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[This corrects the article DOI: 10.1021/acsomega.2c05605.].
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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.
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Bacillus subtilis , Subtilisina , Subtilisina/genética , Subtilisina/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular , Sequência de Aminoácidos , Subtilisinas/metabolismo , Expressão GênicaRESUMO
A green-based approach for the synthesis of silver nanoparticles has gained tremendous attention in biomedical applications. Fungal endophytes have been recognized as a remarkable biological source for the synthesis of potential nanodrugs. The present study focuses on the fabrication of silver nanoparticles using the fungal endophyte Penicillium oxalicum (POAgNPs) associated with the leaf of the Amoora rohituka plant. Sharp UV-visible spectra at 420 nm appeared due to the surface plasmon resonance of POAgNPs and the reduction of silver salt. FT-IR analysis revealed the presence of functional groups of bioactive compounds of P. oxalicum responsible for the reduction of silver salt and validated the synthesis of POAgNPs. A high degree of crystallinity was revealed through XRD analysis, and microscopy-based characterizations such as AFM, TEM, and FESEM showed uniformly distributed, and spherically shaped nanoparticles. Furthermore, POAgNPs showed a potential inhibitory effect against bacterial and fungal strains of pathogenic nature. POAgNPs also exhibited potential antioxidant activity against the synthetically generated free radicals such as DPPH, superoxide, hydroxyl, and nitric oxide with EC50 values of 9.034 ± 0.449, 56.378 ± 1.137, 34.094 ± 1.944, and 61.219 ± 0.69 µg/mL, respectively. Moreover, POAgNPs exhibited cytotoxic potential against the breast cancer cell lines, MDA-MB-231 and MCF-7 with IC50 values of 20.080 ± 0.761 and 40.038 ± 1.022 µg/mL, respectively. POAgNPs showed anticancer potential through inhibition of wound closure and by altering the nuclear morphology of MDA-MB-231 and MCF-7 cells. Further anticancer activity revealed that POAgNPs induced apoptosis in MDA-MB-231 and MCF-7 cells by differential expression of genes related to apoptosis, tumor suppression, and cell cycle arrest and increased the level of Caspase-3. The novel study showed that P. oxalicum-mediated silver nanoparticles exhibit potential biological activity, which can be exploited as nanodrugs in clinical applications.
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Biosurfactants are eco-friendly surface-active molecules recommended for enhanced oil recovery techniques. In the present study, a potential lipopeptide (biosurfactant) encoding the iturin A gene was synthesized from Bacillus aryabhattai. To improvise the yield of the lipopeptide for specific applications, current research tends toward engineering and expressing recombinant peptides. An iturin A gene sequence was codon-optimized, amplified with gene-specific primers, and ligated into the pET-32A expression vector to achieve high-level protein expression. The plasmid construct was transformed into an E. coli BL21 DE3 host to evaluate the expression. The highly expressed recombinant iturin A lipopeptide was purified on a nickel nitrilotriacetic acid (Ni-NTA) agarose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the purity and molecular mass of iturin A was 41 kDa. The yield of recombinant iturin A was found to be 60 g/L with a 6.7-fold increase in comparison with our previously published study on the wild strain. The approach of cloning a functional fragment of partial iturin A resulted in the increased production of the lipopeptide. When motor oil was used, recombinant protein iturin A revealed a biosurfactant property with a 74 ± 1.9% emulsification index (E24). Purified recombinant protein iturin A was characterized by mass spectrometry. MALDI-TOF spectra of trypsin digestion (protein/trypsin of 50:1 and 25:1) showed desired digested mass peaks for the protein, further confirming the identity of iturin A. The iturin A structure was elucidated based on distinctive spectral bands in Raman spectra, which revealed the presence of a peptide backbone and lipid. Recombinant iturin A was employed for enhanced oil recovery through a sand-packed column that yielded 61.18 ± 0.85% additional oil. Hence, the novel approach of the high-level expression of iturin A (lipopeptide) as a promising biosurfactant employed for oil recovery from Bacillus aryabhattai is not much reported. Thus, recombinant iturin A demonstrated its promising ability for efficient oil recovery, finding specific applications in petroleum industries.
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Understanding the biochemical changes in irradiated human mandible after radiotherapy of cancer patients is critical for oral rehabilitation. The underlying mechanism for radiation-associated changes in the bone at the molecular level could lead to implant failure and osteoradionecrosis. The study aimed to assess the chemical composition and bone quality in irradiated human mandibular bone using Raman spectroscopy. A total of 33 bone biopsies from 16 control and 17 irradiated patients were included to quantify different biochemical parameters from the Raman spectra. The differences in bone mineral and matrix band intensities between control and irradiated groups were analyzed using unpaired Student's t-test with statistical significance at p < 0.05. Findings suggest that the intensity of the phosphate band is significantly decreased and the carbonate band is significantly increased in the irradiated group. Further, the mineral crystallinity and carbonate to phosphate ratio are increased. The mineral to matrix ratio is decreased in the irradiated group. Principal component analysis (PCA) based on the local radiation dose and biopsy time interval of irradiated samples did not show any specific classification between irradiation sub-groups. Irradiation disrupted the interaction and bonding between the organic matrix and hydroxyapatite minerals affecting the bone biochemical properties. However, the normal clinical appearance of irradiated bone would have been accompanied by underlying biochemical and microscopical changes which might result in radiation-induced delayed complications.