Chhabra, Meenu

2012-10-23, Garg, Neha, Bieler, Nora, Kenzom, Tenzin, Chhabra, Meenu, Ansorge-Schumacher, Marion, Mishra, Saroj

Background: Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase.Results: In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac.Conclusion: Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L-1 in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation. © 2012 Garg et al.; licensee BioMed Central Ltd.

2017-01-01, Vyas, Sachin, Chhabra, Meenu

Oleaginous yeast closely related to Cystobasidium oligophagum was isolated from soil rich in cellulosic waste. The yeast was isolated based on its ability to accumulate intracellular lipid, grow on carboxymethylcellulose (CMC) and produce lipase. It could accumulate up to 39.44% lipid in a glucose medium (12.45 ± 0.97 g/l biomass production). It was able to grow and accumulate lipids (36.46%) in the medium containing CMC as the sole carbon source. The specific enzyme activities obtained for endoglucanase, exoglucanase, and β-glucosidase were 2.27, 1.26, and 0.98 IU/mg respectively. The specific enzyme activities obtained for intracellular and extracellular lipase were 2.16 and 2.88 IU/mg respectively. It could grow and accumulate lipids in substrates including glycerol (42.04%), starch (41.54%), xylose (36.24%), maltose (26.31%), fructose (24.29%), lactose (21.91%) and sucrose (21.72%). The lipid profile of the organism was suitable for obtaining biodiesel with desirable fuel properties.

2015-04-01, Chhabra, Meenu, Mishra, Saroj, Sreekrishnan, Trichur Ramaswamy

Abstract Background: Laccases have good potential as bioremediating agents and can be used continuously in the immobilized form like many other enzymes. Methods: In the present study, laccase from Cyathus bulleri was immobilized by entrapment in Poly Vinyl Alcohol (PVA) beads cross-linked with either nitrate or boric acid. Immobilized laccase was used for dye decolorization in both batch and continuous mode employing a packed bed column. The products of degradation of dye Acid Red 27 were identified by LC MS/MS analysis. Results: The method led to very effective (90%) laccase immobilization and also imparted significant stability to the enzyme (more than 70% after 5 months of storage at 4°C). In batch decolorization, 90-95% decolorization was achieved of the simulated dye effluent for up to 10-20 cycles. Continuous decolorization in a packed bed bioreactor led to nearly 90% decolorization for up to 5 days. The immobilized laccase was also effective in decolorization and degradation of Acid Red 27 in the presence of a mediator. Four products of degradation were identified by LC-MS/MS analysis. Conclusions: The immobilized laccase in PVA-nitrate was concluded to be an effective agent in treatment of textile dye effluents.

2024, Ritesh Mishra, Sushma Jangra, Abhijit Mishra, Shikha Pandey, Prakash, Ram, Chhabra, Meenu

The effect of non-equilibrium cold plasma (NECP) treatment on functional and nutritional properties of finger millet flour was investigated. Finger millet flour was treated at two different exposure times for 5 min and 10 min, and functional and nutritional properties such as Water and oil holding capacity, water binding capacity, color, and dispersibility of the control and plasma treated flour were studied. There was no significant difference in the color intensity and the whitening index (WI) with the treatment. The functional properties such as water holding capacity (WHC), oil absorption capacity (OAC), foaming and emulsifying capacity have shown significant increment with treatment time. FTIR and DSC results depicted the depolymerization of starch in the treated flour. Plasma treatment was observed to enhance the functional properties of the finger millet flour, whilst the physical properties remained unchanged. Overall plasma treatment can be explored for development of a process to enhance functionality of finger millet flour for applications in food systems. There remains plenty of scope for commercial level expansion.

2018-01-01, Khandelwal, Amitap, Vijay, Ankisha, Dixit, Ambesh, Chhabra, Meenu

In this study, a promising microbial fuel cell (MFC) system has been developed, wherein algae is cultivated in the cathode chamber, algae biomass is harvested and lipids are extracted. The lipid extracted algal (LEA) biomass was then used as an electron donor substrate. The performance of MFCs fed with LEA biomass was compared with that of fruit waste fed MFCs (FP-MFCs), wherein LEA-fed MFC was superior in all aspects. Power density of 2.7 W m−3 was obtained by LEA-fed MFCs which is 145% and 260% higher than FP MFC and control MFC respectively. The volumetric algae productivity of 0.028 kg m−3 day−1 in cathode chamber was achieved. The system was able to generate 0.0136 kWh Kg−1 COD day−1 of electric energy and 0.0782 kWh m−3 day−1 of algal oil energy. The proposed system is a net energy producer which does not rely heavily on the external supply of electron donor substrates.

2015-01-01, Chhabra, Meenu, Mishra, Saroj, Sreekrishnan, Trichur Ramaswamy

Textile effluent is characterized by high colour, chemical oxygen demand and conductivity due to presence of a large number of recalcitrant compounds. The effluent is often discharged without an effective treatment leading to contamination of water bodies. In the present study, a combination of mediator assisted laccase and chemical treatment was used for decolorization of effluent from a local textile mill in a continuous enzyme membrane reactor (EMR). Treatment of the effluent first with laccase and ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) lead to about 60% decolorization but was accompanied by membrane fouling. Addition of alum further coagulated 90% of the residual colour but the process was associated with dye sludge formation and ABTS could not be recovered from the treated effluent. Reversal of the treatment sequence was effective in that 85% decolorization was achieved in the EMR and the process could be operated for over a period of 15 days. No sludge formation was noticed and membrane fouling was negligible. Most importantly, about 60% ABTS could be recovered from the treated effluent. Analysis of the treated effluent by mass spectrometery indicated extensive breakdown of the dye molecules by laccase and ABTS and the breakdown products were neither toxic nor mutagenic as assessed by measurement of the oxygen consumption rate and the standard Ames test.

2016-04-01, Vijay, Ankisha, Vaishnava, Monika, Chhabra, Meenu

Microbial fuel cells (MFCs) are emerging wastewater treatment systems with a proven potential for denitrification. In this study, we have developed a high-rate denitrifying MFC. The anode consisted of cow manure and fruit waste and the cathode consisted of cow manure and soil. The initial chemical oxygen demand (COD)/nitrate nitrogen (NO3−-N) was varied from 2 to 40 at the cathode while keeping the anode ratio fixed at 100. NO3−-N removal rate of 7.1 ± 0.9 kg NO3−-N/m3 net cathodic compartment (NCC)/day was achieved at cathode COD/NO3−-N ratio 7.31 with the current density of 190 ± 9.1 mA/m2 and power density of 31.92 ± 4 mW/m2 of electrode surface area. We achieved an open-circuit voltage (OCV) of 410 ± 20 mV at initial cathodic NO3−-N of 0.345 g/l. The cathode COD/NO3−-N ratio had a significant influence on MFC’s OCV and nitrate removal rate. Lower OCV (<150 mV) and NO3−-N removal rates were observed at COD/NO3−-N ratio >12 and <7. Experiments done at different cathode pH values indicated that the optimum pH for denitrification was 7. Under optimized biochemical conditions, nitrate removal rate of 6.5 kg NO3−-N/m3 net cathodic compartment (NCC)/day and power density of 210 mW/m2 were achieved in a low resistance MFC. The present study thus demonstrates the utility of MFCs for the treatment of high nitrate wastes.

2018-05-01, Vijay, Ankisha, Arora, Shivam, Gupta, Sandeep, Chhabra, Meenu

In this study, Microbial Fuel Cell (MFC) capable of treating saline starch water was developed. Sodium chloride (NaCl) concentrations ranging from 500 mM to 3000 mM were tested at the anode. Nitrate was used as an electron acceptor at the biocathode. The halophilic bacteria were isolated from Sambhar Lake, India. Results indicated successful removal of starch (1.83 kg/m3-d) and nitrate (0.13 kg/m3-d NO3−-N) with concomitant power output of 207.05 mW/m2 at 1000 mM NaCl concentration. An increase in power density from 71.06 mW/m2 to 207.05 mW/m2 (2.92 folds) was observed when NaCl concentration was increased from 500 mM to 1000 mM. A decline in power density was observed when the salt concentrations >1000 mM were used. Concentration of 3000 mM supported power output as well as the highest starch degradation (3.2 kg/m3-d) and amylase activity of 2.26 IU/ml. The halophilic exoelectrogens were isolated and identified. The present study demonstrates the utility of MFC for degrading starch in saline water.

2015-01-01, Kumar, Deepak, Pal, Samanwita, Harinipriya, S., Chhabra, Meenu

Spectroelectrochemicalinvestigation of racemic alanine on poly crystalline metal surfaces was studied in 1M LiClO4 medium, to achieve cost effective separation of biologically important molecules from the racemic mixture. Analysis of the FTIR spectrum of racemic alanine in 1M LiClO4 indicated degradation of the same. Upon prolonged exposure in LiClO4, byproducts from degradation of alanine increasedin concentration as also observed from UV-Vis spectroscopy. Cyclic Voltammetry studies unraveled different adsorption behaviors on different metal surfaces for the racemic and the enantiomeric solution of alanine. Further FTIR analysis of the alanine solution after Cyclic Voltammetry revealed possible presence of cyanide (-C=N) and terminal alkyne as byproducts. As predicted by Monte Carlo simulations earlier, Circular Dichroism implied the dextro rotation of the racemic alanine solution after Cyclic Voltammetry. These results support the preferential adsorption of L-alanine over D-alanine from racemic mixture that could be attributed to the umbrella inversion of the molecule after adsorption on the metal surface in presence of applied electric field in the LiClO4 environment. A plausible mechanism for the formation of terminal alkyne and - C=N byproducts had also been attempted.

2024, Arti Sharma, Prachi Nawkarkar, Vikas U. Kapase, Chhabra, Meenu, Shashi Kumar

Microalgal biorefineries have emerged as significant reservoirs of therapeutic compounds, including pigments and proteins. Facilitating a robust circular bioeconomy necessitates the augmentation of pigment synthesis alongside algae biofuel production. Nevertheless, inherent constraints in ketocarotenoid synthesis exist in naturally fast-growing microalgae strains, such as Chlamydomonas reinhardtii. To address this limitation, we overexpressed two pivotal enzymes in the carotenoid biosynthetic pathway, namely β-carotene hydroxylase (crt) and β-carotene ketolase (bkt), in C. reinhardtii utilizing strong promoters to amplify carotenoid production. The genetically modified (GM) microalgae were validated through PCR, Southern hybridization, and Western blot assays, confirming the presence and expression of both genes in the C. reinhardtii strains. These GM lines exhibited a substantial enhancement over wild-type (WT) algae, showcasing a remarkable 5.39-fold increase in β-carotene concentration and twofold increase in total carotenoids compared to the WT microalgae. Notably, the GM microalgae achieved astaxanthin production up to 1.47 ± 0.063 mg/g DCW, a compound absent in WT C. reinhardtii. These findings indicate the successful functionalization of Hematococcus pluvialis genes through nuclear expression in C. reinhardtii, facilitating ketocarotenoid production. This study presents a valuable strategy to boost carotenoid production in microalgae by stable overexpression of two heterologous genes within the nuclear genome of C. reinhardtii. Graphical abstract: (Figure presented.) Graphical abstract for the study carried out which represents the in silico plasmid vector designing, algae transformation by electroporation, selection on antibiotic plates, PCR amplification for GM confirmation, Southern hybridization to confirm gene integration, Western blotting to check protein expression, pigment quantification, and algae growth determination.