Amaj Ahmed Laskar

Enzyme immobilization and molecular modeling studies on an organic–inorganic polypyrrole–titanium(IV)phosphate nanocomposite

In this work we report the synthesis of an electrically conductive polypyrrole–titanium(IV)phosphate (PPy–TiP) nanocomposite using simple, facile in situ chemical oxidative polymerization of polypyrrole (PPy) in the presence of titanium(IV)phosphate (TiP) for immobilization of the yeast alcohol dehydrogenase enzyme. FTIR, FE-SEM and TGA were employed for the characterization of PPy, TiP and the PPy–TiP nanocomposite. YADH was successfully immobilized on the PPy–TiP nanocomposite with a loading efficiency of 69%. The immobilized YADH showed no change in the pH optima (pH 8.0) and there was a broadening of the peak both at acidic as well as in alkaline pH. The optimum temperature of immobilized YADH was increased by 5 °C with almost the same residual activity. Immobilized YADH showed improved thermal stability at 60 °C and retained about 71% activity after 5 h of incubation. Also, the immobilized YADH showed greater reusability and retained 75% activity after 10th successive use. All the results were compared to those of free YADH, immobilized YADH on PPy and immobilized YADH on TiP. The affinity of immobilized YADH for ethanol was decreased as evident from the Km value (223.71 mM) and also there was a decrease in the maximum velocity (201.53 μM min−1) as compared to soluble YADH. The improved residual activity, stability and reusability of YADH immobilized on the PPy–TiP nanocomposite make the enzyme more suitable for industry-based applications. Molecular docking was used to query the protein – the newly synthesized chemical entity interactions that help in understanding the affinity in enzyme activity.

Activation of Human Salivary Aldehyde Dehydrogenase by Sulforaphane: Mechanism and Significance.

Cruciferous vegetables contain the bio-active compound sulforaphane (SF) which has been reported to protect individuals against various diseases by a number of mechanisms, including activation of the phase II detoxification enzymes. In this study, we show that the extracts of five cruciferous vegetables that we commonly consume and SF activate human salivary aldehyde dehydrogenase (hsALDH), which is a very important detoxifying enzyme in the mouth. Maximum activation was observed at 1 μg/ml of cabbage extract with 2.6 fold increase in the activity. There was a ~1.9 fold increase in the activity of hsALDH at SF concentration of ≥ 100 nM. The concentration of SF at half the maximum response (EC50 value) was determined to be 52 ± 2 nM. There was an increase in the Vmax and a decrease in the Km of the enzyme in the presence of SF. Hence, SF interacts with the enzyme and increases its affinity for the substrate. UV absorbance, fluorescence and CD studies revealed that SF binds to hsALDH and does not disrupt its native structure. SF binds with the enzyme with a binding constant of 1.23 x 107 M-1. There is one binding site on hsALDH for SF, and the thermodynamic parameters indicate the formation of a spontaneous strong complex between the two. Molecular docking analysis depicted that SF fits into the active site of ALDH3A1, and facilitates the catalytic mechanism of the enzyme. SF being an antioxidant, is very likely to protect the catalytic Cys 243 residue from oxidation, which leads to the increase in the catalytic efficiency and hence the activation of the enzyme. Further, hsALDH which is virtually inactive towards acetaldehyde exhibited significant activity towards it in the presence of SF. It is therefore very likely that consumption of large quantities of cruciferous vegetables or SF supplements, through their activating effect on hsALDH can protect individuals who are alcohol intolerant against acetaldehyde toxicity and also lower the risk of oral cancer development.

Colorimetric method for the detection of melamine using in-situ formed silver nanoparticles via tannic acid

Melamine toxicity has recently attracted worldwide attention as it causes renal failure and the death of humans and animals. Therefore, developing a simple, fast and sensitive method for the routine detection of melamine is the need of the hour. Herein, we have developed a selective colorimetric method for the detection of melamine in milk samples based upon in-situ formation of silver nanoparticles (AgNPs) via tannic acid. The AgNPs thus formed were characterized by UV-Visible spectrophotometer, transmission electron microscope (TEM), zetasizer and dynamic light scattering (DLS). The AgNPs were used to detect melamine under in vitro condition and in raw milk spiked with melamine. Under optimal conditions, melamine could be selectively detected in vitro within the concentration range of 0.05-1.4μM with a limit of detection (LOD) of 0.01μM, which is lower than the strictest melamine safety requirement of 1ppm. In spiked raw milk, the recovery percentage range was 99.5-106.5% for liquid milk and 98.5-105.5% for powdered milk. The present method shows extreme selectivity with no significant interference with other substances like urea, glucose, glycine, ascorbic acid etc. This assay method does not utilize organic cosolvents, enzymatic reactions, light sensitive dye molecules and sophisticated instrumentation, thereby overcoming some of the limitations of the other conventional methods.

In vitro activity and stability of pure human salivary aldehyde dehydrogenase

Human salivary aldehyde dehydrogenase (HsALDH) appears to be the first line of defence against toxic aldehydes contained in exogenous sources and is important for maintaining healthy oral cavity and protection from oral cancer. Here, the activity and stability of purified hsALDH has been determined under different conditions such as temperature, in presence of denaturants [Urea and guanidine hydrochloride (GnHCl)] and in the presence of salt (NaCl). The pure enzyme exhibited low stability when stored at room temperature (25°C) as well as at low temperature (4°C). 10% glycerol significantly improved its storage stability, particularly at 25°C. HsALDH was observed to have very low thermal stability. At higher temperatures, the enzyme gets unfolded and loses its activity quite rapidly. Further, the enzyme is unstable in the presence of denaturants like urea and GnHCl which unfold the enzyme. Salt (NaCl) has an activating effect on the enzyme, resulting from perhaps due to some conformational changes in the enzyme which facilitates the catalysis process. HsALDH proved to be a labile enzyme under in vitro conditions and certain additives like glycerol and NaCl can improve the activity/stability of the enzyme. Hence, a stabilizing agent is required to use the enzyme in in vitro studies.

Thymoquinone binds and activates human salivary aldehyde dehydrogenase: Potential therapy for the mitigation of aldehyde toxicity and maintenance of oral health

Human salivary aldehyde dehydrogenase (hsALDH) is a very important anti-oxidant enzyme present in the saliva. It is involved in the detoxification of toxic aldehydes and maintenance of oral health. Reduced level of hsALDH activity is a risk factor for oral cancer development. Thymoquinone (TQ) has many pharmacological activities and health benefits. This study aimed to examine the activation of hsALDH by TQ. The effect of TQ on the activity and kinetics of hsALDH was studied. The binding of TQ with the enzyme was examined by different biophysical methods and molecular docking analysis. TQ enhanced the dehydrogenase activity of crude and purified hsALDH by 3.2 and 2.9 fold, respectively. The Km of the purified enzyme decreased and the Vmax increased. The esterase activity also increased by 1.2 fold. No significant change in the nucleophilicity of the catalytic cysteine residue was observed. TQ forms a strong complex with hsALDH without altering the secondary structures of the enzyme. It fits in the active site of ALDH3A1 close to Cys 243 and the other highly conserved amino acid residues which lead to enhancement of substrate binding affinity and catalytic efficiency of the enzyme. TQ is expected to give better protection from toxic aldehydes in the oral cavity and to reduce the risk of oral cancer development through the activation of hsALDH. Therefore, the addition of TQ in the diet and other oral formulations is expected to be beneficial for health.

In vitro DNA binding studies of therapeutic and prophylactic drug citral

The study of drug-DNA interactions is of great importance, as it paves the way towards the design of better therapeutic agents. Here, the interaction of DNA with a therapeutic and prophylactic drug citral has been studied. We have attempted to ascertain the mode of binding of citral with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of the UV-visible absorbance spectra and fluorescence spectra indicated the formation of a complex between citral and Ct-DNA. Competitive binding assays with ethidium bromide (EB), acridine orange (AO) and Hoechst 33258 reflected that citral possibly intercalates within the Ct-DNA. These observations were further confirmed by circular dichroism (CD) spectral analysis, viscosity measurements, DNA melting and molecular docking studies. This study is expected to contribute to a better understanding of molecular mechanisms of citral, and design of new drugs in the future.

Induced mutation analysis with biochemical and molecular characterization of high yielding lentil mutant lines

Induced mutagenesis generates macromolecular variations which ultimately alters the bio-physiological and morphological nature of the crop genotypes. In the present study, molecular characterization of six high yielding lentil mutant lines, developed from hydrazine hydrates (HZ) and gamma rays mutagenesis, was carried out with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and random amplified polymorphic DNA (RAPD) markers. Activity of nitrate reductase (NR) and content of chlorophyll and carotenoid were found to be significantly high in the mutant lines. Protein and mineral (Fe, Zn & Cu) contents were also increased considerably in the mutant lines compared to their respective parent genotypes. SDS-PAGE profile of seed storage proteins showed 35 unique bands with 97.14% polymorphism. Genetic divergence analysis generated total 41 reproducible RAPD bands with average calculated polymorphic percentage of 63.06%. Among the primers, OPA-10 showed the highest polymorphism with significant PIC value. Genetic divergent analysis revealed that genome of cultivar DPL 62 mutated relatively more than the cultivar Pant L 406 due to the mutagen treatments, while DPL 62-B and Pant L406-A were the most divergent mutants induced in the present study. Biochemical and molecular profile of the induced mutant lines facilitates a basis for future conservation and utilization strategies to widen the genetic base of the current lentil breeding population.

Hepatoprotective Action of Thymoquinone

Nigella sativa (black cumin) seeds and its principal bioactive component, thymoquinone (TQ), have been used for centuries in folk medicine because of their exceptional healing power to a wide range of diseases. Nigella sativa plant has been regarded as nature’s miracle herb. The black cumin seeds have been used for the treatment of wide range of diseases including diabetes, infections, skin diseases, hypertension, cancer, asthma, gastric ulcers, liver diseases, etc. and are consumed regularly for the general well-being. The pharmacological properties possessed by TQ are anticancer, antidiabetic, antioxidant, anti-inflammatory, antimicrobial, gastroprotective, immunomodulatory, cytoprotective, hepatoprotective activity, etc. The hepatoprotective activity of TQ has been studied extensively in many in vitro and in vivo studies. It has been found to be an excellent natural protective agent against hepatotoxicity induced by several chemicals and drug molecules and the liver injury and liver diseases in experimental animal models. The antioxidant capacity of TQ was found to play a major role in the hepatoprotection. Therefore, TQ is a potential natural remedy to protect the liver from many toxic agents and drugs and for the treatment of liver diseases.