Abstract:L-asparaginase is an enzyme used as a chemotherapeutic agent, mainly for treating acute lymphoblastic leukemia. In this study, the gene of L-asparaginase from Zymomonas mobilis was cloned in pET vectors, fused to a histidine tag, and had its codons optimized. The L-asparaginase was expressed extracellularly and intracellularly (cytoplasmically) in Escherichia coli in far larger quantities than obtained from the microorganism of origin, and sufficient for initial cytotoxicity tests on leukemic cells. The in sil… Show more
“…The Zymomonas mobilis species is likely related to ethanol production; it showed a high performance in response to external stresses such as high ethanol concentration, high temperature, and harsh acidic fermentation conditions (Wang et al, 2016;Zhang et al, 2016). In addition, Zymomonas mobilis has anti-leukemic activity, antitumor effects, and prebiotic activity (Einsfeldt et al, 2016;Lorenzetti et al, 2015;Yoon et al, 2004). No Acetobacter was detected in this study, despite representatives of this genus have previously been isolated from other tibicos (Gulitz et al, 2011;Marsh et al, 2013).…”
Chinese Tianshan tibico grains were collected from the rural area of Tianshan in Xinjiang province, China. Typical tibico grains are known to consist of polysaccharide matrix that embeds a variety of bacteria and yeasts. These grains are widely used in some rural regions to produce a beneficial sugary beverage that is slightly acidic and contains low level of alcohol. This work aimed to characterize the microbiota composition of Chinese Tianshan tibicos using the single molecule, real-time sequencing technology, which is advantageous in generating long reads. Our results revealed that the microbiota mainly comprised of the bacterial species of Lactobacillus hilgardii, Lactococcus raffinolactis, Leuconostoc mesenteroides, Zymomonas mobilis, together with a Guehomyces pullulans-dominating fungal community. The data generated in this work helps identify beneficial microbes in Chinese Tianshan tibico grains. Keywords Chinese Tianshan tibicos Á Microbial diversity Á Single molecule Á Real-time sequencing technology Á Leuconostoc mesenteroides Á Guehomyces pullulans
“…The Zymomonas mobilis species is likely related to ethanol production; it showed a high performance in response to external stresses such as high ethanol concentration, high temperature, and harsh acidic fermentation conditions (Wang et al, 2016;Zhang et al, 2016). In addition, Zymomonas mobilis has anti-leukemic activity, antitumor effects, and prebiotic activity (Einsfeldt et al, 2016;Lorenzetti et al, 2015;Yoon et al, 2004). No Acetobacter was detected in this study, despite representatives of this genus have previously been isolated from other tibicos (Gulitz et al, 2011;Marsh et al, 2013).…”
Chinese Tianshan tibico grains were collected from the rural area of Tianshan in Xinjiang province, China. Typical tibico grains are known to consist of polysaccharide matrix that embeds a variety of bacteria and yeasts. These grains are widely used in some rural regions to produce a beneficial sugary beverage that is slightly acidic and contains low level of alcohol. This work aimed to characterize the microbiota composition of Chinese Tianshan tibicos using the single molecule, real-time sequencing technology, which is advantageous in generating long reads. Our results revealed that the microbiota mainly comprised of the bacterial species of Lactobacillus hilgardii, Lactococcus raffinolactis, Leuconostoc mesenteroides, Zymomonas mobilis, together with a Guehomyces pullulans-dominating fungal community. The data generated in this work helps identify beneficial microbes in Chinese Tianshan tibico grains. Keywords Chinese Tianshan tibicos Á Microbial diversity Á Single molecule Á Real-time sequencing technology Á Leuconostoc mesenteroides Á Guehomyces pullulans
“…The role of chemical modifications and protein engineering in improving the effectiveness of medicines is also discussed. Currently, as an alternative to bacterial L-ASNases with chemotherapeutic use, several type II bacterial enzymes are under consideration: Thermus thermophiles , Proteus vulgaris , Pseudomonas fluorescens , Serratia marcescens , Erwinia aroidea , Aspergillus terreus , Mycobacterium tuberculosis , Yersinia pseudotuberculosis , recombinant Saccharomyces cerevisiae , Halomonas elongata , Sarocladium strictum , Streptomyces rochei , Aspergillus terreus , Fusarium culmorum and Zymomonas mobilis [ 62 , 138 , 139 , 140 , 141 , 142 ]. They all have antiproliferative activities toward cancer cells.…”
Section: Alternative Approaches To the Development Of Antitumor L-asn...mentioning
L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity.
“…In the present study we have described the short chain L-asparaginase I for its characteristics and anticancer effects. Genetic engineering tools and techniques have been widely used for the production of enzyme from various resources (Einsfeldt et al, 2016;Saeed et al, 2018).…”
L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G – 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.
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