Heterologous expression of recombinant nattokinase in Escherichia coli BL21(DE3) and media optimization for overproduction of nattokinase using RSM.

Nattokinase, a serine protease, was discovered in Bacillus subtilis during the fermentation of a soybean byproduct. Nattokinase is essential for the lysis of blood clots and the treatment of cardiac diseases including atherosclerosis, thrombosis, high blood pressure, and stroke. The demand for thrombolytic drugs rises as the prevalence of cardiovascular disease rises, and nattokinase is particularly effective for the treatment of cardiovascular diseases due to its long duration of action. In this study, we cloned the nattokinase gene from the Bacillus subtilis strain into the pET32a vector and expressed the protein in the E. coli BL21(DE3) strain. The active recombinant nattokinase was purified using Ni-NTA affinity chromatography and then evaluated for fibrinolytic and blood clot lysis activity. Physiological parameters for optimizing protein production at optimal pH, temperature, IPTG concentration, and incubation time were investigated. A statistical technique was used to optimize media components for nattokinase overproduction, and Central Composite Design-Response Surface Methodology-based optimization was used to select significant components for protein production. The optimized media produced 1805.50 mg/L of expressed nattokinase and 42.80 gm/L of bacterial mass. The fibrinolytic activity obtained from refolded native protein was 58FU/mg, which was five times higher than the available orokinase drug (11FU/mg). The efficiency with which a statistical technique for media optimization was implemented improved recombinant nattokinase production and provides new information for scale - up nattokinase toward industrial applications.

Cloning and characterization of FMN-dependent azoreductases from textile industry effluent identified through metagenomic sequencing.

Azo dyes, when released untreated in the environment, cause detrimental effects on flora and fauna. Azoreductases are enzymes capable of cleaving commercially used azo dyes, sometimes in less toxic by-products which can be further degraded via synergistic microbial cometabolism. In this study, azoreductases encoded by FMN1 and FMN2 genes were screened from metagenome shotgun sequences generated from the samples of textile dye industries' effluents, cloned, expressed, and evaluated for their azo dye decolorization efficacy. At pH 7 and 45°C temperature, both recombinant enzymes FMN1 and FMN2 were able to decolorize methyl red at 20 and 100 ppm concentrations, respectively. FMN2 was found to be more efficient in decolorization/degradation of methyl red than FMN1. This study offers valuable insights into the possible application of azoreductases to reduce the environmental damage caused by azo dyes, with the hope of contributing to sustainable and eco-friendly practices for the environment management. This enzymatic approach offers a promising solution for the bioremediation of textile industrial effluents. However, the study acknowledges the need for further process optimization to enhance the efficacy of these enzymes in large-scale applications.Implications: The study underscores the environmental hazards associated with untreated release of azo dyes into the environment and emphasizes the potential of azoreductases, specifically those encoded by FMN1 and FMN2 genes, to mitigate the detrimental effects. The study emphasizes the ongoing commitment to refining and advancing the enzymatic approach for the bioremediation of azo dye-containing effluents, marking a positive stride toward more sustainable industrial practices.

Screening and identification of azo dye decolorizers from mangrove rhizospheric soil.

Removal of synthetic textile dyes poses a challenge to the textile industry and a threat to the environment's flora and fauna. These dyes are recalcitrant and not very amenable to physical and chemical techniques of degradation. Hence, several studies on alternative bioremediation methods involving plants, plant roots, single microbes, or a consortium of microbes for the decolorization of dyes have been carried out. In the present study, potent bacteria for dye decolorization were isolated from rhizospheric soil of mangrove plants collected from Kamothe, Navi Mumbai, India. Of the 20 isolates obtained after enrichment, seven isolates were used for further screening of efficient decolorization ability in minimal basal media containing 10% glucose, 2.5% trace metal solution, and 0.1% of Methyl Orange (MO) dye concentration. Physiological parameters to optimize the decolorization of dye at optimum pH, temperature, and incubation time were studied for all the seven isolates. UV-vis and Fourier transform infrared spectroscopy were used to investigate dye decolorization. The seven isolates were characterized morphologically, biochemically, and molecular identification of these bacterial isolates was performed by 16S rRNA sequence analysis. The isolates were identified as Bacillus paramycoides, Pseudomonas taiwanensis, Citrobacter murliniae, Acinetobacter pitti, Exiguobacterium acetylicum, Psychrobacter celer, and Aeromonas taiwanensis. Out of these, Aeromonas taiwanensis has shown exceptional capacity by ~ 100% decolorization of azo dye in minimum time.