Virendra S. Bisaria

Biodegradation of cellulosic materials: Substrates, microorganisms, enzymes and products

Abstract Cellulosic materials are the only renewable resources available in large quantities which need to be properly utilized to meet our needs of energy, chemicals, food and feed for a long-range solution. A variety of lignocellulosic materials are available and microorganisms capable of degrading either one or more of the three main constituents, viz. cellulose, hemicellulose and lignin have been studied. At least three different enzymes of the multicomponent cellulase system, i.e. cellobiohydrolase endo-glucanase and β-glucosidase are involved in the degradation of crystalline cellulose into glucose. Their mode of action and the manner in which they bring about hydrolysis of crystalline cellulose is discussed in detail. The involvement of parallel enzymes for hemicellulose degradation is also known to some extent but needs to be studied more elaborately, independently and in combination with cellulases. The potential of cellulosic biomass as a source of fuel and petroleum-sparing substances is also reviewed.

Simultaneous bioconversion of cellulose and hemicellulose to ethanol.

Lignocellulosic materials containing cellulose, hemicellulose, and lignin as their main constituents are the most abundant renewable organic resource present on Earth. The conversion of both cellulose and hemicellulose for production of fuel ethanol is being studied intensively with a view to develop a technically and economically viable bioprocess. The fermentation of glucose, the main constituent of cellulose hydrolyzate, to ethanol can be carried out efficiently. On the other hand, although bioconversion of xylose, the main pentose sugar obtained on hydrolysis of hemicellulose, to ethanol presents a biochemical challenge, especially if it is present along with glucose, it needs to be fermented to make the biomass-to-ethanol process economical. A lot of attention therefore has been focussed on the utilization of both glucose and xylose to ethanol. Accordingly, while describing the advancements that have taken place to get xylose converted efficiently to ethanol by xylose-fermenting organisms, the review deals mainly with the strategies that have been put forward for bioconversion of both the sugars to achieve high ethanol concentration, yield, and productivity. The approaches, which include the use of (1) xylose-fermenting yeasts alone, (2) xylose isomerase enzyme as well as yeast, (3) immobilized enzymes and cells, and (4) sequential fermentation and co-culture process are described with respect to their underlying concepts and major limitations. Genetic improvements in the cultures have been made either to enlarge the range of substrate utilization or to channel metabolic intermediates specifically toward ethanol. These contributions represent real significant advancements in the field and have also been adequately dealt with from the point of view of their impact on utilization of both cellulose and hemicellulose sugars to ethanol.

Regulatory aspects of cellulase biosynthesis and secretion.

The cellulase enzyme system consists of cellobiohydrolase, endoglucanase, and beta-glucosidase and has been extensively studied with respect to its biosynthesis, properties, mode of action, application, and, most recently, secretion mechanisms. A knowledge of the factors governing the biosynthesis and secretion of these enzymes at the molecular level will be useful in maximizing enzyme productivity in extracellular fluid. Among other topics, the regulatory effects of sorbose (a noninducing sugar which is not a product of cellulose hydrolysis) on cellulase synthesis and release are described. Cellulase genes have recently been cloned into a number of microorganisms with a view to understanding the gene structure and expression and to obtaining the enzyme components in pure form. The factors governing biosynthesis and secretion of cellulases in recombinant cells are also discussed. Cellulases are known to be glycoproteins, therefore, the role of O- and N-linked glycosylation on enzyme stability and secretion is also detailed.

Bioprocess Considerations for Production of Secondary Metabolites by Plant Cell Suspension Cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.

Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor.

Submerged cultivation of Podophyllum hexandrum for the production of podophyllotoxin was carried out in a 3l stirred tank bioreactor fitted with a low-shear Setric impeller. The specific requirements of the medium, such as carbon source (sugar) and light, were established for the growth of and podophyllotoxin production by P. hexandrum in suspension cultures. Substitution of sucrose by glucose resulted in higher growth and podophyllotoxin production. The biosynthesis of podophyllotoxin was favored when plant cells were cultivated in the dark. An agitation speed of 100 rpm was sufficient to mix the culture broth in the bioreactor without causing any significant cell damage. Biomass and podophyllotoxin accumulation in 3 l bioreactor under batch growth conditions were 6.5 g/l and 4.26 mg/l, respectively, in 22 d. This resulted in an overall podophyllotoxin productivity of 0.19 mg/(l.d), which represented an increase of 27% in comparison to its productivity in a shake flask. Podophyllotoxin production was found to be a combined growth-associated and non-growth associated process.

Inhibition of the NEMO/IKKβ association complex formation, a novel mechanism associated with the NF-κB activation suppression by Withania somnifera’s key metabolite withaferin A

BackgroundNuclear Factor kappa B (NF-κB) is a transcription factor involved in the regulation of cell signaling responses and is a key regulator of cellular processes involved in the immune response, differentiation, cell proliferation, and apoptosis. The constitutive activation of NF-κB contributes to multiple cellular outcomes and pathophysiological conditions such as rheumatoid arthritis, asthma, inflammatory bowel disease, AIDS and cancer. Thus there lies a huge therapeutic potential beneath inhibition of NF-κB signalling pathway for reducing these chronic ailments. Withania somnifera, a reputed herb in ayurvedic medicine, comprises a large number of steroidal lactones known as withanolides which show plethora of pharmacological activities like anti- inflammatory, antitumor, antibacterial, antioxidant, anticonvulsive, and immunosuppressive. Though a few studies have been reported depicting the effect of WA (withaferin A) on suppression of NF-κB activation, the mechanism behind this is still eluding the researchers. The study conducted here is an attempt to explore NF-κB signalling pathway modulating capability of Withania somnifera’s major constituent WA and to elucidate its possible mode of action using molecular docking and molecular dynamics simulations studies.ResultsFormation of active IKK (IκB kinase) complex comprising NEMO (NF-κB Essential Modulator) and IKKβ subunits is one of the essential steps for NF-κB signalling pathway, non-assembly of which can lead to prevention of the above mentioned vulnerable disorders. As observed from our semi-flexible docking analysis, WA forms strong intermolecular interactions with the NEMO chains thus building steric as well as thermodynamic barriers to the incoming IKKβ subunits, which in turn pave way to naive complex formation capability of NEMO with IKKβ. Docking of WA into active NEMO/IKKβ complex using flexible docking in which key residues of the complex were kept flexible also suggest the disruption of the active complex. Thus the molecular docking analysis of WA into NEMO and active NEMO/IKKβ complex conducted in this study provides significant evidence in support of the proposed mechanism of NF-κB activation suppression by inhibition or disruption of active NEMO/IKKβ complex formation being accounted by non-assembly of the catalytically active NEMO/IKKβ complex. Results from the molecular dynamics simulations in water show that the trajectories of the native protein and the protein complexed with WA are stable over a considerably long time period of 2.6 ns.ConclusionsNF-κB is one of the most attractive topics in current biological, biochemical, and pharmacological research, and in the recent years the number of studies focusing on its inhibition/regulation has increased manifolds. Small ligands (both natural and synthetic) are gaining particular attention in this context. Our computational analysis provided a rationalization of the ability of naturally occurring withaferin A to alter the NF-κB signalling pathway along with its proposed mode of inhibition of the pathway. The absence of active IKK multisubunit complex would prevent degradation of IκB proteins, as the IκB proteins would not get phosphorylated by IKK. This would ultimately lead to non-release of NF-κB and its further translocation to the nucleus thus arresting its nefarious acts. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent NF-κB modulating capability. Moreover the present MD simulations made clear the dynamic structural stability of NEMO/IKKβ in complex with the drug WA, together with the inhibitory mechanism.

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.

Optimization of xylanase production by Melanocarpus albomyces IIS68 in solid state fermentation using response surface methodology

Xylanase production by the thermophilic fungus, Melanocarpus albomyces IIS68, during solid state fermentation of wheat straw was studied and the effects of various variables were observed. Using the response surface methodology and the multivariant statistical approach, the optimum levels of the variables affecting xylanase production were determined. The optimum levels of the variables were 600-850 microm particle size, 43 h inoculum age, 1.37% Tween 80, 86% initial moisture content, 5.1% urea, 0.74% yeast extract and a harvest time of 96 h. Under these optimized conditions, xylanase activity of 7760 U/g initial dry substrate was obtained which was in very good agreement with the value predicted by the quadratic model (7890 U/g initial dry substrate).

Biological efficiency and nutritive value of Pleurotus sajor-caju cultivated on different agro-wastes

Abstract The oyster mushroom, Pleurotus sajor-caju (Fr.) Singer, was cultivated on a number of agro-residues and their mixtures. Biological efficiency, defined as the percentage conversion of substrate into fruit bodies on a dry weight basis, was found to be maximum on paddy straw supplemented with cotton seeds (12·82 g/100 g substrate). Biochemical changes effected in the substrates as a result of mushroom growth, in terms of nitrogen content and degradation of cellulose, hemicellulose and lignin components, were monitored. The proximate compositions of fruit bodies of the mushroom in terms of protein, total carbohydrates, fat, fibre and ash were determined. The crude protein and total carbohydrate contents varied from 26·3% to 36·7% and 41·2% to 47·1%, respectively, on various residues. The variation in the contents of crude fat, crude fibre and ash ranged from 1·7% to 2·0%, 11·7% to 17·0% and 6·1%, respectively, on different residues. The energy value of the fruit bodies was found to be between 275 and 300 kCal/100 g, whereas the energy recovery of the substrate in the fruit bodies was from 5% to 10%.

Hsp90/Cdc37 Chaperone/co-chaperone complex, a novel junction anticancer target elucidated by the mode of action of herbal drug Withaferin A

BackgroundHSPs (Heat shock proteins) are highly conserved ubiquitous proteins among species which are involved in maintaining appropriate folding and conformation of other proteins and are thus referred to as molecular chaperones. Hsp90 (Heat-shock protein 90 kDa) is one of a group of molecular chaperones responsible for managing protein folding and quality control in cell environment. However it is also involved in the maturation and stabilization of a wide range of oncogenic client proteins which are crucial for oncogenesis and malignant progression. Hsp90 requires a series of co-chaperones to assemble into a super-chaperone complex for its function. These co-chaperones bind and leave the complex at various stages to regulate the chaperoning process. Arresting the chaperone cycle at these stages by targeting different co-chaperone/Hsp90 interactions seems to be quite a viable alternative and is likely to achieve similar consequences as that of Hsp90 direct inhibition with added favors of high specificity and reduced side effect profile. The study conducted here is an attempt to explore the potential of Withania somnifera’s major constituent WA (Withaferin A) in attenuating the Hsp90/Cdc37 chaperone/co-chaperone interactions for enhanced tumor arresting activity and to elucidate the underlying mode of action using computational approaches.ResultsFormation of active Hsp90/Cdc37 complex is one of the essential steps for facilitation of chaperone client interaction, non-assembly of which can lead to prevention of the chaperone-client association resulting in apoptosis of tumor cells. From our flexible docking analysis of WA into active Hsp90/Cdc37 complex in which key interfacing residues of the complex were kept flexible, disruption of the active association complex can be discerned. While docking of WA into segregated Hsp90 leaves the interface residues untouched. Thus the molecular docking analysis of WA into Hsp90 and active Hsp90/Cdc37 complex conducted in this study provides significant evidence in support of the proposed mechanism of chaperone assembly suppression by inhibition or disruption of active Hsp90/Cdc37 complex formation being accounted by non-assembly of the catalytically active Hsp90/Cdc37 complex. Results from the molecular dynamics simulations in water show that the trajectories of the protein complexed with ligand WA are stable over a considerably long time period of 4 ns, with the energies of the complex being lowered in comparison to the un-docked association complex, suggesting the thermodynamic stability of WA complexed Hsp90/Cdc37.ConclusionsThe molecular chaperone Hsp90 has been a promising target for cancer therapy. Cancer is a disease marked by genetic instability. Thus specific inhibition of individual proteins or signalling pathways holds a great potential for subversion of this genetic plasticity of cancers. This study is a step forward in this direction. Our computational analysis provided a rationalization to the ability of naturally occurring WA to alter the chaperone signalling pathway. The large value of binding energy involved in binding of WA to the active Hsp90/Cdc37 complex consolidates the thermodynamic stability of the binding. Our docking results obtained substantiate the hypothesis that WA has the potential to inhibit the association of chaperone (Hsp90) to its co-chaperone (Cdc37) by disrupting the stability of attachment of Hsp90 to Cdc37. Conclusively our results strongly suggest that withaferin A is a potent anticancer agent as ascertained by its potent Hsp90-client modulating capability.