Papers by Krishna kant Sharma
Functional analysis of metalloenzymes from human gut microbiota and their role in ulcerative colitis
Journal of Applied Microbiology
AimMetalloenzymes produced by gut microbiota play an essential role in various physiological proc... more AimMetalloenzymes produced by gut microbiota play an essential role in various physiological processes, and maintains homeostasis of gastrointestinal tract. Our study includes functional analysis of microbial metalloenzymes using metagenomics and metatranscriptomics data from Inflammatory Bowel Disease Multiomics Database.Methods and resultsThe distance matrix calculated by using metalloenzymes data produced significant results for bacterial taxonomy, with higher variance compared to HMP analysis in both Western and Indian population. Differential gene expression analysis revealed altered expression of ulcerative colitis (UC)-associated enzymes, increased folds changes in Prevotella and Megamonas transcripts; whereas, low transcripts of Alistipes genera. Further, docking and simulation studies performed on screened UC-associated enzymes revealed changes in catalytic efficiency and ligand interacting residues.ConclusionThe β-diversity using microbes containing metalloenzymes suggests...
Exploring Fungi-Associated Lignocellulose Degradation: Secretomic and Proteomic Approaches
Advancing Frontiers in Mycology & Mycotechnology, 2019
Filamentous fungi of phyla Basidiomycota and Ascomycota are the group of microorganisms that are ... more Filamentous fungi of phyla Basidiomycota and Ascomycota are the group of microorganisms that are capable of secreting a variety of proteins and other secondary metabolites depending on the environment and culture conditions. The protein constitutes the hydrolytic enzymes which cause the deconstruction of the plant cell wall and has applicability in several biotechnological processes including second-generation ethanol production. Secretomic and proteomic analysis of the fungi is an excellent tool to find out the biological mechanisms of lignocellulose degradation. Furthermore, it is also an important tool to search for novel enzymes or metabolites of the biotechnology field. Extracellular secretion of the protein from different fungal species has been studied using different high-throughput techniques such as 2-D PAGE, MALDI-ToF/ToF, LC-MS/MS, iTRAQ technique using LC-MS/MS, and Nano-LC-MS/MS protein mass spectrometry. Bioinformatics tools have equal importance in the prediction and profiling of the expressed proteins, according to the current database. For this reason, publications documenting the fungal secretome and proteome have increased significantly in the past few years. Herein, we have updated the development and evolution of the proteome/secretome technology and its application in the protein profiling and functional genomics of the economically important filamentous fungi.
Applied Microbiology and Biotechnology, 2022
Probiotics have been considered as an economical and safe alternative for the treatment of a larg... more Probiotics have been considered as an economical and safe alternative for the treatment of a large number of chronic diseases and improvement of human health. They are known to modulate the host immunity and protect from several infectious and noninfectious diseases. The colonization, killing of pathogens and induction of host cells are few of the important probiotic attributes which affect several functions of the host. In addition, prebiotics and non-digestible food substances selectively promote the growth of probiotics and human health through nutrient enrichment, and modulation of gut microbiota and immune system. This review highlights the role of probiotics and prebiotics alone and in combination (synbiotics) in the modulation of immune system, treatment of infections, management of inflammatory bowel disease and cancer therapy. • Probiotics and their derivatives against several human diseases. • Prebiotics feed probiotics and induce several functions in the host. • Discovery of novel and biosafe products needs attention for human health.
Pathobionts: mechanisms of survival, expansion, and interaction with host with a focus on Clostridioides difficile
Gut Microbes, 2021
ABSTRACT Pathobionts are opportunistic microbes that emerge as a result of perturbations in the h... more ABSTRACT Pathobionts are opportunistic microbes that emerge as a result of perturbations in the healthy microbiome due to complex interactions of various genetic, exposomal, microbial, and host factors that lead to their selection and expansion. Their proliferations can aggravate inflammatory manifestations, trigger autoimmune diseases, and lead to severe life-threatening conditions. Current surge in microbiome research is unwinding these complex interplays between disease development and protection against pathobionts. This review summarizes the current knowledge of pathobiont emergence with a focus on Clostridioides difficile and the recent findings on the roles of immune cells such as iTreg cells, Th17 cells, innate lymphoid cells, and cytokines in protection against pathobionts. The review calls for adoption of innovative tools and cutting-edge technologies in clinical diagnostics and therapeutics to provide insights in identification and quantification of pathobionts.
Fungal Biology, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Renewable Energy, 2019
Bioethanol production using paper wastes seems a promising approach towards sustainable energy, b... more Bioethanol production using paper wastes seems a promising approach towards sustainable energy, but mainly hindered by hazardous ink. Therefore, in our experimental plan various methods were applied for the deinking of waste newspapers, laser printed papers and examination papers. A newly designed paper pulper reduced the pulping time from 6 h to 2 h. Cellulase (15 FPU/g) from Aspergillus oryzae MDU-4 was found effective for the deinking of newspapers, whereas laccase isozymes (150 U/g) from Ganoderma lucidum MDU-7 along with 2 mM HOBt was preferred for the ink removal and degradation from the examination papers. Ozonation in the presence of Tween-80 was found to be efficient in the removal of toxic toners used in laser printing papers. The biologically and physically deinked papers, studied with the help of SEM, TEM, FTIR, and XRD analysis revealed significant changes in the chemical and surface structure. Moreover, the saccharification of deinked papers with the help of an enzymatic consortium of Trichoderma citrinoviride MDU-1 resulted in 305 mg/g, 377 mg/g, and 409 mg/g release of sugars from the newspaper, examination paper, and laser printed paper, respectively. Finally, the enzymatic hydrolysates fermented with Saccharomyces cerevisiae NCIM-3640 produced 3.35 g/L ethanol, with 40.85% ethanol yield.
Computational biology and chemistry, 2018
Laccase in Cryptococcus neoformans is covalently linked to the carbohydrate moiety of the cell wa... more Laccase in Cryptococcus neoformans is covalently linked to the carbohydrate moiety of the cell wall, which allows it to get access to the different substrates for catalyzing their oxidation and therefore plays a vital role in the virulence. The laccase gene (3.0 kb) from C. neoformans serotype D was amplified, cloned and sequenced for protein modeling, docking and simulation studies. The three dimensional homology models of laccase protein from C. neoformans and other pathogenic gut bacteria were docked with selected biomolecules like prostaglandins (PG), membrane phospholipids, neurotransmitters (serotonin) using GOLD software. The GOLDscore values of laccase from C. neoformans docked with prostaglandinH(59.76), prostaglandinG(59.45), prostaglandinE(60.99), phosphatidylinositol (54.95), phosphatidylcholine (46.26), phosphatidylserine (55.26), arachidonic acid (53.08) and serotonin (46.22) were similar to the laccase from enteropathogenic bacteria but showed a better binding affinit...
Concomitant production of xylanases and cellulases from Trichoderma longibrachiatum MDU-6 selected for the deinking of paper waste
Bioprocess and biosystems engineering, Jan 8, 2016
Sixty fungal cultures were isolated from agricultural soil, industrial soil, forest canopy soil h... more Sixty fungal cultures were isolated from agricultural soil, industrial soil, forest canopy soil having decomposed leaf litter and compost samples collected from different regions of India. Fifteen fungal cultures were selected qualitatively for the production of xylanase and cellulases and were identified employing ITS, NS and MNS primers. The enzyme cocktail consisting of 3811 IU g(-1) of xylanase and 9.9 IU g(-1) of cellulase from Trichoderma longibrachiatum MDU-6 was selected quantitatively for the deinking of diverse paper wastes. The enzyme production increased two fold when produced at tray level in comparison with flasks. The enzyme cocktail was effective in the deinking of old newspaper samples with significant removal of chromophores, phenolics and hydrophobic compounds and less sugar loss. While in case of examination papers and laser printed papers, ink removal was not very significant. Moreover, the sugar loss was significantly high in case of examination papers. The dei...
Biochemical and Biophysical Research Communications, 2014
Multi-copper oxidases (MCOs) are widely distributed in bacteria, where they are responsible for m... more Multi-copper oxidases (MCOs) are widely distributed in bacteria, where they are responsible for metal 28 homeostasis, acquisition and oxidation. Using specific primers, yacK coding for MCO was amplified from 29 different serotypes of Yersinia enterocolitica biovar 1A. Homology modeling of the protein followed by 30 docking with five well-known substrates for different MCO's (viz., 2,2 0 -azino-bis(3-ethylbenzthiazo-31 line-6-sulphonic acid [ABTS], syringaldazine, L-tyrosine, ammonium ferrous sulfate and guaiacol), lignin 32 monomers (Coniferyl alcohol, p-coumaryl alcohol and sinapyl alcohol) and two inhibitors i.e., kojic acid 33 and N-hydroxyglycine was done. The docking gave maximum GoldScore i.e., 91.93 and 72.64 with 34 ammonium ferrous sulfate and ABTS, respectively. Similarly, docking with ICM gave À82.10 and 35 À83.61 docking score, confirming the protein to be true laccase with ferroxidase activity. Further, valida-36 tion with ammonium ferrous sulfate as substrate gave laccase activity of 0.36 Units/L/min. Guaiacol, 37 L-tyrosine, and lignin monomers showed good binding affinity with protein models with GoldScores of 38 35.89, 41.82, 40.41, 41.12 and 43.10, respectively. The sequence study of all the cloned Yack genes 39 showed serotype specific clades in dendrogram. There was distinct discrimination in the ligand binding 40 affinity of Y. enterocolitica laccase, among strains of same clonal groups suggesting it as a tool for 41 phylogenetic studies. 42
Water, Air, & Soil Pollution, 2014
The bacterial communities in the intestinal tracts of earthworm were isolated by culture-dependen... more The bacterial communities in the intestinal tracts of earthworm were isolated by culture-dependent approaches. In total, 72 cultures were isolated and purified from the gut of an earthworm under aerobic culture condition, out of which 25 isolates were laccase positive. Isolate 33, a good laccase producer was identified as Bacillus safensis DSKK5, using both biochemical and molecular approaches. It was found to produce maximum laccase activity at 0.75 % of wheat bran, 37 °C, and pH 6.2. Further, copper sulfate and copper chloride showed a maximum laccase production. In order to understand the affinity of binding and interaction between toxic dyes and bacterial laccase, homology models were generated. The resulted models were further validated and used for docking studies with commonly used industrial dyes. Molecular docking using CCDC GOLD software gave a good score with all the textile dyes. Further, validation using molsoft ICM software showed a good binding energy of -104.25, -106.00, -113.98, and -100.36, with commercial dyes, i.e., procion blue, procion green, procion red, and reactive yellow 86, respectively. Experimental data showed a maximum decolorization with procion green (85.66 %) and procion red (85.58 %), which validate the molsoft ICM results, i.e., -106.00 and -113.98, respectively.
Scientific Reports, 2013
The variables influencing laccase production by white-rot fungus Ganoderma sp. rckk-02 were optim... more The variables influencing laccase production by white-rot fungus Ganoderma sp. rckk-02 were optimized employing response surface methodology. Malt extract (6.0% w/v), lignin (0.5% w/v) and pH (5.5) were found to be the most significant factors for enhanced laccase production by 7 fold (226.0 U/ml) as compared to unoptimized growth conditions (32.0 U/ml). The N-terminal sequence of laccase revealed its distinct amino acid profile (S-I-R-N-S-G), which suggested it as a novel enzyme. The Far-UV CD spectrum of the laccase showed single broad negative trough at around 213 nm, a typical signature of all b proteins. The laccase was found to fall in the range of middle redox potential laccases. Purified laccase at dosage of 2.5 Ug 21 body weight when supplemented with pelleted diet of rats, a significant improvement (p , 0.05) in nutrients digestibility without causing any elevation of blood stress enzymes was observed. hite rot fungi are known to be the potential producer of ligninolytic enzymes i.e. mangnese peroxidase, lignin peroxidase and laccases. Among these, laccases are one of the widely studied oxidoreductases and majority of them contain four copper ions per molecule. They catalyze single-electron oxidation of a wide variety of organic and inorganic substrates including mono-, di-, polyphenols, methoxyphenols, aromatic amines and ascorbate with concomitant four-electron reduction of oxygen to water 1 . This makes laccases useful for their varied applications in several biotechnological processes such as phenolics elimination for stabilization and browning of fruit juices, beer/wine, in bleaching of paper pulp, decolourization of dyes and textile effluents, oxidation-reduction reaction in biosensors development, PAHs and other xenobiotics degradation for soil bioremediation, production of complex polymers in organic synthesis and mild oxidizing agent in cosmetics development 2 . In addition, its being an efficient lignin degrading biocatalyst, has been applied in biopulping but scarcely been tried for its possible application in improving the bioavailability of nutrients and digestibility of animal feed, especially for monogastric diets. Laccase can either act on feed components prior to or after consumption i.e. within the gastrointestinal tract 3 . However, to make use of laccase commercially viable in feed improvement, its production level should be high as well as it should be nontoxic to animals. The critical safety evaluation of the enzymes used for improving animal feed is must and tests involving rat models have been found essential to provide some preliminary indication of possible cellular mutagenicity, potential carcinogenicity and teratogenecity during the developmental stages 3-5 . Furthermore, random addition of enzymes to diets without consideration for specific situations, toxicity and substrate targets will only discourage or delay on-farm adoption of enzyme technology 6 . Therefore, we have also selected the rat model for evaluation of efficiency of laccase in improvement of pelleted diets specifically for monogastric animals. Thus for producing higher amount of such enzymes researchers have gained interest in minimizing the cost of enzyme preparation to be used as ruminants diet supplement 7 . Thereby, designing and optimization of fermentation media for the most economical production is one of the most important task. The objectives of this work were to optimize production of laccase with middle redox potential from an indigenous white rot fungus, Ganoderma sp. rckk-02 using response surface methodology under liquid static cultivation conditions, its characterization and its evaluation for possible application as animal feed supplement.
Laccase from an alkalitolerant basidiomycetes Crinipellis sp. RCK‐1: Production optimization by response surface methodology
Journal of Basic Microbiology, 2011
A newly isolated alkalitolerant basidiomycetous fungus, identified as Crinipellis sp. RCK‐1 was o... more A newly isolated alkalitolerant basidiomycetous fungus, identified as Crinipellis sp. RCK‐1 was observed to produce laccase. The effect of different physicochemical factors on laccase production was studied. The identification of the important factors (initial pH of the medium, copper and tryptophan) with simple screening experiment involving optimization using single factor at a time strategy, was followed by application of complex response surface design for further maximizing the laccase production and was helpful in defining the effects and interactions of the physiological and nutritional factors. The statistical optimization by response surface methodology resulted in a 27.0‐fold (619.9 U ml–1) increase in the production of laccase from Crinipellis sp. RCK‐1 when compared to laccase production in unoptimized medium (23.0 U ml–1). The results from the response surface curve suggested that there was interaction between tryptophan and copper in a way that might resulted in positi...
Biotechnology and Applied Biochemistry, 2006
The lack of an efficient transformation system in filamentous fungi is one of the major problems ... more The lack of an efficient transformation system in filamentous fungi is one of the major problems in carrying out cloning and expression of genes to produce a value‐added product at the commercial level. Therefore an attempt has been made to develop an efficient, convenient and expeditious genetic transformation system for successful transfer of transferred DNA, carrying the genes coding for GUS (β‐glucuronidase) (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of Phanerochaete chrysosporium, a widely studied lignin‐degrading white‐rot fungus. The transformation of the marker gene was confirmed through GUS, PCR and Southern‐blot hybridization. Transformation with acetosyringone, in the presence of light with partial bleaching for 1 h, were found to be a better combination for fungal transformation with 48% efficiency. The agro‐transformation method to transform the modified mycelial pellets offers a practical means for exploiting t...
Bioresource Technology, 2008
The empirical models developed through two independent RSM (RSM-I, 2 3 ; RSM-II, 2 5 ) in terms o... more The empirical models developed through two independent RSM (RSM-I, 2 3 ; RSM-II, 2 5 ) in terms of effective operational factors of inoculum age, inoculum volume, wheat bran-to-moisture ratio (RSM-I) and contact time, extraction temperature, agitation, fermented bran-to-solvent ratio and SDS (RSM-II) were found adequate to describe the optimization of exo-polygalacturonase from Bacillus subtilis RCK under solid-state fermentation (SSF) conditions. Through the analysis of RSM-I, wheat bran-to-moisture ratio and inoculum volume were found to be the most significant factors and an increment in both had a positive effect in enhancing enzyme yield, while in RSM-II all the factors significantly affected enzyme recovery except fermented bran-to-solvent ratio, which had the least impact within the ranges investigated in enhancing enzyme recovery. Based on contour plots and variance analysis, optimum operational conditions for maximum exo-polygalacturonase yield were achieved when 1.5% (v/w) of 24 h old (OD 600 nm % 2.7 ± 0.2) B. subtilis RCK cells were inoculated on moistened wheat bran (1:7 solid substrate-to-moisture ratio) and enzyme was harvested by addition of solvent (1:6 fermented bran-to-solvent ratio) under shaking conditions (200 rpm) in presence of SDS (0.25% w/v) for 15 min at 35 °C. An over all 3.4 fold (1.7-fold RSM-I; 2.0 fold RSM-II) increase in enzyme production was attained because of optimization by RSM.
Bioresource Technology, 2009
Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosi... more Prosopis juliflora (Mesquite) is a raw material for long-term sustainable production of cellulosics ethanol. In this study, we used acid pretreatment, delignification and enzymatic hydrolysis to evaluate the pretreatment to produce more sugar, to be fermented to ethanol. Dilute H 2 SO 4 (3.0%, v/v) treatment resulted in hydrolysis of hemicelluloses from lignocellulosic complex to pentose sugars along with other byproducts such as furfural, hydroxymethyl furfural (HMF), phenolics and acetic acid. The acid pretreated substrate was delignified to the extent of 93.2% by the combined action of sodium sulphite (5.0%, w/v) and sodium chlorite (3.0%, w/v). The remaining cellulosic residue was enzymatically hydrolyzed in 0.05 M citrate phosphate buffer (pH 5.0) using 3.0 U of filter paper cellulase (FPase) and 9.0 U of b-glucosidase per mL of citrate phosphate buffer. The maximum enzymatic saccharification of cellulosic material (82.8%) was achieved after 28 h incubation at 50 °C. The fermentation of both acid and enzymatic hydrolysates, containing 18.24 g/L and 37.47 g/L sugars, with Pichia stipitis and Saccharomyces cerevisiae produced 7.13 g/L and 18.52 g/L of ethanol with corresponding yield of 0.39 g/g and 0.49 g/g, respectively.
Fed batch enzymatic saccharification of newspaper cellulosics improves the sugar content in the hydrolysates and eventually the ethanol fermentation by Saccharomyces cerevisiae
Biomass and Bioenergy, 2010
The newspaper is comprised of (ww− 1) holocellulose (70.0%) with substantial amount of lignin (16... more The newspaper is comprised of (ww− 1) holocellulose (70.0%) with substantial amount of lignin (16.0%). Bioconversion of the carbohydrate component of newspaper to sugars by enzymatic saccharification, and its fermentation to ethanol was investigated. Of various ...
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Papers by Krishna kant Sharma