Cloning and characterization of Halomonas elongata L-asparaginase, a promising chemotherapeutic agent

Abstract

L-asparaginase has been used in the treatment of patients with acute lymphoblastic leukemia (ALL) for more than 30 years. Rapid clearance of the enzyme from blood stream and its L-glutaminase-dependent neurotoxicity has led to searching for new L-asparaginases with more desirable properties. In the present study, L-asparaginase coding gene of Halomonas elongata was isolated, expressed in Escherichia coli, purified, and characterized. The purified protein was found to have a molecular mass of 39.5 kDa and 1000-folds more activity towards L-asparagine than L-glutamine. Enzyme-specific activity towards L-asparagine was determined to be 1510 U/mg, which is among the highest reported values for microbial L-asparaginases. K , V, and k values were 5.6 mM, 2.2 μmol/min, and 1.96 × 10 1/S, respectively. Optimum temperature was found to be 37 °C while the enzyme showed maximum activity at a wide pH range (from 6 to 9). Enzyme half-life in the presence of human serum at 37 °C was 90 min which is three times higher when compared with reported values for E. coli L-asparaginase. Enzyme showed cytotoxic effects against Jurkat and U937 cell lines with an IC of 2 and 1 U/ml, respectively. Also, no toxic effects on human erythrocytes and Chinese hamster ovary cell lines were detected, and just minor inhibitory effects on human umbilical vein endothelial cells were observed. This is the first report describing the therapeutic potentials of a recombinant L-asparaginase isolated from a halophilic bacterium as an anticancer agent.

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

“…1 and 2). Current therapeutic Lasparaginase enzymes originating from E. coli and Erwinia chrysanthemi respectively lose 40 and 80 percent of their activity in blood salinity (0.9%) [15,45]. Here we target the brackish microbiome of the Caspian Sea for in-silico metagenomics exploration of novel putative L-asparaginase.…”

“…The high metabolic diversity of prokaryotes renders them a promising resource for L-asparaginase screening campaigns. However, all screening efforts so far have only targeted cultivated prokaryotes [15,16]. One major limitation of this approach is due to cultivation bottleneck; majority of prokaryotes (up to 99%) are still evading the bound of culture [17], consequently; their novel genetic contents remain inaccessible.…”

“…In this study, we leverage the availability of metagenomic datasets derived from the brackish samples of the Caspian Sea for in-silico screening of putatively novel L-asparaginase genes and further verify their activity via cloning and expression analysis. As the salinity and osmolality of brackish Caspian water are akin to blood serum [15], it is expected that secretory proteins screened from this ecosystem would represent optimal activity in physiologic condition. Additionally, cultureindependent in-silico screening of metagenomes provide an untapped reservoir of unexplored genes already available in nature [23].…”

Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine

“…From these strains, the anti-cancer activity of a novel recombinant l-asparaginase enzyme produced by Halomonas elongata strain IBRC M10216 was assayed against human lymphoblastic and myeloid leukemia cell lines, Jurkat and U937 (Table 4). This enzyme enhanced the viability of these cancer cell lines with IC50 values of 2 and 1 U/mL, respectively, but at these concentrations had no effect on the viability of normal HUVEC cell line [124]. Abbreviations: Not reported (NR).…”

Screening of Bacteria Producing Asparaginase Free of Glutaminase and Urease from Hot Springs in West Sulawesi

“…However, Lasparaginase therapy has various side effect including hepatitis, neurotoxicity, liver disfunction, coagulopathy, and leucopenia. These side effects are caused by the L-glutaminase activity of Lasparaginase (Ghasemi et al, 2017;Lee et al, 2016;Pourhossein & Korbekandi, 2014). Bano and Sivaramakrishnan were successfully purifying L-asparaginase from green chillies but still showed glutaminase and urease activity.…”