Browsing by Author "Sharma, Pankaj"

Now showing 1 - 12 of 12

Biochemical characteristics of a novel vegetative tissue geraniol acetyltransferase from a monoterpene oil grass (Palmarosa, Cymbopogon martinii var. Motia) leaf
(Elsiever, 2013-04) Sharma, Pankaj
Plants synthesize volatile alcohol esters on environmental insult or as metabolic induction during flower/fruit development. However, essential oil plants constitutively produce them as the oil constituents. Their synthesis is catalyzed by BAHD family enzymes called alcohol acyltransferases (AATs). However, no AAT has been characterized from plant foliage synthesizing acyclic monoterpenoids containing essential oils. Therefore, we have purified and biochemically characterized a geraniol: acetyl coenzyme A acetyltransferase (GAAT) from Palmarosa aroma grass (Cymbopogon martinii) leaf. MALDI-assisted proteomic study of the 43 kDa monomeric enzyme revealed its sequence motif novelties e.g. relaxed conservation at Phe and Trp in DFGWG’. This suggests permissiveness of variations in the conserved motif without loss of catalytic ability. Also, some new conserved/semi-conserved motifs of AATs were recognized. The GAAT kcat/Km values (300–700 M−1 s−1) were low (a generic characteristic for secondary metabolism enzyme) but higher than those of some floral AATs. Wide substrate acceptability for catalyzing acetylation of diverse primary alcohols (chain of ≥C6) implied its catalytic description as a ‘primary aliphatic alcohol acetyltransferase’. It signifies metabolic ability to deliver diverse aroma esters, should the acceptor alcohols be available in planta. To our knowledge, this is the first report of detailed kinetics of a vegetal monoterpenol acyltransferase
Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plant
(Wiley, 2007) Sharma, Pankaj
Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH ~3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efﬁcient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide proﬁling. The process may also be helpful in the reliable analysis of other acidic plant tissues
Coherent ontogenic dynamics of geraniol acetyltransferase activity and geranyl acetate concentration in flowers and leaves of aroma grass Cymbopogon martinii var. Motia
(Springer, 2009) Sharma, Pankaj
We have investigated the correlation between the concentration of geranyl acetate (GA) and acetyl CoA: geraniol acetyltransferase (GAAT) activity in palamarosa (Cymbopogon martini var. Motia) inflorescence and leaves at their different physiological stages. The results on GA concentration and the GAAT activity have been expressed on per gram fresh weight, per spikelet pair or leaf and per unit area of the phylloplane also incase of leaf. The percentage of geranyl acetate and geraniol in the volatile oil has also been considered. GA concentration was found to be highest in unopened floral spikelets and on the decline in fully open spikelets matching the trend of GAAT activity. Similarly, highest concentration of GA and maximum GAAT activity were found in leaves at mid-stage of development (stage II). The regression analysis curve between GA concentration (mg gFw−1) and GAAT activity (IU 10−3 gFw−1) gave an estimate of correlation coefficient (at 95% confidence) value of 0.79 for flowers and 0.92 for leaf. The results suggest that volatile ester (like geranyl acetate) synthesis in foliage and flowers of the aroma oil plant is controlled by the existent catalytic levels GAAT rather than the availability of geraniol. The study also indicates that the GAAT to be a good target to over-express for improvement of oil quality in terms of GA linked to fruit-fresh olfactory note of the oil.
Combining Diverse Pedagogical Tools for Effective Teaching of General Biology to a Large Class Comprising of First Year Science and Engineering Undergraduates
(Divya Enterprise, 2017-12) Sharma, Pankaj
Getting students interested in learning science and opting for a science-based career has always been a dream, despite the associated challenges in the form of dropout and attenuated student interest, for the university teacher. Secondly, in college or university academic curriculum, large classes often become a necessity to ensure uniformity in concept delivery. As a consequence, the best teacher strives to become something akin to an all-rounder, where the huge diversity concomitant with large crowds (classes) needs to be addressed to succeed. The present work was carried out to tackle the above challenges by introducing certain innovations in the teaching methodology. In the present study, the author experimented with the different fields from which analogies could be picked up to deliver relevant biological concepts; other pedagogical tools used were variety in language usage; student-teacher distance; colourful and pictorial slides with least text, musical concept summaries, drama, story, humor; interruption in lecture delivery was often done by asking questions of higher Bloom's taxonomy levels from the students, and allowing them time for peer discussion. Clapping for correct answers was encouraged to break the monotony of sitting sedentarily in class. Analogy pertaining to a real life inspiring figure was deemed the most effective by student community. Among other pedagogical tools, questions from previous years’ exam papers were most effective. It was also realized that timing of the non-academic element (such as story or music) was important for effective lectures. The present study, although simple and small in scale, could help guide further classroom innovations where introduction of variety in the traditional lecture mode in biology (science) classes is the ultimate goal.
Comparative analysis of antioxidant and antiproliferative activities of crude and purified flavonoid enriched fractions of pods/seeds of two desert legumes Prosopis cineraria and Cyamopsis tetragonoloba
(Elsiever, 2021-06) Sharma, Pankaj
Cyamopsis tetragonoloba and Prosopis cineraria are two legumes of the semi-arid region of Indian subcontinent which are unexplored with respect to their medicinal potential. Moreover, there is considerable lack in the comparative analysis of the biological properties of crude and enriched fractions obtained from the pods and seeds. Therefore, this study aims in investigating the effect of purification on the antioxidant and anticancerous activities of the extracts from the two legumes. This is the first study to purify an enriched methanolic fraction using Amberlite XAD7HP column chromatography followed by analysis using Thin Layer Chromatography. This matrix provided an economic and time efficient isolation of flavonoids and isoflavonoids from the seeds and pods of the above mentioned legumes. In addition, antioxidant activity carried out using DPPH assay showed that purification process did not contributed to enhanced antioxidant potential. However, inverse results were obtained during anticancerous activity assay on Huh-7 cell lines.
Geranyl acetate esterase is commonly present but linalyl acetate esterase occurrence is highly limited in plants
(Pascal Francis, 2007) Sharma, Pankaj
Identification of an isoflavone glycoside hydrolyzing-glucosidase in the seeds of the desert legume Prosopis cineraria
(Divya Enterprise, 2018) Sharma, Pankaj
Legumes, in general, are rich in glycosidicisoflavone conjugates, wherebyspecific β-glucosidases act to release the free aglycones that could serve in plant-microbe interactions and defense. Prosopis cineraria is an important legume tree, bearing medicinally useful and edible pods, generally growing in extreme arid/semi-arid zones of Rajasthan. However, specific phytochemical-cum-metabolic profiles are lacking for the same. Therefore, the present investigation undertook phytochemical and metabolic screening of the pods/seeds of P. cineraria for the presence of putative isoflavonoids viz. genistein and daidzein, their glycosides and β-glucosidase(s) capable of catalyzing the glycoside hydrolysis. Extraction and identification of these two aglycone forms of the above isoflavonoids were performed with solvent partition chromatography and Fluorescent/High Performance Thin Layer Chromatography, respectively. Furthermore, optimization of the isoflavone conjugate-specific β-glucosidase activity with respect to pH optima, temperature and time were carried out. The partially purified enzyme showed a temperature optima of 50°C and pH optima of 4.5. The enzyme also demonstrated activity towards natural substrates daidzin and genistin which are glycosides of isoflavonoids daidzein and genistein respectively. The methanolic extracts of the seeds of P. cineraria indicated the presence of related isoflavonoids which needs to be further validated.
Nonlinear Optimization of Enzyme Kinetic Parameters
(Science Alert, 2008) Sharma, Pankaj
In the analysis of enzyme kinetics data, Km and Vmax play a very important role. Linearization of kinetic equation is still a common practice for determining these parameters. Although graphical methods help in understanding the kinetic behavior of enzymes, they have certain shortcomings associated with them due to which they sometimes lead to an anomalous estimation of the kinetic parameters. In order to yield a more accurate estimate of parameters, minimization of least square error can be quite useful. However, since the least square error determination is a non linear function, the usual methods may not be fruitful. This research recommends the use of two simple and fast evolutionary optimization techniques such as Genetic Algorithms (GA) and Particle Swarm Optimization (PSO) which may be applied for the determination of Michaelis Menten (MM) enzyme analysis. We have shown the working of these methods on a set of six enzymes taken from literature along with a unique enzyme, geraniol acetyltransferase (GAAT), purified from the aromatic grass palmarosa. The entire study shows that GA and PSO can be used efficiently for determining accurate values for Km and Vmax.
Purification and characterization of an isoflavones conjugate hydrolyzing β-glucosidase (ICHG) from Cyamopsis tetragonoloba (guar)
(Elsiever, 2019-12) Sharma, Pankaj
A β-glucosidase with high specific activity towards isoflavone glycosidic conjugates was purified from seeds of Guar (Cyamopsis tetragonoloba) by ammonium sulphate precipitation followed by size exclusion and ion exchange chromatography. The pH and temperature optima of the purified Isoflavones conjugate hydrolyzing β-glucosidase (ICHG) were found to be pH 4.5 and 37 °C, respectively. The enzyme was relatively stable at higher temperatures. Effect of different divalent metal ions was studied and it was found that Cobalt and Mercury ions completely inhibited the enzyme activity. Km and Vmax of the purified isoflavones conjugates hydrolyzing β-glucosidases (ICHG) was 0.86 mM and 6.6 IU/mg respectively. The enzyme was most likely a trimer (approximate Mr 150 kDa) with potential subunits of 50 kDa. The purified enzyme showed activity against isoflavone conjugate glycosides viz daidzin and genistin but was inactive towards other flavonoid conjugates. The product conversion was confirmed by HPTLC and HRMS analysis. The MALDI-TOF analysis of the ICHG showed a score greater than 78 with 20 matches in MASCOT software. The five resultant peptides obtained had highest similarity in sequence with β-glucosidase from Cicer arietinum. The β-glucosidase from the C. arietinum has also been reported to exhibit the isoflavone conjugate hydrolyzing properties thus confirming the nature of the enzyme purified from the Guar seeds.
Release of iron from ferritin by metabolites of benzene and superoxide radical generating agents
(Elsiever, 2001) Sharma, Pankaj
The release of iron from ferritin in the presence of benzene metabolites, viz. phenol (P), catechol (CT), hydroquinone (HQ) and superoxide radical generating compounds, viz. pyrogallol (PL), phloroglucinol (PG), phenylhydrazine (PH) or phenylenediamine (PD) was studied in acetate buffer, pH 5.6. Monitoring the formation of the iron–ferrozine complex quantitated the release of iron from ferritin. The presence of P (125 μM) did not result in the release of iron from ferritin, whereas the same concentration of CT, HQ, PL, PH or PD resulted in the release of significant amounts of iron from ferritin and a marginal amount of iron in the presence of PG, CT, HQ, PL, PH or PD concentration and time-dependent increase in iron release from ferritin were observed although the increase was not linear as a function of time and concentration of the compounds studied. The presence of superoxide dismutase inhibited significantly the release of iron from ferritin by CT, HQ, PL, PH or PD. The iron released from ferritin by CT, HQ, PL, PH or PD enhanced lipid peroxidation in rat brain homogenate and released aldehydic products from bleomycin-dependent degradation of DNA and also caused single strand nicks to pUC18 DNA. These studies indicate that CT and HQ, the two principal polyphenolic metabolites of benzene and PL, PH or PD, the superoxide radical generating compounds were capable of reducing ferric iron from ferritin and also mobilizing and releasing iron from ferritin core. The release of iron from ferritin by these compounds is a result of direct reduction of ferritin iron by electron transfer and also reduction via superoxide radical. The release of iron from ferritin by CT and HQ may have toxicological implications in relation to benzene toxicity. The release of iron by superoxide radical generating agents suggests that oxidative stress may play a role as this could lead to disruption of intracellular iron homeostasis.
Trichloroacetic acid-facilitated coenzyme A-SH-stabilization-based end-point spectrophotometric 5,5 -dithiobis-2-nitrobenzoic acid assay for plant terpene alcohol acetyltransferases
(Elsiever, 2005) Sharma, Pankaj
Acyltransferases catalyze critical acylation of metabolic intermediates in metabolic pathways and are important in metabolome diversiWcation in secondary metabolism. Terpenes form the largest and most diverse Wed group of secondary metabolites (>35,000 compounds). Terpene alcohol acetyltransferases (AATs)1 are responsible for biogeneration of volatile esters that attribute unique and Wne olfactory aromas to herbs, Xowers, fruits, and foods. AATs catalyze transfer of an acyl group from acetyl/acyl coenzyme A to an alcohol, stoichiometrically generating ester and coenzyme A-SH (CoASH). Recently, the signiWcance of AATs in the Xavor and fragrance of phytogenic foods, drinks, and aromatic oils and in developmental physiology of Xowers and fruits has attracted their biochemical characterization in plants [1–5] for metabolic engineering initiatives. Radioactivity [5] or GC-based quantitation [1,3,5] of ester formed by the reaction are employed to monitor the enzyme activity.
Water quality management of a stretch of river Yamuna: An interactive fuzzy multi-objective approach
(Springer, 2006-07-08) Singh, Ajit Pratap; Sharma, Pankaj
This paper presents an Interactive Fuzzy Multi-Objective Linear Programming (IFMOLP) model for water quality management in a river basin. The IFMOLP model formulated will first evaluate dissolved oxygen (DO) concentrations or DO deficits at a point in different reaches depending on the overall Biochemical Oxygen Demand (BOD) concentration present in the respective drain. Subsequently, the model incorporates the aspirations and conflicting objectives of the decision maker (DM) by taking into consideration the aspects relevant for pollution control boards as well as dischargers responsible for generating wastewater. The uncertainty associated with specifying the water quality criteria (based on DO concentration or DO deficit) and treatment cost to remove pollution level is incorporated by interacting the decision maker. In this process DM is asked to specify the reference aspiration levels of achievement for the values of all membership functions generated with respect to each objective. This provides flexibility for the pollution control authorities and dischargers to specify their aspirations. IFMOLP model developed herein is then used in a case study for the evaluation of optimal BOD removal in different drains located across the river Yamuna at New Delhi, India. The presented model will simulate the allocation of waste load efficiencies with satisfactory results which will indicate usefulness of the model in managing more complex river basins along with better flexible policies of water management.