Isolation and Purification of High Efficiency L-asparaginase by Quantitative Preparative Continuous-elution SDS PAGE Electrophoresis

Abstract

An Unique extracelluar glutaminase free L-asparaginase from novel marine Actinomycetes was isolated to perceptible homogeneity in agro industrial wastes. Quantitative Preparative Continuous-Elution SDS PAGE Electrophoresis is a high-resolution method for the preparative isolation of L-Asparaginase in biological samples. The enzyme was purified 248.68-fold and showed a final specific activity of 5035.28 IU/mg with an 80.71% yield. The homotetramer enzyme has a molecular mass of 133.25 kDa and an isoelectric point of approximately 5.4.Kinetic parameters, Km and Vmax of purified L-asparaginase from Streptomyces radiopugnans MS1 were found to be 0.0598, 3.5478 IU µg -1 respectively. The de novo sequencing strategy presented here provides a rapid and reliable means to identify proteins in Streptomyces radiopugnans MS1. The purified L-asparaginase has no glutaminase activity, which can diminish the leeway of side effects during the itinerary of anti-malignancy therapy.

Submerged fermentation, purification, and characterization of L-asparaginase from Streptomyces sp. isolated from soil

“…), and chemicals, namely, dithiothreitol (DTT), SDS, ethylene diamine tetra acetic acid (EDTA), and mercaptoethanol (ME) were used to determine their effect on enzymatic activity. This mixture was incubated at room temperature for 30 min, and then enzyme assay was performed [17][18][19]21].…”

“…The gel was stained with Coomassie Brilliant Blue R-250. Standard molecular weight marker (Bangalore Genei, India) was used to compare the molecular weight of L-asparaginase [21]. The effect of temperature on L-asparaginase activity was studied using Berthelot reaction [18,30].…”

“…The pH was varied between 3 and 9 with a step increase of 1 unit. Acetate buffer (pH 3.0-5.0), phosphate buffer (pH 6.0-7.0), and Tris-HCl buffer (pH 8.0-9.0) at a concentration of 100 mM were used during the study [14,18,21]. Commercial L-asparagine of concentration between 0.2 mM and 2 mM was used as the standard substrate.…”

“…Enzyme was characterized with respect to various parameters such as temperature, pH, and molecular weight. Furthermore, studies were undertaken to assess the effect of metal ions and kinetic parameters [14,15,[17][18][19][20][21][22][23]. Production and purification of extracellular L-asparaginase are easier compared to intracellular enzymes.…”

“…Absorbance measurement (A 280 ) method was used to estimate protein content. L-asparaginase active fractions were pooled and used for further analysis [13,17,21,23]. …”