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

Abstract

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 <1IU/ml) against leukemia cell line Jurkat while no cytotoxic effect on human erythrocytes or human umbilical vein endothelial cells was detected. Similar salinity and ionic concentration of the Caspian water to the human serum highlights the potential of secretory L-asparaginases recovered from these metagenomes as potential treatment agents.

Metagenomic Approaches as a Tool to Unravel Promising Biocatalysts from Natural Resources: Soil and Water

“…Table 4 compiles all the data considered in the category of raw resources. [198] Brackish water basin, Caspian Sea (Iran) --Sequence-based --L-asparaginases 0 [199] * Obtained from the international database Scopus (21 March 2022).…”

Advances on Delivery of Cytotoxic Enzymes as Anticancer Agents

“…Different approaches, such as obtaining protein fusions, conjugating with polymers, encapsulations in cell membranes or liposomes, forming dendrimers, and immobilizing with nanoparticles, have been used to protect and increase the anticancer efficacy of asparaginase [116,117]. Not all asparaginases result in cytotoxic effects in cancer cells due to reasons such as the optimum pH for some of these being around 7.5 [116].…”

“…Different approaches, such as obtaining protein fusions, conjugating with polymers, encapsulations in cell membranes or liposomes, forming dendrimers, and immobilizing with nanoparticles, have been used to protect and increase the anticancer efficacy of asparaginase [116,117]. Not all asparaginases result in cytotoxic effects in cancer cells due to reasons such as the optimum pH for some of these being around 7.5 [116]. Genetically modified forms of asparaginase, expressed in E. coli and other microorganisms [112,117,118], enabled the conservation of enzymatic activity at higher temperatures [119], lower glutaminase and conserved asparaginase activity [120], and enhanced resistance to degradation by trypsin [121].…”

Enzymative activity of technogenic surface formations of Kuzbass

“…Ферменты участвуют в расщеплении сложных органических и неорганических соединений, переводят их в легкоусвояемые и нетоксичные для растений формы. Наиболее значимыми являются [7,8,9,10]:…”

“…Оценку аспарагиназной активности проводили с целью изучения приспособленности аборигенной микрофлоры к условиям техногенных поверхностных образований (к действию полизагрязнения ТМ) [9]. Среднее значение аспарагиназной активности техногенных поверхностных образований представлено на рисунке 5.…”