Production, purification and characterization of L-Glutaminase from Streptomyces sp. isolated from soil

Abstract

L-Glutaminase, an amidohydrolase enzyme has been a choice of interest in the treatment of lymphoblastic leukaemia. The present study reports production, purification and characterization of extracellular glutaminase enzyme from Actinomycetes. Screening was performed for twenty Actinomycetes isolates from soil; one isolate (Isolate 2) was finally selected based on the activity of glutaminase (32.5 U/ml).The isolate was identified as Streptomyces sp. Effect of physicochemical factors namely temperature, pH, NaCl concentration, and supplementary carbon & nitrogen sources on the production of L-glutaminase from the Streptomyces sp. was carried out. The enzyme production was found to be optimum with glucose as carbon source (33 U/ml), Lglutamine as nitrogen source (33.1 U/ml), at 7 pH (32.8 U/ml), temperature 30 o C (32.4 U/ml) and for 0.1% NaCl concentration (32.5 U/ml). The L-glutaminase produced from Streptomyces sp. was purified by ammonium sulphate precipitation, dialysis method and ion exchange chromatography. After the purification of the enzyme by ion exchange chromatography, it has been purified 46-fold from cell-free extract and yield was 3.25%. Characterization of extracellular L-glutaminase showed that the enzyme shown optimal activity at temperature of 30°C, pH 7, at 2% NaCl and for 0.04M substrate and the Km value was calculated to be 2.8mM and Vmax was 7.57 U/ml. The molecular weight of enzyme as determined by sodium dodecyl sulphate polyacrylamide electrophoresis (SDS-PAGE) was found to be 50 kDa.

Purification, characterization and anticancer efficiency of L-glutaminase from <i>Aspergillus flavus</i>

“…The produced enzyme was best precipitated with 70% saturation of ammonium sulfate. This finding is in agreement with that reported by Desai et al (2016) who recorded the same concentration of ammonium sulphate for L-glutaminase purification from Streptomyces sp. In a trial to eliminate the other enzyme activities in the partially purified L-glutaminase preparation of A. flavus, it was further purified by gel filtration and ion exchange chromatography and an overall recovery of 60.25% with 6.3 fold purification was obtained.…”

Evaluation of antitumor activity of fungal L-glutaminase produced by egyptian isolates

“…The maximum enzyme productivity by the identified fungal isolates was obtained after 7 days of cultivation at 30ºC and showed an increase with time and also with increasing the amount of L-glutamine as a carbon source. These results were in good agreement with Savitha et al [33] where they reported that the ideal temperature for the enzyme activity was observed to be 30 to 40 ºC at which the enzyme activity was the highest and they were different from Koibuchi [34] and Das and Agsar [35] where Lglutaminase production by isolates gradually expanded from 24 hours to 120 hours and afterward no more increase was watched. This showed that in the present study, the L-glutaminase enzyme produced from the three fungal isolates was stable and active for more than 7 days.…”

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization

“…A01 toward its natural substrate L-glutamine. Glutaminases from Aspergillus oryzaezae AJ11728 3 , Streptomyces sp 35 , Rhizobium etli 36 have K m near to the rSAM. As shown in Supplementary Table S1 the changes in activation energy (ΔE * ), enthalpy (ΔH * ), entropy (ΔS * ) for rSAM thermo-activation according to the Arrhenius plot were respectively 4 kJ/mol, 1.3 kJ/mol and −0.17 J/mol°K while for L-glutaminase from E. coli were respectively 13.2 kcal/mol (55.44 kJ/mol), 8.2 kcal/mol (34.44 kJ/mol) and −14.1 e.u (59 J/mol°K).…”