Baljit Kaur

Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain

A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant 64, when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol.

Proteome-based profiling of hypercellulase-producing strains developed through interspecific protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis.

Thirty heterokaryons, formed by protoplast fusion of Aspergillus nidulans and Aspergillus tubingensis, were selected on the basis of their ability to grow on 2-deoxyglucose (0.2xa0%, w/v) and intermediate spore color. These heterokaryons were studied for cellulase production using shake flask and solid substrate cultures at 40xa0°C. Fusants 51 and 28 exhibited appreciably higher levels of endoglucanase, cellobiohydrolase, β-glucosidase, and FPase activities when compared with parental strains. Employing proteomic-based approaches, the differential expression of proteins in secretome of fusants and parental strains were analyzed using two-dimensional electrophoresis. The expression of some of the proteins in the fusants was found to be up/downregulated. The upregulated proteins in the fusant 51 were identified by liquid chromatography–mass spectroscopy as endoxylanase, endochitinase, β-glucosidase, as well as hypothetical proteins. The cellulases produced by fusants 28 and 51 showed improved saccharification of alkali treated rice straw when compared with the parental strains.

Elevated frequency of Micronuclei in uterine smears of cervix cancer patients

Abstract Epithelial scrapes of the cervix from patients with cervix cancer (n=30) and without the diseased condition (n=23) were processed for the micronucleus test. Statistical analysis of the data revealed significantly elevated frequency of micronucleated (MNd) cells in all the patients irrespective of the stage of cancer. The damage within the stage-types was however non-significant. Significantly elevated percent frequencies of MNd cells were also observed for pre-treated cervix cancer patients compared with the control data; for different ageat marriage groups; in aged patients; in the weaker socio-economic groups and in those with more child-bearing and/or total number of pregnancies; though within group differences were not always significant.

Evaluation of secretome of highly efficient lignocellulolytic Penicillium sp. Dal 5 isolated from rhizosphere of conifers

Penicillium sp. (Dal 5) isolated from rhizosphere of conifers from Dalhousie (Himachal Pradesh, India) was found to be an efficient cellulolytic strain. The culture under shake flask on CWR (cellulose, wheat bran and rice straw) medium produced appreciably higher levels of endoglucanase (35.69U/ml), β-glucosidase (4.20U/ml), cellobiohydrolase (2.86U/ml), FPase (1.2U/ml) and xylanase (115U/ml) compared to other Penicillium strains reported in literature. The mass spectroscopy analysis of Penicillium sp. Dal 5 secretome identified 108 proteins constituting an array of CAZymes including glycosyl hydrolases (GH) belonging to 24 different families, polysaccharide lyases (PL), carbohydrate esterases (CE), lytic polysaccharide mono-oxygenases (LPMO) in addition to swollenin and a variety of carbohydrate binding modules (CBM) indicating an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. Further, the culture extract was evaluated for hydrolysis of alkali treated rice straw, wheat straw, bagasse and corn cob at 10% substrate loading rate.

Mycothermus thermophilus (Syn. Scytalidium thermophilum): Repertoire of a diverse array of efficient cellulases and hemicellulases in the secretome revealed

Mycothermus thermophilus (Syn. Scytalidium thermophilum/Humicola insolens), a thermophilic fungus, is being reported to produce appreciable titers of cellulases and hemicellulases during shake flask culturing on cellulose/wheat-bran/rice straw based production medium. The sequential and differential expression profile of endoglucanases, β-glucosidases, cellobiohydrolases and xylanases using zymography was studied. Mass spectrometry analysis of secretome (Q-TOF LC/MS) revealed a total of 240 proteins with 92 CAZymes of which 62 glycosyl hydrolases belonging to 30 different families were present. Cellobiohydrolase I (17.42%), β glucosidase (8.69%), endoglucanase (6.2%), xylanase (4.16%) and AA9 (3.95%) were the major proteins in the secretome. In addition, carbohydrate esterases, polysaccharide lyases, auxiliary activity and a variety of carbohydrate binding modules (CBM) were identified using genomic database of the culture indicating to an elaborate genetic potential of this strain for hydrolysis of lignocellulosics. The cellulases from the strain hydrolyzed alkali treated rice straw and bagasse into fermentable sugars efficiently.

Tailoring the Substitution Pattern on 1,3,5-Triazine for Targeting Cyclooxygenase-2: Discovery and Structure–Activity Relationship of Triazine–4-Aminophenylmorpholin-3-one Hybrids that Reverse Algesia and Inflammation in Swiss Albino Mice

Here, we report analgesic and anti-inflammatory activity of a series of compounds obtained by appending 4-aminophenylmorpholin-3-one and acyclic, cyclic, or heterocyclic moieties on 1,3,5-triazine. The structures of compounds 4b and 6b are optimized for the best inhibition of COX-2 with IC50 values of 0.06 and 0.08 μM, respectively, and selectivity over COX-1 of 166 and >125, respectively. At the dose of 5 mg kg-1, these compounds significantly reduced acetic acid induced writhings, and their ED50 values were found to be 2.2 and 1.9 mg kg-1, respectively. Besides the cell-based and animal-based experiments showing the modes of action of these compounds targeting COX-2, the interaction behavior of 4b with COX-2 was also characterized, with physicochemical experiments including ITC, NMR, UV-vis, and molecular-modeling studies. Characteristically, these compounds interact with R120, Y355, and W385, the residues responsible for holding the substrate and mediating the process of electron transfer during the metabolic phase of the enzyme.

Rational modification of semaxanib and sunitinib for developing a tumor growth inhibitor targeting ATP binding site of tyrosine kinase

Analysis of the crystal structure of tyrosine kinase in complexation with an ATP analogue, supplemented with the molecular docking studies of semaxanib and sunitinib in the ATP binding site of the enzyme enabled us to make design of a series of tyrosine kinase inhibitors. The combination of pyrrole and indolinone in one molecule and placement of appropriate substituent thereof made the molecule compatible for the hydrophobic sub-pocket of the enzyme. Screening of the compounds over 60 cell line panel of human tumor cell lines identified compound 3a that exhibited GI50 35u202fnM and 63u202fnM against MCF7 and MDA-MB-468 cell lines of breast cancer.

Approaches for Bioprospecting Cellulases

Cellulases are industrially important enzymes with a market share of 500 million dollars that is expected to rise to 1.5 billion dollars by 2018. Cellulases play a crucial role in generating sugar feedstock for lignocellulosic based biorefinery platform. In addition, their demand in textile, paper, feed and food industries is rising steadily. However, these industrial applications require thermostable, catalytically highly efficient cellulases for making the processes commercially viable. Therefore, search and discovery for novel sources of cellulases is continued area of research. This chapter highlights some of the approaches for bioprospecting for novel cellulases.