Debendra Kumar Sahoo

The marine environment is known for its vast diversity of the microbial population; however, less explored for bioactive compounds. In this study, an AMP produced by a new marine isolate, Vibrio proteolyticus MT110, showed broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. The AMP was purified to homogeneity using ethyl acetate extraction followed by RP-HPLC, and LC-MS analysis showed its molecular weight as 980 Da. The MIC of AMP (peptide-MT110) was obtained in the 7.81-31.25 µg/mL range against different indicator strains. Peptide-MT110 showed stability of its antimicrobial activity at 15-121 °C and pH 4-10 and in the presence of various hydrolytic enzymes. The peaks at 1536 cm^-1 and 1712 cm^-1 wavenumbers in FTIR spectra confirmed the peptidic nature of AMP, and its amino acid analysis confirmed the presence of tyrosine and isoleucine. The antibacterial activity of peptide-MT110 is confirmed by PI assay and TEM. The optimization of peptide-MT110 production using statistical methods resulted in a 2.64-fold higher production. The physicochemical properties and stability in wide pH and temperature ranges showed the potential of peptide-MT110 for its development as a drug candidate. This is believed to be the first report on an AMP from Vibrio proteolyticus.

The present study reports the structural and functional characterization of a new glutaminase-free recombinant L-asparaginase (PrASNase) from Pseudomonas resinovorans IGS-131. PrASNase showed substrate specificity to L-asparagine, and its kinetic parameters, K _m , V _max , and k _cat were 9.49×10 ^-3 M, 25.13 IUmL ^-1 min ^-1 , and 3.01×10 ³ s ^-1 , respectively. The CD spectra showed that PrASNase consists of 30.9% α-helix and 69.1% other structures in its native form. FTIR was used for the functional characterization, and molecular docking predicted that the substrate interacts with serine, alanine, and glutamine in the binding pocket of PrASNase. Different from known asparaginases, structural characterization by small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) unambiguously revealed PrASNase to exist as a monomer in solution at low temperatures and oligomerized to a higher state with temperature rise. Through SAXS studies and enzyme assay, PrASNase was found to be mostly monomer and catalytically active at 37°C. Furthermore, this glutaminase-free PrASNase showed killing effects against WIL2-S and TF-1.28 cells with IC ₅₀ of 7.4 µg.mL ^-1 and 5.6 µg.mL ^-1 , respectively. This is probably the first report with significant findings of fully active L-asparaginase in monomeric form using SAXS and AUC and demonstrates the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate. HIGHLIGHTS: A new L-asparaginase (PrASNase) was structurally and functionally characterized. SAXS revealed PrASNase is functionally active in monomeric form and oligomerizes with temperature rise. Monomeric PrASNase showed an IC ₅₀ value of 7.4 and 5.6 µg mL ^-1 against WIL2-S and TF-1.28 cells. Cytotoxicity of PrASNase against leukemic cell lines showed its potential as a biotherapeutic. GRAPHICAL

Epidermal growth factor receptor (EGFR) is the primary target for the treatment of colorectal cancer, the third most diagnosed cancer worldwide. In recent years, regulatory changes have facilitated the approval of biosimilars aimed to bring more access to biologics to patients. However, it has also expended the requirements of non-clinical characterisation data using state-of-the-art and orthogonal methodologies to demonstrate similarity between proposed biologic and its reference medicinal product (RMP). The current study was aimed to develop a stable CHO-S cell line producing panitumumab biosimilar candidate, P-mAb, a fully human IgG2 anti-EGFR monoclonal antibody and assess its physicochemical and functional similarity with RMP, Vectibix. The single-cell clone from stably transfected CHO-S cell pools was used for the production of P-mAb. This was followed by purification and comparative physicochemical and biological characterisation of P-mAb and RMP using SDS-PAGE, LC/MS, MALDI, MS/MS, CD spectrometry, DSF, SAXS, ITF, MTT assay and binding affinity. SAXS and MST assays are being used for first time in biosimilarity analysis of therapeutic monoclonal antibody. The results of structural and functional analysis of anti-EGFR P-mAb, produced by stable CHO-S cell line revealed high similarity between P-mAb and RMP, vectibix, thus providing the scientific basis of its potential for therapeutic applications.

An increase in antibiotic resistance has led to escalating the need for the development of alternate therapy. Antimicrobial peptides (AMPs) are at the forefront of replacing conventional antibiotics, showing slower development of drug resistance, antibiofilm activity, and the ability to modulate the host immune response. The ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that jeopardize most conventional antibiotics are known to be involved in severe respiratory tract, bloodstream, urinary tract, soft tissue, and skin infections. Among them, S. aureus is an insidious microbe and developed resistance against conventional antibiotics. In the present study, an AMP (named as peptide-Ba49) isolated from Bacillus subtilis subsp. spizizenii strain from Allium cepa (the common onion) exhibited strong antibacterial efficacy against S. aureus ATCC 25923. The mode of action of this peptide-Ba49 on S. aureus was deciphered through various sensitive probes, i.e., DiSC₃ (5) and H₂DCFDA, suggesting the peptide-Ba49 to be acting upon through change in membrane potential and by triggering the production of reactive oxygen species (ROS). This induced disruption of the cell membrane was further supported by morphological studies using scanning electron microscopy (SEM). Investigations on a possible post-antibiotic effect (PAE) of peptide-Ba49 showed prolonged PAE against S. aureus. Furthermore, the peptide-Ba49 prevented the formation of S. aureus biofilm at low concentration and showed its potential to degrade the mature biofilm of S. aureus. The peptide-Ba49 also exhibited intracellular killing potential against S. aureus ATCC 25923 in the macrophage cells, and moreover, peptide-Ba49 was found to bolster the fibroblast cell migration in the scratch assay at low concentration, exhibiting a wound healing efficacy of this peptide. These studies demonstrated that peptide-Ba49 isolated from the strain B. subtilis subsp. spizizenii could be a therapeutic candidate to combat the pathogenic S. aureus infections.

Extensive usage of antibiotics has led to the emergence of drug-resistant strains of pathogens and hence, there is an urgent need for alternative antimicrobial agents. Antimicrobial Peptides (AMPs) of bacterial origin have shown the potential to replace some conventional antibiotics. In the present study, an AMP was isolated from Bacillus subtilis subsp. spizizenii strain Ba49 present on the Allium cepa, the common onion and named as peptide-Ba49. The isolated AMP was purified and characterized. The purified peptide-Ba49, having a molecular weight of ~ 3.3 kDa as determined using mass spectroscopy, was stable up to 121 °C and in the pH range of 5–10. Its interaction with protein degrading enzymes confirmed the peptide nature of the molecule. The peptide exhibited low minimum inhibitory concentration (MIC) against Staphylococcus aureus and its (Methicillin-resistant Staphylococcus aureus) MRSA strains (MIC, 2–16 µM/mL). Further, time kill kinetic assay was performed and analysis of the results of membrane depolarization and permeabilization assays (TEM, DiBAC₄ (3) and PI) suggested peptide-Ba49 to be acting through the change in membrane potential leading to disruption of S. aureus membrane. Additionally, cytotoxicity studies of peptide-Ba49, carried out using three mammalian cell lines viz. HEK 293T, RAW 264.7, and L929, showed limited cytotoxicity on these cell lines at a concentration much higher than its MIC values. All these studies suggested that the AMP isolated from strain Ba49 (peptide-Ba49) has the potential to be an alternative to antibiotics in terms of eradicating the pathogenic as well as drug-resistant microorganisms.

In the current study, the production of novel glutaminase free l-asparaginase from a new microbial source (Pseudomonas resinovorans IGS-131) is reported. Optimization of l-asparaginase production using conventional and statistical optimization techniques resulted in an enzyme yield of 37.63 IU/mL, which was 3.45-fold higher than the initial enzyme activity (i.e., 10.91 IU/mL). l-Asparaginase production from P. resinovorans IGS-131 was successfully carried out at the bioreactor level and investigations on the effect of agitation rates showed a maximum asparaginase yield of 38.88 IU/mL after 24 h fermentation at 400 rpm. The l-asparaginase gene from this source, showing 78% identity with a reported sequence in GenBank, was expressed in Escherichia coli rosetta DE3. The molecular weight of the recombinant protein was determined as 35.6 kDa. Downstream processing of recombinant l-asparaginase resulted in a purified protein concentration of 62.53 mg/L, which showed good free radical scavenging activity of 62%. The current findings provide promising results for a process of l-asparaginase production from P. resinovorans IGS-131. Furthermore, the recombinant production of this enzyme could help in avoiding the complexity of down streaming processes associated with the purification of this enzyme from wild-type organisms.