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Summary By screening 27,000 publicly available prokaryotic genomes, we recovered ca. 6300 type I and ca. 5200 type II putative L-asparaginase highlighting the vast potential of prokaryotes. Caspian water with similar salt composition to the human serum was targeted for in silico L-asparaginase screening. We screened ca. three million predicted genes of its assembled metagenomes that resulted in annotation of 87 putative L-asparaginase genes. The L-asparagine hydrolysis was experimentally confirmed by synthesizing and cloning three selected genes in E. coli. Catalytic parameters of the purified enzymes were determined to be among the most desirable reported values. Two recombinant enzymes represented remarkable anti-proliferative activity (IC50, Graphical Abstract, Highlights • In silico screening finds 87 putative L-asparaginases from brackish Caspian Sea • Three candidate genes, cloned and expressed in E. coli, showed the desired activity • In silico screening coupled with functional validation bypasses cultivation bottleneck, Biological Sciences; Cell Biology; Cancer; In Silico Biology