Molecular expression, purification and structural characterization of recombinant L-Glutaminase from Streptomyces roseolus.

The present research reports the anti-cancer potential of recombinant L-Glutaminase from Streptomyces roseolus. L-Glutaminase gene was synthesized by codon-optimization, cloned and successfully expressed in E. coli BL21 (DE3). Affinity purified recombinant L-Glutaminase revealed a molecular mass of 32 kDa. Purified recombinant L-Glutaminase revealed stability at pH 7.0-8.0 with optimum activity at 70 °C further indicating its thermostable nature based on thermodynamic characterization. Recombinant L-Glutaminase exhibited profound stability in the presence of several biochemical parameters and demonstrated its metalloenzyme nature and was also found to be highly specific towards favorable substrate (l-Glutamine) based on kinetics. It demonstrated antioxidant property and pronounced cytotoxic effect against breast cancer (MCF-7 cell lines) in a dose dependent behavior with IC50 of 40.68 µg/mL. Matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of desired mass peaks ascertained the recombinant L-Glutaminase identity. N-terminal amino acid sequence characterization through Edman degradation revealed highest resemblance for L-glutaminase within the Streptomyces sp. family. The purified protein was characterized structurally and functionally by employing spectroscopic methods like Raman, circular dichroism and nuclear magnetic resonance. The thermostability was assessed by thermogravimetric analysis. The outcomes of the study, suggests the promising application of recombinant L-Glutaminase as targeted therapeutic candidate for breast cancer.

Biochemical characterization of immobilized recombinant subtilisin and synthesis and functional characterization of recombinant subtilisin capped silver and zinc oxide nanoparticles.

This pioneering research explores the transformative potential of recombinant subtilisin, emphasizing its strategic immobilization and nanoparticle synthesis to elevate both stability and therapeutic efficacy. Achieving an impressive 95.25 % immobilization yield with 3 % alginate composed of sodium along with 0.2 M CaCl2 indicates heightened pH levels and thermal resistance, with optimal action around pH 10 as well as 80 °C temperature. Notably, the Ca-alginate-immobilized subtilisin exhibits exceptional storage longevity and recyclability, affirming its practical viability. Comprehensive analyses of the recombinant subtilisin under diverse conditions underscore its adaptability, reflected in kinetic enhancements with increased Vmax (10.7 ± 15 × 103 U/mg) and decreased Km (0.19 ± 0.3 mM) values post-immobilization using N-Suc-F-A-A-F-pNA. UV-visible spectroscopy confirms the successful capping of nanoparticles made of Ag and ZnO by recombinant subtilisin, imparting profound antibacterial efficacy against diverse organisms and compelling antioxidant properties. Cytotoxicity was detected against the MCF-7 breast cancer line of cells, exhibiting IC50 concentrations at 8.87 as well as 14.52 µg/mL of AgNP as well as ZnONP, correspondingly, indicating promising anticancer potential. Rigorous characterization, including FTIR, SEM-EDS, TGA and AFM robustly validate the properties of the capped nanoparticles. Beyond therapeutic implications, the investigation explores industrial applications, revealing the versatility of recombinant subtilisin in dehairing, blood clot dissolution, biosurfactant activity, and blood stain removal. In summary, this research unfolds the exceptional promise of recombinant subtilisin and its nanoparticles, presenting compelling opportunities for diverse therapeutic applications in medicine. These findings contribute substantively to biotechnology and healthcare and stimulate avenues for further innovation and exploration.

Optimization of subtilisin production from Bacillus subtilis strain ZK3 and biological and molecular characterization of synthesized subtilisin capped nanoparticles.

The increase and dissemination of multi-drug resistant bacteria have presented a major healthcare challenge, making bacterial infections a significant concern. The present research contributes towards the production of bioactive subtilisin from a marine soil isolate Bacillus subtilis strain ZK3. Custard apple seed powder (raw carbon) and mustard oil cake (raw nitrogen) sources showed a pronounced effect on subtilisin production. A 7.67-fold enhancement in the production was evidenced after optimization with central composite design-response surface methodology. Subtilisin capped silver (AgNP) and zinc oxide (ZnONP) nanoparticles were synthesized and characterized by UV-Visible spectroscopy. Subtilisin and its respective nanoparticles revealed significant biological properties such as, antibacterial activity against all tested pathogenic strains with potential against Escherichia coli and Pseudomonas aeruginosa. Prospective antioxidant behavior of subtilisin, AgNP and ZnONP was evidenced through radical scavenging assays with ABTS and DPPH. Subtilisin, AgNP and ZnONP revealed cytotoxic effect against cancerous breast cell lines MCF-7 with IC50of 83.48, 3.62 and 7.57 µg/mL respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the structure, surface and thermostability properties. The study proposes the potential therapeutic applications of subtilisin and its nanoparticles, a way forward for further exploration in the field of healthcare.

Gene expression and molecular characterization of recombinant subtilisin from Bacillus subtilis with antibacterial, antioxidant and anticancer properties.

This study investigated the multifunctional attributes such as, antibacterial, antioxidant and anticancer potential of recombinant subtilisin. A codon-optimized subtilisin gene was synthesized from Bacillus subtilis and was successfully transformed into E. coli DH5α cells which was further induced for high level expression in E. coli BL21 (DE3). An affinity purified ~40 kDa recombinant subtilisin was obtained that revealed to be highly alkali-thermostable based on the thermodynamic parameters. The kinetic parameters were deduced that indicated higher affinity of N-Suc-F-A-A-F-pNA substrate towards subtilisin. Recombinant subtilisin demonstrated strong antibacterial activity against several pathogens and showed minimum inhibitory concentration of 0.06 µg/mL against B. licheniformis and also revealed high stability under the influence of several biochemical factors. It also displayed antioxidant potential in a dose dependent manner and exhibited cell cytotoxicity against A549 and MCF-7 cancerous cell lines with IC50 of 5 µM and 12 µM respectively. The identity of recombinant subtilisin was established by MALDI-TOF mass spectrum depicting desired mass peaks and N-terminal sequence as MRSK by MALDI-TOF-MS. The deduced N- terminal amino acid sequence by Edman degradation revealed high sequence similarity with subtilisins from Bacillus strains. The structural and functional analysis of recombinant antibacterial subtilisin was elucidated by Raman, circular dichroism and nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results contribute to the development of highly efficient subtilisin with enhanced catalytic properties making it a promising candidate for therapeutic applications in healthcare industries.