Enhanced production of glutaminase-free L-asparaginase by marine Bacillus velezensis and cytotoxic activity against breast cancer cell lines

Background: The increasing rate of breast cancer globally requires extraordinary efforts to discover new effective sources of chemotherapy with fewer side effects. Glutaminase-free L-asparaginase is a vital chemotherapeutic agent for various tumor malignancies. Microorganisms from extreme sources, such as marine bacteria, might have high L-asparaginase productivity and efficiency with exceptional antitumor action toward breast cancer cell lines. Results: L-Asparaginase-producing bacteria, Bacillus velezensis isolated from marine sediments, were identified by 16S rRNA sequencing. L-Asparaginase production by immobilized cells was 61.04% higher than that by free cells fermentation. The significant productivity of enzyme occurred at 72 h, pH 6.5, 37°C, 100 rpm. Optimum carbon and nitrogen sources for enzyme production were glucose and NH4Cl, respectively. L-Asparaginase was free from glutaminase activity, which was crucial medically in terms of their severe side effects. The molecular weight of the purified enzyme is 39.7 KDa by SDS-PAGE analysis and was ideally active at pH 7.5 and 37°C. Notwithstanding, the highest stability of the enzyme was found at pH 8.5 and 70°C for 1 h. The enzyme kinetic parameters displayed Vmax at 41.49 µmol/mL/min and a Km of 3.6 × 10−5 M, which serve as a proof of the affinity to its substrate. The anticancer activity of the enzyme against breast adenocarcinoma cell lines demonstrated significant activity toward MDA-MB-231 cells when compared with MCF-7 cells with IC50 values of 12.6 ± 1.2 µg/mL and 17.3 ± 2.8 µg/mL, respectively. Conclusion: This study provides the first potential of glutaminase-free L-asparaginase production from the marine bacterium Bacillus velezensis as a prospect anticancer pharmaceutical agent for two different breast cancer cell lines.

Management of toxicities associated with pegaspargase in treatment of patients with lymphoid malignancy: experience from 443 cases in a single center / 中华血液学杂志

Objective: To summarize the adverse effects of pegaspargase in the treatment of lymphoid malignancies and management experience. Methods: Clinical data of patients who received chemotherapy including pegaspargase in the Department of Hematology of Beijing Tongren hospital during August 2011 to December 2015 were retrospective analyzed, and the adverse effects of pegaspargase and the management experience was summarized. Results: A total of 129 patients with 443 times of pegaspargase used during this period. The common adverse reactions included allergic reactions in 2 cases (1.6%), acute pancreatitis in 19 (14.7%) including 6 acute symptomatic pancreatitis and 13 chemical pancreatitis with elevated pancreatin, hypertriglyceridemia in 15 cases(11.6%), hyperglycemia in 85 (65.9%), hypoglycemia in 7 (5.4%), elevated aminotransferase in 25 (19.4%), hyperbilirubinemia in 21 (15.5%), hypoalbuminemia in 62 (48.1%), prolonged APTT in 61 (47.3%), prolonged PT in 22 (17.1%), prolonged TT in 15 (11.6%), hypofibrinogen in 75 (58.1%), thrombus in 11 (8.5%) and bleeding in 3 (2.3%). The above adverse reactions were improved by symptomatic treatment of anti allergy, inhibition of secretion of pancreatic juice, lipid lowering, hypoglycemic, liver preservation, supplementation of plasma and hemostasis, respectively. Some serious adverse reactions affected the application of pegaspargase, even lead to discontinuation of the aspartate. Conclusion: Though adverse effects associated with pegaspargase are extensive, most patients can successfully complete the chemotherapy containing the pegaspargase with close monitoring and timely treatment.

Studies on L-asparaginase enzyme of actinomycetes isolated from estuarine fishes.

Actinomycetes were isolated from different organs viz. skin, gills and gut contents of three species of fishes viz. Mugil cephalus (Linnaeus, 1758), Chanos chanos (Forskal, 1775) and Etroplus suratensis (Bloch, 1780) using three different media from the Vellar estuary, situated along the southeast coast of India. Among the three fishes, M. cephalus harboured highest number of actinomycetes population in all the three body parts examined followed by C. chanos and E. suratensis. Out of the three body parts of all fishes, gut contents had highest actinomycetes population followed by gills and skin. Among the three media used for isolation of actinomycetes, Kuster's agar medium was found to be suitable than the starch casein agar and glucose asparagine agar media. Out of the 40 strains isolated, only six strains (LA-2, LA-8, LA-15, LA-20, LA-29 and LA-35) showed significant L-asparagianse activity and were taken up for further studies. Impact of various physical and chemical factors such as pH, temperature, sodium chloride concentration, carbon sources and amino acids on the growth of actinomycetes and L-asparaginase activity was also studied. Optimum growth and enzyme activity was noticed under pH 7 to 8, temperature 37 degrees C, 1-2% sodium chloride concentration, sucrose as carbon source and without any amino acids. Analysis of the cell components of the isolated strains has revealed the wall type-I (the wall type-I is typical for the genus Streptomyces) and the strains were micromorphologically similar to the genus Streptomyces. Hence, the morphological, physiological and biochemical along with the micromorphological results obtained for the L-asparaginase producing strains were compared and the strains were tentatively identified as Streptomyces aureofasciculus (LA-2), S. chattanoogenesis (LA-8), S. hawaiiensis (LA-15), S. orientalis (LA-20), S. canus (LA-29) and S. olivoviridis (LA-35).

L-asparaginase activity in Aeromonas sp. isolated from freshwater mussel.

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.

Metabolism of proteins and glycoproteins in tumour bearing mice treated with Aeromonas L-asparaginase.

L-asparaginase, isolated in our laboratory, from Aeromonas had been found to be antileukaemic. In the present study, changes in the levels of proteins and glycoproteins in leukaemic mice and under treatment with Aeromonas L-asparaginase have been compared. Levels of protein bound hexose, fucose and sialic acid which were increased during leukaemia attained normal levels when treated with L-asparaginase. The increased blood urea level declined significantly during enzyme therapy. Effects of L-asparaginase are compared with 'Leunase', a commercially available drug used in the treatment of leukaemia.