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Purpose: L-asparaginase has been widely recognized as a critical component in the treatment of various types of lymphoproliferative disorders, since its introduction in 1960s. However, its use in some cases leads to allergic reactions rendering the continuation of treatment unfeasible. Thus, the development of L-asparaginase from alternative sources or the production of engineered enzymes have always been considered. This study aimed to produce and evaluate a novel enzyme designed based on the sequence of L-asparaginase from Escherichia coli bacteria with Y176F/S241C mutations. Methods: The Y176F/S241C mutant L-asparaginase was successfully expressed as the GST-fusion protein in E. coli , and then was subjected to affinity and size exclusion chromatography. The activity of the purified enzyme was determined based on the released ammonia as the result of substrate hydrolysis using Nessler's reagent. Chemical denaturation experiment in the presence of increasing concentration of guanidinium chloride was applied to determine the folding stability of the purified enzyme. Results: The mutant enzyme was purified with an efficiency of 77-fold but at a low recovery of 0.7%. The determined kinetic parameters Km, Vmax, kcat, specific activity and catalytic efficiency were 13.96 (mM), 2.218 (mM/min), 273.9 (min-1), 237.8 (IU/mg) and 19.62 (mM-1 min-1), respectively. Moreover, unfolding free energy determined by guanidinium chloride induced denaturation for mutated and commercial L-asparaginase enzymes were 8421 J/mol and 5274 J/mol, respectively. Conclusion: The mutant enzyme showed improved stability over the wild-type. Although the expression level and recovery were low, the mutant L-asparaginase demonstrated promising activity and stability, with potential clinical and industrial applications. [ABSTRACT FROM AUTHOR]

Ultraviolet (UV) irradiation was used to induce mutagenesis in Aspergillus niger to provide a suitable mutant strain for overproduction of cellulase. Response surface methodology was successfully utilized to evaluate the effect of the mutation conditions, namely: UV exposure time (60–300 s) and distance of the strain from the UV source (0–20 cm) on created clear zone area around the strains. A maximum clear zone area (600.525±15.537 mm2) was obtained under the optimal mutation conditions of UV exposure time and distance of the 9 cm and 220 s. Rice and wheat straw were used as cellulosic substrates to produce cellulase by using the mutant strain through solid state fermentation. The results indicated that the total cellulase activity of the produced cellulase during 10 days of fermentation was 4.159 IU/ml, which was approximately twofold higher than that of the cellulase for the wild strain.

Background: Despite tremendous efforts made by scientific community during the outbreak of COVID‐19 pandemic, this disease still remains as a public health concern. Although different types of vaccines were globally used to reduce the mortality, emergence of new variants of SARS‐CoV‐2 is a challenging issue in COVID‐19 pharmacotherapy. In this context, target therapy of SARS‐CoV‐2 by small ligands is a promising strategy. Methods: In this investigation, we applied ligand‐based virtual screening for finding novel molecules based on nirmatrelvir structure. Various criteria including drug‐likeness, ADME, and toxicity properties were applied for filtering the compounds. The selected candidate molecules were subjected to molecular docking and dynamics simulation for predicting the binding mode and binding free energy, respectively. Then the molecules were experimentally evaluated in terms of antiviral activity against SARS‐CoV‐2 and toxicity assessment. Results: The results demonstrated that the identified compounds showed inhibitory activity towards SARS‐CoV‐2 Mpro. Conclusion: In summary, the introduced compounds may provide novel scaffold for further structural modification and optimization with improved anti SARS‐CoV‐2 Mpro activity. [ABSTRACT FROM AUTHOR]