Department of Biotechnology

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    Biological Evaluation of Indigenous Medicinal Plants from Darjeeling Himalayan Region of West Bengal for Natural Compound(s) As Inhibitor of Cancer Cell Proliferation and Studies on their Molecular Mechanism
    (University of North Bengal, 2023) Rai, Vijeta; Ghosh, Shilpi; Kumar, Anoop
    Cancer remains one of the major world health issue after cardiovascular diseases. The area of cancer research is continually expanding with better understanding of molecular mechanism of progression of the disease, which has resulted in development of new drug targets for more efficient cancer therapy. In addition, the search for new anticancer lead compounds is a great challenge due to the development of drug resistance, deleterious side effects and unaffordable cost of current chemotherapeutic drugs. The enormous success of plant based natural products as anticancer agent represents medicinal plants as an important pool for the identification of novel drug. Darjeeling Himalayan region has a rich diversity of therapeutic plants that can be utilized for development of novel drugs. In this study some ethnomedicinally important plants of the Darjeeling Himalayan region, such as Astilbe rivularis, Tupistra nutans, Zanthoxylum oxyphyllum, Bergenia ciliata, Artemesia vulgaris and Eupatorium cannabium were screened for various phytochemicals, and antioxidant, antimicrobial activities, and cytotoxic potential against cancer cell lines, and finally, Astilbe rivularis was selected futher studies on isolation of active compound with anti-cancer potential. A steroid ester compound, spectrometrically characterized as Stigmasta-5(6), 22(23)-dien-3-beta–yl acetate, designated as A11, was isolated for the first time from the plant rhizome in a bioassay guided approach. The catalytic inhibition and structural alteration of human dihydrofolate reductase (hDHFR) by A11 was evaluated using methotrexate (MTX), a DHFR inhibitor anticancer drug as a reference. The compound was found to inhibit the in vitro activity of hDHFR) with IC50 values of 1.20 μM. A11 interacted with hDHFR as revealed by concentration dependent quenching of the tryptophan fluorescence of the enzyme suggesting its effect on structural alteration of the enzyme. Molecular docking of A11 on crystal structure of hDHFR revealed significant interaction with free energy of binding and Ki values of -10.86 kcal mol-1 and 11 nM, respectively. Subsequent in vitro studies at cellular level showed a relatively greater cytotoxic effect of A11 against human kidney (ACHN, IC50 60 μM) and liver (HepG2, IC50 70 μM) cancer cells than their respective normal cells (HEK-293, IC50 350 μM and WRL-68, IC50 520 μM). Scanning electron microscopy of A11 treated cells revealed the morphological feature of apoptosis, like cell rounding and surface detachment, membrane blebbing, loss of cilia and increased number of pores of decreased sizes. A11 mediated apoptosis of cancer cells was found to be correlated with induction of intracellular of reactive oxygen species (ROS) level and fragmentation of genomic DNA, which is a hallmark of apoptosis. A11 mediated induction of apoptotic feature of ACHN cells was found to be correlated with increased accumulation of cleaved active form of the pro-apoptotic proteins, like caspase 3, caspase 7, caspase 9 and PARP1. The cleaving of caspace 3 and caspase 7 was further confirmed by western blot analysis. The results thus provide an insight into the anti-tumorigenic potential of A11. The function of A11 in both inhibition of hDHFR and induction of apoptosis suggest that the compound could act via diverse signaling pathways of cancer cells without affecting normal cells. However, a possible link between hDHFR inhibition and cell cycle regulation needs to be illustrated in future studies. The outcomes of this research contribute to the growing field of natural product-based drug discovery and highlight the significance of traditional medicinal knowledge in the context of cancer treatment.
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    Studies on biodegradation of petroleum hydrocarbons by a soil bacterium
    (University of North Bengal, 2015) Sarkar, Payel; Ghosh, Shilpi
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    Studies on cellulase-producing bacteria isolated from a vermicompost-derived consortium and to evaluate their synergism in depolymerization of agricultural residues
    (University of North Bengal, 2023) Basak, Arijita; Ghosh, Shilpi
    The microorganisms growing in lignocellulose-degrading environment produce multiple enzyme systems that act synergistically to break down the polysaccharide constituents of JjgnocelluLosic material, namely cellulose, and bemicellulose. Vennicompost has taxonomically distinct microorganisms that could be used for the production of biocatalysts for the decomposition of industrial agroresidues. In this study, the microbial communities present in vermicompost were gradually adapted to grow at higher temperature of 60 °C while utilizing chopped rice straw as carbon source. The lignocellulolytic consortium RSV so developed exhibited the ability to degrade cellulose and hemicellulose. This was backed by quantitative estimation whereby RSV produced glycoside hydrolases belonging to cellulases such as exoglucanase, endoglucanase and ~-glucosidase with maximum activity of 5.8 ± 0.05, 19.56 ± 0.07 and 5.8 ± 0.03 ill/mg, respectively; and xylanases such as endoxylanase, ~xylosidase and a -L-arabinofuranosidase with highest activity 54 ± 15. 76, 5.2 ± 0.04, and 4.8 ± 0.03 IU/mg, respectively, on Day 2 of growth at pH 6 and 60 °C. The extracellular enzyme concentrate of RSV showed the ability to saccharify rice straw pretreated by glacial acetic acid, hydrogen peroxide, acid-peroxide combinations and heat The saccharification yield of reducing sugar equivalents was significantly greater from acid-peroxide combinations of 1: l (460 mg/g of rice straw), 2:1 (360 mg/g ofR and 4:1 (290 mg/g of rice straw on Day 5). The total genomic DNA of RSV was isolated, sequenced and the raw reads were trimmed and assembled into a metagenome for taxonomic and CAZyme profiling. The RSV consortium was comprised of a majority of bacteria from the phyla Finnicutes (56.91 %), Proteobacteria (28.32 %) and Bacteroidetes ( 12.22 %) and included members of the genera Pseudoc/osrridium (21.29 %), Chelatococcus ( I 6. l 3 %), Thermoanaerobacrerium (14.8 %), Algoriphagus (10.45 %), Bacillus (3.95 %), Pseudoxanthomonas (3.88 %), Geobacillus (3.57 %), Halomonas (3.29 %), Aeribacillus (3 .26 %) and Syrnbiobacterium (1.77 %) as the most abundant bacteria. Apart from producing GHs, RSV also produced enzymes belonging to CAZymes groups GH, CE and AA and auxiliary active enzymes such as multi copper oxidase, catalase/peroxidase, glycolate oxidase, GMC oxidoreductase and quinone oxidoreductase. Lignocellulolytic bacteria were obtained from RSV by dilution plating technique. Eight of them were capable of cellulose degradation while one was predominantly ligninolytic. Phylogenetic analysis of the isolates based on l 6S rRNA gene sequence identified these bacteria as belonging to genera Geobacillus, Parageobacillus, Aeribacil/us and Micrococcus. Among the bacterial isolates two cellulolytic isolates exhibiting significantly higher cellulase activity, Parageobacil!us thermoglucosidasius NBCB 1 (23.94±1 .34 TU/mg on Avicel, Day3), and Aeribacillus composti XLN l (22. 75± 1.50 ill/mg on A vicel, Day3 ), and the ligninolytic isolate Micrococcus yunnanensis B4 with laccase ()40.81±10.3 l μIU/mg on Day 3), lignin peroxidase (191. I 9±26.66 μTU/mg on Day 3) and manganese peroxidase (3239.73±177.11 μIU/mg oa Day 3) activities, were selected for the development of synthetic triculture consortium. The synthetic triculture consortium was further assessed for cellulase production and activity. Inoculation of the three cultures simultaneously to the celluJase production medium MSM-RS gave 53.24±0.96 TU/mg of cellulase production on Day 3 with the highest degree of synergy among all synthetic consortia (1.26±0.04). The cellulase production of the triculture was further optimized by OFAT (94.00±4.52 ill/mg), RSM-CCRD (115.68±0.01 ill/mg) and ANN-GA (139.00±0.00 IU/mg). Of all of them, ANN was found to give better process precision with optimized culture parameters of 3.33 days of incubation, medium pH of 6.06, 0.89 % sorbitol and 1.09 % peptone. The enzyme preparation obtained from triculture on the optimized MSM-RS medium was then utilized in the saccbarification of alkali, peroxy acetic acid, moist heat pretreated or untreated rice straw which gave reducing sugar equivalent yields of l 70.40±2.13 mglg of pretreated rice straw (Day 5), 156.00± l.25 mg/g (Day 5), 113.54±2.49 mg/g (Day 5) and 82.07±0.00 mg/g (Day 5), respectively, which were ~53.47, ~58.01 , ~65.87 and - 137.10 % of the yields from commercial cellulase Celluclast. The cellulase produced by Parageobacillus thermoglucosidasius NBCB 1 was purified to homogeneity, characterized and the encoding gene was cloned and sequenced. Before purifying, the enzyme production by the bacterium was enhanced to 78.47±2.23 IU/mg through ANN-GA. The cellulase enzyme, designated as PtCell , was purified through ammoniwn sulphate precipitation, gel filtration and DEAE-Sephacel anion exchange chromatography with 4 1.95-fold purification and final y ield 21 .52 %. The specific activity of PtCel 1 was 184 TU/mg on CMC and 305 IU/mg on A vicel thereby qualifying it as a processive endoglucanase. It was functional within a broad range of pH (4.5-8.5) and temperature (4-80 °C) with optimum activity at pH 5.5 and 60 °C. PtCel 1 showed enhanced activity in presence of Zn2+ (~369.14 %), Mg2+ (~245.15 %), Ca2 i (~261.10 %), Na+ (~ 179.77 %), Sn2+ (~ 150.80 %), SDS (- 132.8 %) and P-ME (~ 186.81 %). It was resistant to and retained activity in presence of galactose (~85.53 %), xylose (~74.28 %), cellobiose (~63.37 %), EDTA (- 62.18 %) and H202 (~33.33 %). PtCell had Km and Ymax at 0.363 mg/ml and 308.64 IU/mg, respectively. The putative gene encoding PtCell it was cloned and sequenced. It was characterized to be a Zn•dependeut endoglucanase/metallopeptidase from the M42 family.
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    Thermophilic lignocellulose deconstructing microbial consortium: Mining of Cellulolytic glycoside hydrolases for saccharification of agro residues
    (University of North Bengal, 2023) Lepcha, Khusboo; Ghosh, Shilpi
    Plant cell wall lignocellulose is recognized as one of the most abundant source of fennentable sugars with potential use in the production of different value added products like biofuel and other chemicals. Deconstruction of the complex lignocellulose into useable monomers by microbial enzymes has been long adapted; however a major bottleneck in efficient bioconversion of cellwall polysaccharide by enzymes is that plant cell walls have evolved to resist enzymatic deconstruction - a factor collectively known as "biomass recalcitrance". Using a broader suite of enzymes for hydrolysis of cellulose and hemicelluloses to fennentable sugars would be a better approach to combat biomass recalcitrance. Ln nature also, lignocellulosic biomass is degraded with the cooperation of many microorganisms producing a variety of cellulolytic and hemicellulolytic enzymes under aerobic and anaerobic conditions. The biodegradation of cellulosic biomass through the use of microbial co-cultures or complex communities has been proposed as a highly efficient approach for biotechnological application, since it avoids the problems of feedback regulation and metabolite repression posed by isolated single strains. In this study microbial communities of lignocellulosic habitat of forest soil (FS) and goat rumen contents (GR) were adapted to grow and survive at higher temperatures in destarched wheat bran /rice straw as sole carbon source to generate four thennophilic microbial consortia amongst which the consortium developed from goat rumen contents bred on rice straw (GR~ RS) was selected for further analysis due to its better hydrolytic potential on the basis of enzyme assays and in-gel analysis. Characterisation of cellulolytic and xylanolytic potential of GR-RS revealed both the cellulolytic and xylanolytic potential to be thennophilic and thermostable over a broad range of pH. The enzymes were tolerant to and activated in the presence of many metal ions such as Magnesium, Calcium, Barium, Sodium etc. whereas it was greatly reduced to 10% by Hg2+. Comparative Taxonomic profiling of GR-RS-T (developed at 60°C) and GR-RS-M (developed at 37°C) by meta.genomic analysis revealed GR-RS-M had a majority of Proteobacteria (48.67%), Finnicutes (30.34%), Verrucomicrobia (5.45%), Bacteroidetes (5. 12%), Planctomyecetes (3.13%), wheras GR-RS-T exhibited majority of bacteria belonging to phyla Proteobacteria (34.65%), Bacteroidetes (31 %) and Finnicutes (30.83%). Analysis of differential transcriptional expression of cellulolytic and xylanolytic enzymes in GR-RR-T revealed that expression of cellulases and xylanases were mostly from Paenibacillus , Thermobacillus, Pseudoclostridium, Geobacillus, Clostridium etc in case of GR-RS-T whereas its expression were mostly from Klebsiella, Cellulomonas and Pseudoxanthomonas in case of GR-RS-M. The analysis of transcriptome for distribution of different classes of CAZymes revealed that GR-RS-T had GH (34%), GT (38%), CBM (17%), AA (I%), CE (9%) and PL (1 %) whereas GR-RS-M had GH (29%), GT (43%), CBM (16%), AA (3%), CE (8%) and PL (I%). It was also revealed that most families of GH, PL and AA had higher abundance in GRRS- M as compared to GR-RS-T based on the number of reads detected. The study highlights the significance of sub-culturing the lignocellulolytic population from goat rumen contents at 60°C which selects and maintains the therrnophiJic members for production of thermophilic GHs as also evidenced by in vitro analysis. Comparison of saccharification potential of Conso,tiurn Enzyme Preparation (CEP) from GR-RS-T with commercial cellulase blend (CCB) on biologically pretreated rice straw (Spent rice straw) revealed a much higher release by CCB as compared to CEP indicating the supreme potential of the former which has been designed and developed for commercial applications. SEM image of SRS control showed prominent holes and cracks on its surface as compared to the intact strucrure of URS control indicating the delignification of rice straw and loosening of its structure during the growth of P!eurotus sp on rice straw. The structure of URS and SRS after saccharification showed prominently higher level of disorganisation when CCB was used as compared to CEP which cotTelated with the release of reducing sugars from the substrates. The release of reducing sugars in case of SRS-CEP ( 175 mg/gm) was comparable to, in fact slightly higher than URS-CCB (158 mg/gm) indicating that the saccharification potential of CEP could be made comparable to CCB if biologically pretreated rice straw was used. The outcome of this research contributes to the growing interest in lignocellulose deconstruction by enzymes, and highlights the potential of biological pretreatment in enhancing sacbharification by GHs.