for the treatment of acute lymphoblastic leukemia (ALL) for more than 30 years. However, efforts continued to find new enzymes with more desirable properties due to the immunogenicity, short half-life, rapid clearance and L-glutaminase side activity of the existing commercial enzymes. Screening for novel L-asparaginases in prokaryotes as a promising resource has been mainly hampered by the cultivation/expression bottleneck. Results By screening 27000 publicly available prokaryotic genomes, we recovered ca. 6300 type I and ca. 5200 type II putative L-asparaginase in 36 and 42 bacterial phyla respectively highlighting the vast potential of prokaryotes for L-asparaginase activity. Caspian water with similar salt composition to the human serum was targeted for in-silico screening of L-asparaginase. We screened ca. three million predicted Open Reading Frames of the assembled Caspian Sea metagenomes. In-silico screening resulted in 87 putative L-asparaginase genes from the Caspian Sea datasets. The L-asparagine hydrolysis was experimentally confirmed by cloning three selected genes (1092, 1218 and 1011 bp) in E. coli. Catalytic parameters of the purified enzymes including Km, Vmax and catalytic efficiency were determined to be among the most desirable reported values of microbial L-asparaginases. Two of the recombinant enzymes represented remarkable anti-proliferative activity (IC50 less than 1 IU/ml) against leukemia cell line Jurkat while no cytotoxic effect on human erythrocytes or human umbilical vein endothelial cells was detected. Conclusions Similar salinity and ionic concentration of the Caspian water samples to the human serum highlights the secretory L-asparaginases recovered from these metagenomes as potential treatment agents.