Thermoalkalophilic polygalacturonase from a novel Glutamicibacter sp.: Bioprospecting, strain improvement, statistical optimization and applications.

The current research discusses a multidimensional bioprocess development, that includes bioprospecting, strain improvement, media optimisation, and applications of the extracted enzyme. A potent alkalophilic polygalacturonase (PG) producing bacterial strain was isolated and identified as a novel Glutamicibacter sp. Furthermore, strain improvement by UV and chemical mutagenesis not only improved the enzyme (PGmut) production but also enhanced its temperature optima from 37 °C to 50 °C. The use of solid substrate fermentation, followed bystatistical optimisation through PB and RSM, substantially increasedPGmut production. A 10-fold increase in enzyme production (632 U/gm) was observed when sugarcane bagasse with a pH of 10.5, 66.8 % moisture, and an inoculum size of 10.15 % was used. The model's accuracy was supported by p-value (p < 0.0001), and an R2 of 0.9940. A pilot-scale experiment, demonstrated ≈ 62,229 U/100 gm PG activity. Additionally, the enzyme's efficacy in demucilization of coffee beans, and bioscouring of jute fibre indicated that it is a valuable biocatalyst.

Statistical bioprocess optimization for enhanced production of a thermo alkalophilic polygalacturonase (PGase) from Pseudomonas sp. 13156349 using solid substrate fermentation (SSF).

In this study, a polygalacturonase (PGase) producing bacterial strain was isolated and identified as Pseudomonas sp. 13159349 from fruit market soils, and TLC analysis confirmed its pectinolytic activity. Additionally, SSF, Plackett-Burman design (PB), and response surface methodology (RSM) were used to optimize the production of this thermostable and alkalophilic PGase. Wheat bran demonstrated the highest activity (60.13 ± 3.39 U/gm) among the various agricultural wastes used as solid substrates. To further enhance the enzyme production, statistical optimization of media components was investigated using the PB design. Among the 11 variables tested, pH (p < 0.0001), inoculum size (p < 0.0001), incubation time (p < 0.0001), and temperature (p < 0.0041) were found to have a positive effect on the production. The interaction and concentration of the selected factors were examined by RSM, which demonstrated the optimal conditions for maximum production (315.65 U/gm) of the enzyme using wheat bran as the solid substrate were pH 10.5, 61-66 h of incubation, and 6-7.5% inoculum size. The model was highly significant, with a p-value of <0.0001, an F-value of 95.33, and a low CV of 2.31. The RSM model was validated by a laboratory-scale experiment showing 30600 ± 400.32 U/100 gm PGase activity. Thus, SSF and the statistical design of media components resulted in a significant 5.2-fold increase in PGase output solely by using agro waste and optimizing the physical parameters, making this a highly cost-effective bioprocess.

Identification of a potential inhibitor for New Delhi metallo-ß-lactamase 1 (NDM-1) from FDA approved chemical library- a drug repurposing approach to combat carbapenem resistance.

Superbugs producing New Delhi metallo-ß-lactamase 1 (NDM-1) enzyme is a growing crisis, that is adversely affecting the global health care system. NDM-1 empowers the bacteria to inactivate entire arsenal of ß-lactam antibiotics including carbapenem (the last resort antibiotic) and remains ineffective to all the available ß lactamase inhibitors used in the clinics. Limited therapeutic option available for rapidly disseminating NDM-1 producing bacteria makes it imperative to identify a potential inhibitor for NDM-1 enzyme. With drug repurposing approach, in this study, we used virtual screening of available Food and Drug Administration (FDA) approved chemical library (ZINC12 database) and captured 'adapalene' (FDA drug) as a potent inhibitor candidate for NDM-1 enzyme. Active site docking with NDM-1, showed adapalene with binding energy -9.21 kcal/mol and interacting with key amino acid residues (Asp124, His122, His189, His250, Cys208) in the active site of NDM-1. Further, molecular dynamic simulation of NDM-1 docked with the adapalene at 100 ns displayed a stable conformation dynamic, with relative RMSD and RMSF in the acceptable range. Subsequently, in vitro enzyme assays using recombinant NDM-1 protein demonstrated inhibition of NDM-1 by adapalene. Further, the combination of adapalene plus meropenem (carbapenem antibiotic) showed synergistic effect against the NDM-1 producing carbapenem (meropenem) resistant clinical isolates (Escherichia coli and Klebsiella pneumoniae). Overall, our data indicated that adapalene can be a potential inhibitor candidate for NDM-1 enzyme that can contribute to the development of a suitable adjuvant to save the activity of carbapenem antibiotic against infections caused by NDM-1 positive gram-negative bacteria. Communicated by Ramaswamy H. Sarma.

Synthesis of nano hydroxyapatite from Hypopthalmichthys molitrix (silver carp) bone waste by two different methods: a comparative biophysical and in vitro evaluation on osteoblast MG63 cell lines.

More than a thousand tonnes of fish bone wastes can be transformed into biomedical products annually. Alkaline hydrolysis and thermal calcification were used to create nanosized hydroxyapatite (HAp) crystals from Silver carp bone wastes. Biophysical tests were used to determine the nano size and chemical composition of synthesised hydroxyapatite. Alkaline hydrolysis hydroxyapatite (AH-HAp) was 58.3 nm, while Thermal calcination hydroxyapatite (TC-HAp) was 64.3 nm in size, confirmed by Atomic Force Microscopy. Energy Dispersive X-ray Analysis studies showed Ca/P (Calcium phosphate) ratio of AH-HAp to be 1.65, whereas TC-HAp as 1.45, confirming AH-HAp to be organically rich along with a similar Ca/P ratio as natural HAp. Fourier Transform Infrared Spectroscopy spectra indicated HAp formation from both procedures, however AH-HAp had superior crystallinity than TC-HAp confirmed from X-Ray Diffraction spectra. MG63 osteoblast cell lines showed 91% cell viability in cytotoxicity studies and 70.1% proliferation efficiency in Alkaline Phosphatase assay, which was higher than TC-HAp. The present study shows that HAp produced via alkaline hydrolysis has better biocompatibility which enhances its applicability as a biomaterial, than HAp synthesized through thermal calcination, which tends to incinerate organic moieties.

A comparative assessment of collagen type 1 from silver carp (fresh water) and milk shark(marine) fish waste.

In this study, a comparative structural and bioactive analysis of collagen extracted from the skin of bony and cartilaginous fishes. The acid-soluble method was followed to extract the collagen from Hypophthalmichthys molitrix (silver carp) and Rhizoprionodon acutus (milk shark) followed by purification using Ion exchange chromatography. A higher yield of collagen was obtained from the skin of SCsk (69.45%) as compared to SHsk (55.29%). SDS PAGE displayed the characteristic α, ß bands of the collagen type1. The native conformation and secondary structure stability of collagen were confirmed by FTIR, XRD and CD studies. The SEM micrographs exhibited the layered and fibrillar nature of the collagen from SHsk and SCsk, respectively. Relative solubility and thermal denaturation analysis showed SCsk to be more stable, but SHsk could withstand higher temperatures. 53.65% of antioxidant activity was observed in SCsk collagen as compared to SHsk (45.9%). Haemocompatibility, cell viability and adhesion results also displayed silvercarp skin to be a better source than Shark skin collagen. The results establish the potential of silver carp collagen as a biomaterial that can have many commercial applications in tissue engineering, cosmetics and food industries.

Large-scale production of enzymes for biotechnology uses.

Enzymes are biocatalysts that speed up the chemical reaction to obtain the final valuable product/s. Biotechnology has revolutionized the use of traditional enzymes to be applicable in industries such as food, beverage, personal and household care, agriculture, bioenergy, pharmaceutical, and various other segments. With respect to the exponential growth of enzymes in biotech industries, it becomes important to highlight the advancements and impact of enzyme technology over recent years. In this review article, we discuss the existing and emerging production approaches, applications, developments, and global need for enzymes. Special emphasis is given to the predominantly utilized hydrolytic microbial enzymes in industrial bioprocesses.

Conformational features of the Staphylococcus aureus AgrA-promoter interactions rationalize quorum-sensing triggered gene expression.

The intracellular trigger for the quorum sensing response mechanism in Staphylococcus aureus involves the phosphorylation of the response regulator AgrA by the membrane anchored histidine kinase AgrC. AgrA activates transcription from three promoter sequences (P1-P3). The promoter strength, conditional association of AgrA with these promoter elements and temporal delay in AgrA-mediated changes in gene expression contribute to the diversity of the quorum sensing response in different S. aureus strains. AgrA promoters comprise of imperfect direct repeats of DNA with a consensus sequence- [TA][AC][CA]GTTN[AG][TG]. Here we describe crystal structures of the DNA-binding (LytTR) domain of AgrA with different cognate DNA sequences that reveal a hitherto unanticipated feature of AgrA-DNA interactions. AgrA promoter interactions are asymmetric with fewer interactions at the binding site proximal to the -35 promoter element. Biochemical assays to evaluate AgrA-promoter interactions suggests that phosphorylation induced dimerization of AgrA can compensate for the asymmetry in AgrA-DNA interactions. The structures also provide a basis to rationalize mutations that were noted to alter AgrA activity without affecting protein-DNA interactions. Put together, the structural data, gene expression and mutational analysis reveal that promoter strength and AgrA phosphorylation enable quorum-sensing triggered transcriptional changes leading to a transition from the persistent to virulent phenotype.

S137 phosphorylation of profilin 1 is an important signaling event in breast cancer progression.

BACKGROUND: Profilins are actin-modulating proteins regulating many intracellular functions based on their multiple and diverse ligand interactions. They have been implicated to play a role in many pathological conditions such as allergies, cardiovascular diseases, muscular atrophy, diabetes, dementia and cancer. Post-translational modifications of profilin 1 can alter its properties and subsequently its function in a cell. In the present study, we identify the importance of phosphorylation of profilin 1 at serine 137 (S137) residue in breast cancer progression. METHODS/PRINCIPAL FINDINGS: We found elevated profilin 1 (PFN) in human breast cancer tissues when compared to adjacent normal tissues. Overexpression of wild-type profilin 1 (PFN-WT) in breast cancer MCF7 cells made them more migratory, invasive and adherent independent in comparison to empty vector transfected cells. Mutation in serine phosphorylation site (S137) of profilin 1 (PFN-S137A) significantly abrogated these properties. Mutation affecting actin-binding ability (PFN-R74E) of profilin 1 enhanced its tumorigenic function whereas mutation affecting its poly-L-proline binding function (PFN-H133S) alleviated these mechanisms in breast cancer cells. PFN-WT was found to activate matrix metalloproteinases by zymography, MMP2 and MMP9 in presence of PDBu (phorbol 12, 13 dibutyrate, PI3K agonist) to enhance migration and invasion in MCF7 cells while PFN-S137A did not. Phosphorylation increased migration and invasion in other mutants of profilin 1. Nuclear profilin levels also increased in the presence of PDBu. CONCLUSIONS: Previous studies show that profilin could be executing a dual role in cancer by either suppressing or promoting tumorigenesis in a context dependent manner. In this study we demonstrate for the first time that phosphorylation of profilin 1 at serine 137 enhances oncogenic properties in breast cancer cells. Inhibitors targeting profilin 1 phosphorylation directly or indirectly through inhibition of kinases that phosphorylate profilin could be valuable therapeutic agents that can alter its activity and thereby control the progression of cancer.

Influence of the AgrC-AgrA complex on the response time of Staphylococcus aureus quorum sensing.

The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrCCyto) and the response regulator domain of AgrA (AgrARR) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrCCyto provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections.

Phorbol ester mediated activation of inducible nitric oxide synthase results in platelet profilin nitration.

Nitric oxide is an important precursor for peroxynitrite production under in vivo conditions leading to cell injury and cell death. In platelets, a number of cytosolic and actin binding proteins were shown to be nitrated [K.M. Naseem, S.Y. Low, M. Sabetkar, N.J. Bradley, J. Khan, M. Jacobs, K.R. Bruckdorfer, The nitration of platelet cytosolic proteins during agonist-induced activation of platelets. FEBS Lett. 473 (1) (2000) 199-122 and M. Sabetkar, S.Y. Low, K.M. Naseem, K.R. Bruckdorfer, The nitration of proteins in platelets: significance in platelet function, Free Radic. Biol. Med. 33 (6) (2002) 728-736]. We investigated the possible mechanism that regulates profilin (an actin binding protein) nitration in platelets. Activation of bovine platelets with arachidonic acid, thrombin, and phorbol 12,13-dibutyrate resulted in nitration of profilin on tyrosine residue. In vivo profilin nitration showed a four- and eight-fold increase in the presence of thrombin and phorbol 12,13-dibutyrate, respectively. Analysis of nitroprofilin levels in the presence of NOS inhibitors (1400W and EGTA), indicated that profilin nitration in phorbol 12,13-dibutyrate treated platelets is mediated by inducible nitric oxide synthase. Phorbol ester treated platelets exhibited higher levels by inducible nitric oxide synthase (491% over control), while total nitric oxide synthase activity increased by 5% over control. Higher levels of peroxynitrite in platelets treated with phorbol 12,13-dibutyrate indicated that profilin nitration is mediated by peroxynitrite. Increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity in platelets treated with thrombin and phorbol 12,13-dibutyrate indicates that nitration of platelet profilin could be mediated by PI 3-kinase. A decrease in the level of nitroprofilin in PDBu treated platelets in the presence of inducible nitric oxide synthase inhibitor, 1400W, was observed suggesting that profilin nitration is mediated by PI 3-kinase dependent activation of inducible nitric oxide synthase.