Debendra K. Sahoo

Molecular mechanism of polyethylene glycol mediated stabilization of protein.

The effect of different molar ratios of polyethylene glycol (PEG) on the conformational stability of protein, bovine serum albumin (BSA), was studied. The binding of PEG with BSA was observed by fluorescence spectroscopy by measuring the fluorescence intensity after displacement of PEG with chromophore ANS and had further been confirmed by measuring the intrinsic fluorescence of tryptophan residues of BSA. Co-lyophilization of BSA with PEG at optimum BSA:PEG molar ratio led to the formation of the stable protein particles. Circular dichroism (CD) spectroscopy study suggested that a conformational change had occurred in the protein after PEG interaction and demonstrated the highest stability of protein at the optimum BSA:PEG molar ratio of 1:0.75. Additional differential scanning calorimetry (DSC) study suggested strong binding of PEG to protein leading to thermal stability at optimum molar ratio. Molecular mechanism operating behind the polyethylene glycol (PEG) mediated stabilization of the protein suggested that strong physical adsorption of PEG on the hydrophobic core of the protein (BSA) along with surface adsorption led to the stability of protein.

Production of soluble recombinant proteins in Escherichia coli: Effects of process conditions and chaperone co-expression on cell growth and production of xylanase

In this study, effects of temperature, inducer concentration, time of induction and co-expression of molecular chaperones (GroEL-GroES and DnaKJE), on cell growth and solubilization of model protein, xylanases, were investigated. The yield of soluble xylanases increased with decreasing cultivation temperature and inducer level. In addition, co-expression of DnaKJE chaperone resulted in increased soluble xylanases though the time of induction of chaperone and target protein had a bearing on this yield. A combination of chaperone co-expression and partial induction resulted in ∼40% (in DnaKJE) and 33% (in GroEL-GroES) of total xylanase yield in soluble fraction. However, the conditions for maximum yield of soluble r-XynB and maximum % soluble expression of r-XynB were different. Higher expression of soluble xylanases in a scalable semi-synthetic medium showed potential of the process for soluble enzyme production.

Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture

Fed-batch culture strategy is often used for increasing production of heterologous recombinant proteins in Escherichia coli. This study was initiated to investigate the effects of dissolved oxygen concentration (DOC), complex nitrogen sources and pH control agents on cell growth and intracellular expression of streptokinase (SK) in recombinant E. coli BL21(DE3). Increase in DOC set point from 30% to 50% did not affect SK expression in batch culture where as similar increase in fed-batch cultivation led to a significant improvement in SK expression (from 188 to 720 mg l(-1)). This increase in SK could be correlated with increase in plasmid segregational stability. Supplementation of production medium with yeast extract and tryptone and replacement of liquid ammonia with NaOH as pH control agent further enhanced SK expression without affecting cell growth. Overall, SK concentration of 1120 mg l(-1) representing 14-fold increase in SK production on process scale-up from flask to bioreactor scale fed-batch culture is the highest reported concentration of SK to date.

Sonorensin: an Antimicrobial Peptide, Belonging to the Heterocycloanthracin Subfamily of Bacteriocins, from a New Marine Isolate, Bacillus sonorensis MT93

ABSTRACT Marine environments are the greatest fronts of biodiversity, representing a resource of unexploited or unknown microorganisms and new substances having potential applications. Among microbial products, antimicrobial peptides (AMPs) have received great attention recently due to their applications as food preservatives and therapeutic agents. A new marine soil isolate producing an AMP was identified as Bacillus sonorensis based on 16S rRNA gene sequence analysis. It produced an AMP that showed a broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The peptide, named sonorensin, was purified to homogeneity using a combination of chromatographic techniques. The intact molecular mass of the purified peptide, 6,274 Da, as revealed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF), was in agreement with Tricine-SDS-PAGE analysis. A PCR array of primers was used to identify AMP structural genes, which allowed the successful amplification of the related genes from strain MT93. The putative open reading frame of sonorensin was amplified, cloned into the pET-32a(+) vector, expressed as a thioredoxin (Trx) fusion protein in Escherichia coli, and then purified. Sequence alignment analysis revealed that the bacteriocin being reported could belong to new subfamily of bacteriocins, heterocycloanthracin. The peptide indicated its potential as a biocontrol agent or food antimicrobial agent, due to its antimicrobial activity against bacteria such as Listeria monocytogenes and Staphylococcus aureus. This is the first report of the production, purification, and characterization of wild-type and recombinant bacteriocin by B. sonorensis and the first bacteriocin of the heterocycloanthracin subfamily to be characterized.

Sonorensin: A new bacteriocin with potential of an anti-biofilm agent and a food biopreservative

The emergence of antibiotic resistant bacteria has led to exploration of alternative therapeutic agents such as ribosomally synthesized bacterial peptides known as bacteriocins. Biofilms, which are microbial communities that cause serious chronic infections, form environments that enhance antimicrobial resistance. Bacteria in biofilm can be upto thousand times more resistant to antibiotics than the same bacteria circulating in a planktonic state. In this study, sonorensin, predicted to belong to the heterocycloanthracin subfamily of bacteriocins, was found to be effectively killing active and non-multiplying cells of both Gram-positive and Gram-negative bacteria. Sonorensin showed marked inhibition activity against biofilm of Staphylococcus aureus. Fluorescence and electron microscopy suggested that growth inhibition occurred because of increased membrane permeability. Low density polyethylene film coated with sonorensin was found to effectively control the growth of food spoilage bacteria like Listeria monocytogenes and S. aureus. The biopreservative effect of sonorensin coated film showing growth inhibition of spoilage bacteria in chicken meat and tomato samples demonstrated the potential of sonorensin as an alternative to current antibiotics/ preservatives.

Studies on xylitol production by metabolic pathway engineered Debaryomyces hansenii

Debaryomyces hansenii is one of the most promising natural xylitol producers. As the conversion of xylitol to xylulose mediated by NAD(+) cofactor dependent xylitol dehydrogenase (XDH) reduces its xylitol yield, xylitol dehydrogenase gene (DhXDH)-disrupted mutant of D. hansenii having potential for xylose assimilating pathway stopping at xylitol, was used to study the effects of co-substrates, xylose and oxygen availability on xylitol production. Compared to low cell growth and xylitol production in cultivation medium containing xylose as the only substrate, XDH disrupted mutants grown on glycerol as co-substrate accumulated 2.5-fold increased xylitol concentration over those cells grown on glucose as co-substrate. The oxygen availability, in terms of volumetric oxygen transfer coefficient, kLa (23.86-87.96 h(-1)), affected both xylitol productivity and yield, though the effect is more pronounced on the former. The addition of extra xylose at different phases of xylitol fermentation did not enhance xylitol productivity under experimental conditions.

Novel surface antigen based impedimetric immunosensor for detection of Salmonella typhimurium in water and juice samples

A specific surface antigen, OmpD has been reported first time as a surface biomarker in the development of selective and sensitive immunosensor for detecting Salmonella typhimurium species. The OmpD surface antigen extraction was done from Salmonella typhimurium serovars, under the optimized growth conditions for its expression. Anti-OmpD antibodies were generated and used as detector probe in immunoassay format on graphene-graphene oxide (G-GO) modified screen printed carbon electrodes. The water samples were spiked with standard Salmonella typhimurium cells, and detection was done by measuring the change in impedimetric response of developed immunosensor to know the concentration of serovar Salmonella typhimurium. The developed immunosensor was able to specifically detect S. typhimurium in spiked water and juice samples with a sensitivity upto 10(1)CFUmL(-1), with high selectivity and very low cross-reactivity with other strains. This is the first report on the detection of Salmonella typhimurum species using a specific biomarker, OmpD. The developed technique could be very useful for the detection of nontyphoidal Salmonellosis and is also important from an epidemiological point of view.

Bioprocess development for the production of sonorensin by Bacillus sonorensis MT93 and its application as a food preservative

Media composition and environmental conditions were optimized using statistical tools, Plackett Burman design and response surface methodology, to maximize the yield of a bacteriocin, named as sonorensin, from a new marine isolate Bacillus sonorensis MT93 showing broad spectrum of antimicrobial activity. Under optimized conditions, MT93 produced 15-fold higher yield of sonorensin compared to that under initial fermentation conditions. As oxygen supply is a critical parameter controlling growth and product formation in aerobic bioprocesses and used as a parameter for bioprocess scale up, the effects of oxygen transfer, in terms of volumetric oxygen transfer coefficient (kLa), on production of sonorensin was investigated using optimized medium composition in a bioreactor. Studies on effectiveness of sonorensin against Staphylococcus aureus and Listeria monocytogenes in fruit juice and as a preservative in pasteurized milk demonstrated its potential as a biopreservative in fruit products and shelf life extender of the pasteurized milk.

Molecular mechanism of improved structural integrity of protein in polymer based microsphere delivery system.

Polymer-based delivery systems provide a promising alternative to multidose intake of many drugs/vaccines. Protein aggregation and inactivation, however, are major problems associated with the encapsulation of proteins in microspheres. With this in mind, we investigated the structural integrity of a model protein bovine serum albumin (BSA) released from poly(lactide-co-glycolide) (PLGA) based microspheres. BSA was encapsulated using solid-in-oil-in-water (S/O/W) double emulsification method with different mixtures of surfactants (carboxymethyl cellulose (CMC):Tween 20/CMC:Tween 80/Tween 20:Tween 80) and with or without polyethylene glycol (PEG). The morphology of BSA-loaded microspheres was analyzed using dynamic light scattering (DLS) and scanning electron microscopy (SEM). BSA released from lyophilized microspheres was evaluated for the structural, conformational and thermal stability by using various spectroscopic and calorimetric techniques. Conformational analysis showed greater increase in secondary structural content of BSA in sample containing PEG and surfactant mixture of CMC and Tween 20 as compared to that containing other two mixtures of surfactants. The differential scanning calorimetric (DSC) analysis of released BSA from all PEG containing samples showed an increase in thermal stability of the protein. Furthermore, fluorescence spectra showed compactness of BSA. In conclusion our studies suggest macromolecular crowding, molecular confinement and increase in Gibbs free energy with strong electrostatic forces of repulsion between microspheres, the last phenomenon due to chosen surfactants, to be responsible for making the protein more compact and structurally integrated and result in a potential encapsulation process for improved protein integrity in final formulation.

Molecular Mechanism of Poly(vinyl alcohol) Mediated Prevention of Aggregation and Stabilization of Insulin in Nanoparticles

It is a challenge to formulate polymer based nanoparticles of therapeutic proteins as excipients and process conditions affect stability and structural integrity of the protein. Hence, understanding the protein stability and complex aggregation phenomena is an important area of research in therapeutic protein delivery. Herein we investigated the comparative role of three kinds of surfactant systems (Tween 20:Tween 80), small molecular weight poly(vinyl alcohol) (SMW-PVA), and high molecular weight PVA (HMW-PVA) in prevention of aggregation and stabilization of hexameric insulin in poly(lactide-co-glycolide) (PLGA) based nanoparticle formulation. The nanoparticles were prepared using solid-in-oil-in-water (S/O/W) emulsification method with one of the said surfactant system in inner aqueous phase. The thermal unfolding analysis of released insulin using circular dichroism (CD) indicated thermal stability of the hexameric form. Insulin aggregation monitored by differential scanning calorimetry (DSC) suggested the importance of nuclei formation for aggregation and its prevention by HMW-PVA. Additional guanidinium hydrochloride based equilibrium unfolding and in silico (molecular docking) studies suggested maximum stability of released insulin from formulation containing HMW-PVA (F3). Furthermore, in vivo studies of insulin loaded nanoparticle formulation (F3) in diabetic rats showed its bioactivity. In conclusion, our studies highlight the importance of C-terminal residues of insulin in structural integrity and suggest that the released insulin from formulation containing HMW-PVA in inner aqueous phase was conformationally and thermodynamically stable and bioactive in vivo.