Anticancer Activity of Extremely Effective Recombinant L-Asparaginase fromBurkholderia pseudomallei

Abstract

L-asparaginase (E.C. 3.5.1.1) purified from bacterial cells is widely used in the food industry, as well as in the treatment of childhood acute lymphoblastic leukemia. In the present study, the Burkholderia pseudomallei L-asparaginase gene was cloned into the pGEX-2T DNA plasmid, expressed in E. coli BL21 (DE3) pLysS, and purified to homogeneity using Glutathione Sepharose chromatography with 7.26 purification fold and 16.01% recovery. The purified enzyme exhibited a molecular weight of ~33.6 kDa with SDS-PAGE and showed maximal activity at 50°C and pH 8.0. It retained 95.1, 89.6%, and 70.2% initial activity after 60 min at 30°C, 40°C, and 50°C, respectively. The enzyme reserved its activity at 30 o C and 37 o C up to 24 h. The enzyme had optimum pH of 8 and reserved 50% activity up to 24 h. The recombinant enzyme showed the highest substrate specificity towards L-asparaginase substrate, while no detectable specificity was observed for L-glutamine, urea, and acrylamide at 10 mM concentration. THP-1, a human leukemia cell line, displayed significant morphological alterations after being treated with recombinant L-asparaginase and the IC 50 of the purified enzyme was recorded as 0.8 IU. Furthermore, the purified recombinant Lasparaginase improved cytotoxicity in liver cancer HepG2 and breast cancer MCF-7 cell lines, with IC 50 values of 1.53 and 18 IU, respectively.

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes

“…According to our knowledge, there are only a few reports on antiproliferative or proapoptotic effects induced by novel source L-asparaginases in AML (e.g., THP-1) and promyelocytic (e.g., HL-60) cell lines. It has been reported, for example, that L-asparaginase from Burkholderia pseudomallei (GenBank: ABA50799.1, class 1, type II), which has no detectable L-glutaminase activity, affects the viability of THP-1 cells [51]. As THP-1 cells are more sensitive to the depletion of L-Gln than L-Asn, these data conflict with our observations.…”

Biosynthesis, Partial Purification and Characterization of Extracellular L-Asparaginase From Novel Bacillus Subtilis Subsp. Spizizenii Tu-B-10 Isolated From Gcu Garden Soil Microflora

“…Moreover, EDTA acted as inhibitors of LA from Streptomyces brollosae NEAE-115 activity reducing its activity by 37.55 (El-Naggar et al, 2018). it was revealed that EDTA decreased the activity of B. pseudomallei LA by 60.7 and 41.2%, respectively, at concentrations of 1 mM and 5 mM (Darwesh et al, 2022).…”

“…Similarly, Abbas et al (2015) reported the highest LA activity after 10 min of incubation at 37 º C, and observed the gradual decrease in enzyme activity by increasing the time to incubation beyond 10 min. Darwesh et al, (2022) reported optimum pH of 8 for enzyme action from Burkholderia pseudomallei. LA from Bacillus licheniformis, T. viride HK01 and Pseudomonas sp.…”

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

“…FLAG, poly-His, poly-Arg, Stre-taq, c-Myc, and S-taq are the most commonly used affinity peptide tags because their small size interferes with the protein of interest. Small ubiquitin-related modifier (SUMO) [109], maltose-binding protein (MBP) [110], N-utilization protein A (NusA), thioredoxin (Trx) [111], glutathione-S-transferase (GST) [112], disulfide isomerase I (PDI), Fasciola hepatica antigen (Fh8) and Superfolder green fluorescent protein (sfGFP) are examples of protein tags that improve protein solubility. Examples of insolubility-enhancing tags are N-terminal autoprotease (Npro), β-barrel outer membrane protein (PagP) and two smaller sulfur carrier proteins (ThiS) and (MoaD).…”