Studies on a new antimicrobial peptide from Vibrio proteolyticus MT110.

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.

Development of continuous processing platform utilizing aqueous two-phase extraction for purification of monoclonal antibodies.

Monoclonal antibody downstream processing typically entails chromatography-based purification processes beginning with Protein A chromatography, accounting for 50 % of the total manufacturing expense. Alternatives to protein A chromatography have been explored by several researchers. In this paper, aqueous two-phase extraction (ATPE) has been proposed for continuous processing of monoclonal antibodies (mAbs) as an alternative to the traditional protein A chromatography. The PEG-sulfate system has been employed for phase formation in ATPE, and the mAb is separated in the salt phase, while impurities like high molecular weight (HMW) and host cell proteins (HCPs) are separated in the PEG phase. Following ATPE of clarified cell culture harvest, yield of ≥ 80 % and purity of ≥ 97 % were achieved in the salt phase. Considerable (28 %) reduction in consumable cost has been estimated when comparing the proposed platform to the traditional protein A based platform. The outcomes demonstrate that ATPE can be a potentially effective substitute for the traditional Protein A chromatography for purification of mAbs. The proposed platform offers easy implementation, delivers comparative results, and offers significantly better economics for manufacturing mAb-based biotherapeutics.

Assessment of structural behaviour of a new L-asparaginase and SAXS data-based evidence for catalytic activity in its monomeric form.

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, Km, Vmax, and kcat were 9.49 × 10-3 M, 25.13 IUmL-1 min-1, and 3.01 × 103 s-1, respectively. The CD spectra showed that PrASNase consisted of 18.5 % helix, 21.5 % antiparallel sheets, 4.2 % parallel sheets, 14 % turns, and rest other structures. 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. Differing 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 IC50 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 demonstrated the potential of PrASNase in inhibiting cancerous cells, making it a potential therapeutic candidate.

Description of lipase producing novel yeast species Debaryomyces apis f.a., sp. nov. and a modified pH indicator dye-based method for the screening of lipase producing microorganisms.

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23HT showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095T, 7.8% from Debaryomyces udenii JCM 7855T, and Debaryomyces coudertii JCM 2387T in the D1/D2 domain. In the ITS region sequences, strain CIG-23HT showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095T and Debaryomyces coudertii JCM 2387T. In 18S rRNA gene sequence, the strain CIG-23HT showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23HT can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23HT is a novel species of the genus Debaryomyces, and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297T, and the isotypes are MTCC 12914T and KCTC 37024T. The MycoBank number of Debaryomyces apis f.a., sp. nov. is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis.

Evaluation of physicochemical and functional similarity of a new CHO derived anti-EGFR antibody P-mAb to its reference medicinal product.

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.

Studies on efficient production of a novel l-asparaginase by a newly isolated Pseudomonas resinovorans IGS-131 and its heterologous expression in Escherichia coli.

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.

A new pH indicator dye-based method for rapid and efficient screening of l-asparaginase producing microorganisms.

l-asparaginase is a pharmaceutically and industrially important enzyme as it has potential to treat different cancers and inhibit acrylamide formation in fried and baked food products. In the present study, an attempt was made to screen for new and novel l-asparaginase producers using a widely applied phenol red and bromothymol blue (BTB)1 dye-based plate assay. Screening of four different soil samples for l-asparaginase producers resulted in the isolation of three new potential l-asparaginase producing bacteria. These three strains identified (by 16S rRNA sequencing) as a Pseudomonas resinovorans strain IGS-131, a Bacillus safensis strain IGS-81, and a Glutamicibacter arilaitensis strain ICS-13 with enzyme activities of 10.91 IU/ml, 6.65 IU/ml, and 1.47 IU/ml, respectively. These three strains of bacteria have not been reported as l-asparaginase producers previously. Also, we developed a new pH indicator dye-based plate assay for the screening of l-asparaginase producers after testing eight different pH indicator dyes. This cresol red dye-based method gave a better differentiable zone of hydrolysis and consistent results as compared to previously reported phenol red and BTB-based plate assay. It was also found to be efficient in comparison to all other dyes studied. It produced a bright yellow color at acidic pH (5.5) and turned into a dark red or maroon color when pH was increased (above 7.5). This finding is expected to make screening of all kinds of l-asparaginases more comfortable, rapid, and efficient.