Purification, Characterization and Comparison between Two New L-asparaginases from PG03 and PG04

Abstract

Background:L-asparaginase has been used as a chemotherapeutic agent in treatment of lymphoblastic leukemia. In the present investigation, Bacillus sp. PG03 and Bacillus sp. PG04 were studied.Methods:L- asparaginases were produced using different culture media and were purified using ion exchange chromatography.Results:Maximum productivity was obtained when asparagine was used as the nitrogen source at pH 7 and 48 h after cultivation. New intracellular L-asparaginases showed an apparent molecular weight of 25 kDa and 30 kDa by SDS-PAGE respectively. These enzymes were active in a wide pH range (3-9) with maximum activity at pH 6 for Bacillus PG03 and pH 7 for Bacillus PG04 L-asparaginase. Bacillus PG03 enzyme was optimally active at 37 ˚C and Bacillus PG04 maximum activity was observed at 40˚C. Kinetic parameters km and Vmax of both enzymes were studied using L-asparagine as the substrate. Thermal inactivation studies of Bacillus PG03 and Bacillus PG04 L-asparaginase exhibited t1/2 of 69.3 min and 34.6 min in 37 ˚C respectively. Also T50 and ∆G of inactivation were measured for both enzymes.Conclusion: The results revealed that both enzymes had appropriate characteristics and thus could be a potential candidate for medical applications.

Production and Anticancer Activity of an L-Asparaginase from Bacillus licheniformis Isolated from the Red Sea, Saudi Arabia

“…Maximum enzyme productivity of the ASNase evaluated in this study was obtained at 37 °C, similar to the ASNase from Bacillus subtilis 36 , whereas the optimal temperature for the production of enzyme by Streptomyces olivaceus was 35 °C 38 . In contrast, the optimal production of ASNases from Bacillus PG02 and Bacillus PG04 occurred at pH 6–7.5, which is close to physiological pH 40 . This feature is a major requirement for the antitumor activity of ASNases with optimal temperatures of 37 °C.…”

Caracterización bioinformática y producción de L-asparaginasa de Bacillus sp. M62 aislado de las salinas de Maras, Cusco, Perú

“…La L-asparagina fue la única fuente de nitrógeno usada para la producción de L-asparaginasa por Bacillus sp. M62, debido a la mayor actividad L-asparaginasa obtenida en comparación con L-arginina, NaNO 3 y (NH 4 ) 2 SO 4 en otras cepas de Bacillus (Rahimzadeh et al 2016). Su efecto como inductor de L-asparaginasa se verificó en P. aeruginosa WCHPA075019 con la obtención de 170.70 U/mg, superior a lo obtenido con extrac-to de levadura, peptona, glutamina y arginina (Amany et al 2021).…”

Production of highly cytotoxic and low immunogenic L-asparaginase from Stenotrophomonas maltophilia EMCC2297

“…L-asparaginase was purified up to 5.48 folds with specific activity of 36.251 IU/mg protein, Hussien et al (2016) purified L-asparaginase from Enterobacter cloacae by ammonium sulfate precipitation, ion exchange chromatography and gel exclusion chromatography to 105 IU/mg protein and 119 folds increase enzyme activity (Husain et al 2016 ). While Rahimzadeh et al ( 2016 ) and Naggar et al, (2018) purified intracellular and extracellular L-asparaginase from Bacillus PG03, Bacillus PG04 and Streptomyces brollosae by sonication of the cell followed by anion exchange chromatography using DEAE Sepharose column. The results of the purified L-asparaginase from Bacillus PG03 and Bacillus PG04 were 1.2 mmole/min activity and 7.8 fold purification with 69 IU/mg protein (Rahimzadeh et al 2016 ).…”

“…Different molecular weight was estimated for L-asparaginase from different microorganisms as it was purified from Streptomyces brollosae , Fusarium foetens, Enterobacter cloacae and Streptomyces fradiae with molecular weights of 67 KDa, 37 KDa, 52 KDa, 53 KDa, respectively (El-Naggar et al 2016 ; Husain et al 2016 ). Moreover a 25 KDa and 30 KDa L-asparaginases were purified from Bacillus PG03, Bacillus PG04, respectively (Rahimzadeh et al 2016 ).…”

“…While Rahimzadeh et al (2016) andNaggar et al, (2018) purified intracellular and extracellular L-asparaginase from Bacillus PG03, Bacillus PG04 and Streptomyces brollosae by sonication of the cell followed by anion exchange chromatography using DEAE Sepharose column. The results of the purified L-asparaginase from Bacillus PG03 and Bacillus PG04 were 1.2 mmole/min activity and 7.8 fold purification with 69 IU/ mg protein (Rahimzadeh et al 2016). Likewise Parashiva et al, (2023 purified L-asparaginase from Fusarium foetens by ammonium sulfate fractionation, dialysis and ion exchange chromatography using DEAE cellulose column with specific activity of 231 IU/mg protein and 15.6 fold purification (MAZLUMOĞLU 2023).…”