Structural stability and functional analysis of L-asparaginase from Pyrococcus furiosus

Abstract

We report studies on an L-asparaginase from Pyrococcus furiosus, cloned and expressed in Escherichia coli and purified to homogeneity. Protein stability and enzyme kinetic parameters were determined. The enzyme was found to be thermostable, natively dimeric, and glutaminase-free, with optimum activity at pH 9.0. It showed a K(m) of 12 mM and a substrate inhibition profile above 20 mM L-asparagine. Urea could not induce unfolding and enzyme inactivation; however, with guanidine hydrochloride (GdnCl) a two-state unfolding pattern was observed. Reduced activity and an altered near-UV-CD signal for protein at low GdnCl concentration (1 M) suggested tertiary structural changes at the enzyme active site. A homology three-dimensional model was developed and the structural information was combined with activity and stability data to give functional clues about the asparaginase.

Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88, isolated from Taptapani hotspring of Odisha, India

“…The medical utilization of LAse from the reported sources suffer the limitations of eliciting immunological responses leading to hypersensitivity in the long-term usage, allergic reactions, anaphylaxis and instance of spontaneous resistance of the tumor cells [17] . So the present investigation was under taken with an aim and objective to search for a novel bacterium from an ecologically unexplored and extreme habitat for the production of cost effective, eco-friendly, thermostable and a potent LAse with new antigenic properties.…”

Overview on L-Asparaginase

“…In a study the Lasparaginase producing Streptomyces gulbargensis was subjected to mutagenesis by UV light and the mutant strains showed a higher yield in asparaginase production. Similarly Prema et al [19] reported 0.6 fold increase in L-asparaginase activity by the UV treated mutant strain of Pseudomonas fluorescens as compared to its wild strain. [19] it was observed that EMS mutated strains of Pseudomonas fluorescens showed increased L-asparaginase activity compared to its wild strains.…”

“…Similarly Prema et al [19] reported 0.6 fold increase in L-asparaginase activity by the UV treated mutant strain of Pseudomonas fluorescens as compared to its wild strain. [19] it was observed that EMS mutated strains of Pseudomonas fluorescens showed increased L-asparaginase activity compared to its wild strains. Ethidium Bromide is one of the intercalating mutagens that cause frameshift mutation.…”

“…Although the method is simple it uses toxic components and the low enzyme activity cannot be measured. Another spectrophotometic method has been reported by Balco et al [19] where the ammonia produced is degraded by glutamate dehydrogenase accompanied by oxidation of beta-NADH.…”

“…L-asparaginase (EC 3.5.1.1) is a tetramer protein that belongs to a group of homologous amidohydrolases family that catalyze the hydrolysis L-asparaginase (L-Asn) to L-aspartate (L-Asp) and ammonia (NH 3 , Erwinia chrysanthemi [12,13,14] , Escherichia coli [15,16] , Rhodosporidium toruloides [17] , Serratia marcescens [18] , Thermus thermophilus [19] , Zymomonas mobilis [20] ; and from fungal origin Aspergillus terreus [21,22.23] , Aspergillus nidulans [24] , Candida utilis [25] and Tetrahymena pyriformis. [26] Among these microbial sources, the most commercially notable ones are Erwinia carotovora, Escherichia coli and Serratia marcescens.…”