Valorization of rice biomass by a green approach to release phenolic compounds and their antioxidant activities.

In the present context, we have assessed the green approach for the extraction of phenolics from agro-residues of rice viz., rice bran, and rice straw using water as an extracting solvent. The extraction was optimized with respect to time, temperature, pH, and solid (agro-residues) to liquid (water) ratio. The hydrolysates obtained were determined for phenolics and their antioxidant activities. The maximum total phenolic content (61.32 mg/100 g GAE), flavonoid content (13.19 mg/100 g QE), and tannin content (58.33 mg/100 g TAE) were obtained for rice bran followed by rice straw at pH 5, 1:20 (solid: liquid) for 10 min of extraction. Also, higher antioxidant properties (78.03% for DPPH, 86.45% for ABTS, and 0.85 absorbance at 700 nm for FRAP) were observed for the extracts of rice bran. Caffeic acid, gallic acid, p-coumaric acid, syringic acid, ferulic acid, 2,5-dihydroxy benzoic acid, kaemferol, quercetin, and epicatechin were analyzed by HPLC in both the rice biomass used. This study significantly converts rice biomass to antioxidative phenolic compounds under simple extraction conditions favoring the waste management process and also adding value to the waste biomass.

Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac.

An extracellular laccase enzyme secreted from Sphingobacterium ksn-11 was purified to electrophoretic homogeneity, showing a molecular weight of 90 kDa. The purified enzyme was monomeric in nature confirmed by sodium dodecyl gel electrophoresis. The optimum temperature and pH were found to be 40 °C and 4.5 respectively. The enzyme showed highest substrate specificity for 2,2 azino-bis (ethylthiozoline-6-sulfonate) (ABTS), followed by syringaldazine. The Km value for ABTS was 2.12 mM with a Vmax value of 33.33 U/mg which was higher when compared with syringaldazine and guaiacol substrates. Sodium azide and EDTA inhibited the activity by 30%, whereas presence of Ca2+ and iron increased activity by 50%. The purified enzyme was immobilized in sodium alginate-silicon dioxide-polyvinyl alcohol beads and evaluated for diclofenac transformation studies. LC-MS analysis confirmed that immobilized laccase transformed diclofenac to 4-OH diclofenac after 4 h of incubation. 45 % of diclofenac was able to transform even at 3rd cycle of immobilized laccase use. Therefore, immobilized laccase can be used to transform or degrade several recalcitrant compounds from industrial effluents.

Publisher Correction: Purification, Biochemical Characterization, and Facile Immobilization of Laccase from Sphingobacterium ksn-11 and its Application in Transformation of Diclofenac.

The original version of this article unfortunately contained a mistake in the equation under "Immobilized Laccase Activity and its Storage Stability" section.

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition.

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2 mM CaCO3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55 U/ml, 79.22 U/ml, 12.43 U/ml, 64.66 U/ml, and 21.12 U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.

Determination of quinalphos in human whole blood samples by high-performance thin-layer chromatography for forensic applications.

A simple and rapid procedure for the determination of quinalphos in human whole blood using liquid-liquid extraction and high-performance thin-layer chromatography was developed and validated. Seven different organic solvents were tested for optimum extraction of quinalphos from spiked blood samples. The effect of pH on the extraction yield of quinalphos was also examined. An average recovery of 93.61% was achieved from diethyl ether solvent at pH 3. Chromatographic separation was performed on silica gel 60F254 plates using mobile phase n-hexane-acetone in the ration 9:1 (v/v). Densitometric detection was carried out at 325 nm in absorbance mode. The interference of other organophosphorus pesticides of forensic relevance was not observed. The linear regression analysis in spiked whole blood samples resulted in linear calibration plot in the range 1 to 100 µg mL-1 with r2 = 0.9981. Sensitivity was represented by LLOQ at 1 µg mL-1. The within-day precision and between-day precision ranged from 0.18 to 1.04%, and 0.14 to 0.79% with an overall average recovery of 91.06% at three concentrations 1, 10, and 50 µg mL-1. No significant decrease in the concentration of quinalphos was observed for samples under different storage conditions. Finally, the developed procedure was applied to postmortem blood samples obtained in three fatal cases of poisoning by quinalphos.

Imidacloprid impedes mitochondrial function and induces oxidative stress in cotton bollworm, Helicoverpa armigera larvae (Hubner: Noctuidae).

Neonicotinoids have high agonistic affinity to insect nicotinic acetylcholine receptors (nAChR) and are frequently used as insecticides against most devastating lepidopteran insect pests. Imidacloprid influenced dose-dependent decline in the state III and IV respiration, respiration control index (RCI), and P/O ratios, in vitro and in vivo. The bioassay indicated its LD50 value to be 531.24 µM. The insecticide exhibited a dose-dependent inhibition on F0F1-ATPase and complex IV activity. At 600 µM, the insecticide inhibited 83.62 and 27.13% of F0F1-ATPase and complex IV activity, respectively, and induced the release of 0.26 nmoles/min/mg protein of cytochrome c. A significant dose- and time-dependent increase in oxidative stress was observed; at 600 µM, the insecticide correspondingly induced lipid peroxidation, LDH activity, and accumulation of H2O2 content by 83.33, 31.51 and 223.66%. The stress was the maximum at 48 h of insecticide treatment (91.58, 35.28, and 189.80%, respectively). In contrast, catalase and superoxide dismutase were reduced in a dose- and time-dependent manner in imidacloprid-fed larvae. The results therefore suggest that imidacloprid impedes mitochondrial function and induces oxidative stress in H. armigera, which contributes to reduced growth of the larvae along with its neurotoxic effect.

Evaluation of flubendiamide-induced mitochondrial dysfunction and metabolic changes in Helicoverpa armigera (Hubner).

Phthalic acid diamide insecticides are the most effective insecticides used against most of the lepidopteran pests including Helicoverpa armigera, a polyphagous pest posing threat to several crops worldwide. The present studies were undertaken to understand different target sites and their interaction with insect ryanodine receptors (RyR). Bioassays indicated that flubendiamide inhibited the larval growth in dose-dependent manner with LD50 value of 0.72 µM, and at 0.8 µM larval growth decreased by about 88%. Flubendiamide accelerated the Ca2+ -ATPase activity in dose-dependent trend, and at 0.8 µM, the activity was increased by 77.47%. Flubendiamide impedes mitochondrial function by interfering with complex I and F0 F1 -ATPase activity, and at 0.8 µM the inhibition was found to be about 92% and 50%, respectively. In vitro incubation of larval mitochondria with flubendiamide induced the efflux of cytochrome c, indicating the mitochondrial toxicity of the insecticide. Flubendiamide inhibited lactate dehydrogenase and the accumulation of H2 O2 , thereby preventing the cells from lipid peroxidation compared to control larvae. At 0.8 µM the LDH, H2 O2 content and lipid peroxidation was inhibited by 98.44, 70.81, and 70.81%, respectively. Cytochrome P450, general esterases, AChE, and antioxidant enzymes (catalase and superoxide dismutase) exhibited a dose-dependent increasing trend, whereas alkaline phosphatase and the midgut proteases, except amino peptidase, exhibited dose-dependent inhibition in insecticide-fed larvae. The results suggest that flubendiamide induced the harmful effects on the growth and development of H. armigera larvae by inducing mitochondrial dysfunction and inhibition of midgut proteases, along with its interaction with RyR.

Modulation of P-glycoprotein ATPase of Helicoverpa armigera by cholesterol: effects on ATPase activity and interaction of insecticides.

Purified P-glycoprotein ATPase from Helicoverpa armigera (Ha-Pgp), reconstituted in proteoliposomes composed of phospholipids and cholesterol, shows higher ATPase activity in the presence of cholesterol than in its absence. The Ha-Pgp ATPase activity was increased 30-40% with cholesterol. The KM for ATP was found to be 1 and 0.8 mM in the absence and presence of cholesterol, respectively. The insecticide-stimulated Ha-Pgp ATPase activity was increased by 10-20% for all the insecticides in the reconstituted proteoliposomes containing cholesterol compared to those with no cholesterol. The effects of cholesterol on KM and Vmax values of insecticide-stimulated Ha-Pgp ATPase activity were unrelated to the size of the insecticide. Ha-Pgp tryptophan fluorescence displayed a red shift of 3 and 8 nm in emission spectra upon binding of insecticides. Cholesterol enhances the interaction of insecticides with Ha-Pgp. Kd values of different insecticides for binding to Ha-Pgp were found to be lower in the presence of cholesterol in the proteoliposomes compared to its absence. Results suggest that cholesterol plays a role in the recognition and interaction of insecticides by modulating Ha-Pgp ATPase and may be involved in efflux of insecticides from cells by the transporter.

Modulatory effects of natural curcuminoids on P-glycoprotein ATPase of insecticide-resistant pest Helicoverpa armigera (Lepidopetera: Noctüidae).

Three major curcuminoids (I, II and III) were purified from turmeric and tested for their ability to modulate the function of P-glycoprotein ATPase of the insecticide-resistant pest Helicoverpa armigera (Ha-Pgp). The curcumin mixture inhibited the activity of Ha-Pgp ATPase by 80-90% at 100 µM concentration. Along with curcuminoids I, II and III, it inhibited the verapamil- and ethylparaoxon-stimulated Ha-Pgp ATPase activity. Curcuminoid binding was quantitated by quenching the intrinsic Trp fluorescence of purified Ha-Pgp ATPase. Transport was monitored in proteoliposomes containing Ha-Pgp ATPase using the high-affinity fluorescent substrate tetramethylrosamine (TMR) in real time. Addition of the curcuminoid mixture collapsed the TMR concentration gradient generated by Ha-Pgp ATPase. Inhibition studies on Ha-Pgp ATPase activity are important to develop strategies to overcome insecticide resistance in this pest.

Purification and characterization of extreme alkaline, thermostable keratinase, and keratin disulfide reductase produced by Bacillus halodurans PPKS-2.

Two alkaline keratinases-I and II secreted by Bacillus halodurans PPKS-2 were purified and characterized. Both the keratinases were purified using ammonium sulfate, DEAE-Sephadex followed by Sephadex G-200 column chromatography. The purification was 21.5-fold and 11.17% yield for keratinase-I and 23.7-fold with yield 18.46 for keratinase-II and its molecular weights 30 and 66 kDa. Both purified enzymes were relatively stable over a broad pH range 7.0-13.0 and optimally active at pH 11.0 and 60-70 degrees C. Keratinase-II was found to be more stable at 70 degrees C for 3 h and retained 100% of its activity, whereas keratinase-I lost 10% activity. Keratinase-I had high keratin disulfide reductase activity with low keratinase activity whereas keratinase-II had high keratinase activity with low keratin disulfide reductase activity. Keratinase activities of both the enzymes were completely inhibited by PMSF at 1 mM, whereas keratin disulfide reductase activity of keratinase-I was not affected. Enzymes were active and stable in the presence of the surfactants, bleaching agents (20% H(2)O(2)), commercial detergents (1%), and SDS (20%). Both the enzymes were partially sequenced and found that keratinase-I and II had a homology with disulfide reductases and serine type of proteases, respectively.

P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: purification, characterization and effect of various insecticides on its transport function.

Helicoverpa armigera is a major pest of agricultural crops and has developed resistance to various insecticides. A P-glycoprotein (Pgp) with ATPase activity likely to be involved in insecticide resistance was purified and characterized from insecticide-resistant H. armigera. The purification was 18-fold with 3% yield. The optimum pH and temperature were found to be 7.4 and 30-40 degrees C, respectively. Kinetic studies indicated that this enzyme had a K(m) value of 1.2mM for ATP. Pgp from H. armigera was partially sequenced and found to be homologous to conserved sequences of mammalian Pgps. Pesticides stimulated H. armigera Pgp ATPase activity with a maximum stimulation of up to 40%. Quenching of the intrinsic tryptophan fluorescence of purified Pgp was used to quantitate insecticide binding. Using the high-affinity fluorescent substrate, tetramethylrosamine, transport was monitored in real time in proteoliposomes containing H. armigera Pgp. The presence of Pgp could be one of the reasons for insecticide resistance in this pest.

Production of keratinase by free and immobilized cells of Bacillus halodurans strain PPKS-2: partial characterization and its application in feather degradation and dehairing of the goat skin.

An extremely alkaliphilic bacterial strain, Bacillus sp. PPKS-2, was isolated from rice mill effluents and screened for the production of extracellular keratinase. The maximum production of keratinase occurred after 48 h in shaking culture at pH 11.0 and 37 degrees C in a medium containing 0.5% soybean flour. The strain grew and produced alkaline keratinase using chicken feather and horn meal as the sole source of carbon and nitrogen. An addition of 0.1% soybean flour or feather hydrolysate and sodium sulfite to feather medium increased the production and complete solubilization of feather took place within 5 days under solid-state fermentation conditions. The partially purified enzyme displayed maximum activity at pH 11.0 and 60 degrees C in a broad range of NaCl, 0-16%, and was not inhibited by sodium dodecyl sulfate (10%), ethylenediaminetetraacetic acid (10 mM), H2O2 (15%), and other commercial detergents. Immobilization of the whole cells proved to be useful for continuous production of keratinase and feather degradation. The enzyme was effectively used to remove hair from goat hide. The strain PPKS-2 can be effectively used for solid waste management of poultry feather in submerged as well as solid-state fermentation.

Differential elicitation of proteases and protease inhibitors in two different genotypes of chickpea (Cicer arietinum) by salicylic acid and spermine.

Elicitation of proteases and protease inhibitors (PIs) by salicylic acid (SA) and spermine (Spm) was investigated in roots and shoots of two different genotypes of chickpea cultivars ICCV10 and L550, which were resistant and susceptible to wilt disease, respectively. SA and Spm were found to suppress the elicitation of proteases in the resistant cv, whereas they induce it in susceptible cv. Elicitation of new trypsin and chymotrypsin inhibitors was observed in the roots and shoots of resistant cv treated with SA and Spm. However, no such elicitation was observed in susceptible cv. These results show for the first time that SA and Spm could elicit synthesis of new PIs capable of inhibiting the proteases of insect Helicoverpa armigera and Fusarium oxysporum, wilt causing pathogen. Antifungal property of root extract of resistant cv increased following treatment of seedling with SA and Spm compared with susceptible cv.

Stimulatory effect of insecticides on partially purified P-glycoprotein ATPase from the resistant pest Helicoverpa armigera.

A P-glycoprotein-like protein (Ha-Pgp) was detected in a membrane preparation from the insecticide-resistant pest Helicoverpa armigera (Lepidoptera: Noctüidae) using C219 antibodies that are directed towards an epitope in the nucleotide-binding domains. This protein was partially purified and found to be a glycoprotein displaying ATPase activity. SDS-PAGE confirmed that a high molecular mass glycoprotein (150 kDa) was overexpressed in resistant pests, but was not detected in susceptible pests. The partially purified Ha-Pgp ATPase was reconstituted into proteoliposomes and it was found that some insecticides, namely, monocrotophos, endosulfan, cypermethrin, fenvalerate, and methylparathion, stimulated the ATPase activity. The effect of various inhibitors on partially purified Ha-Pgp showed that orthovanadate is a potent inhibitor of its ATPase activity, inhibiting it by 90% at a concentration of 2 mmol/L. Other inhibitors, such as EDTA, sodium azide, and molybdate resulted in only a 20% decrease in activity. Details of the structure and function of Ha-Pgp will be important in the development of strategies to overcome insecticide resistance in this pest.