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.

Comparative assessment of solvents and lignocellulolytic enzymes affiliated extraction of polyphenols from the various lignocellulosic agro-residues: identification and their antioxidant properties.

The present investigation was aimed to utilize lignocellulosic agro-residues and compare the extraction of polyphenols utilizing lignocellulolytic enzymes secreted by Sphingobacterium sp. ksn and with that of the solvents (ethanol, methanol) affiliated methods. The maximum amount of polyphenols, flavonoids and tannins were 94.29, 11.36, and 79.21 g 100 g-1 respectively, found in the extracts obtained by enzymes affiliated extraction of coffee cherry husk (CCH). The phenolics namely, gallic acid, caffeic acid, coumaric acid, 1-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, p-hydroxybenzaldehyde were commonly found whereas syringic acid, quercetin, kaempferol, and epicatechin were hardly found in the extracts of agro-residues. The extracts of CCH shown maximum antioxidant properties for DPPH, ABTS, and FRAP. The present study reports that the affiliation of enzymes for the extraction of polyphenols from agro-residues is more efficient than that of the solvents affiliation and CCH as the good source of polyphenols.

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.

Polyphenols from different agricultural residues: extraction, identification and their antioxidant properties.

Agricultural residues like sugarcane bagasse (SCB), corn husk (CH), peanut husk (PNH), coffee cherry husk (CCH), rice bran (RB) and wheat bran (WB) are low-value byproducts of agriculture. They have been shown to contain significant levels of phenolic compounds with demonstrated antioxidant properties. In this study, the effects of two types of solvent extraction methods: solid-liquid extraction (SLE) and hot water extraction on the recovery of phenolic compounds from agricultural residues were investigated to optimize the extraction conditions based on total phenolic content (TPC), total tannin content (TTC) and total flavonoids content (TFC). Methanol (50 %) was found to be the most efficient solvent for the extraction of phenolics with higher DPPH, nitric oxide radical scavenging and reducing power activity, followed by ethanol and water. The phenolic compounds of methanolic extracts (50 %) were determined by reverse phase high performance liquid chromatography; in addition gallic acid became the major phenolic acid present in all the agricultural residues whereas ferulic acid, epicatechin, catechin, quercitin and kampferol present in lesser amounts. The present investigation suggested that agricultural residues are potent antioxidants. The overall results of this research demonstrated the potential of agricultural residues to be an abundant source of natural antioxidants suitable for further development into dietary supplements and various food additives.

Salicylic acid and salicylic acid sensitive and insensitive catalases in different genotypes of chickpea against Fusarium oxysporum f. sp. ciceri.

Differential expression of catalase isozymes in different genotypes of chickpea resistant genotypes- A1, JG-315, JG-11, WR-315, R1-315, Vijaya, ICCV-15017, GBS-964, GBM-10, and susceptible genotypes- JG-62, MNK, ICCV-08321, ICCV-08311, KW-104, ICCV-08123, ICC-4951, ICC-11322, ICC-08116 for wilt disease caused by Fusarium oxysporum. f. sp. ciceri (Foc) was analyzed. Salicylic acid (SA) and H2O2 concentrations were determined in control as well as in plants infected with F. ciceri and found that the high and low levels of salicylic acid and H2O2 in resistant and susceptible genotypes of chickpea respectively. Catalase isozyme activities were detected in the gel and found that no induction of new catalases was observed in all the resistant genotypes and their some of the native catalase isozymes were inhibited; whereas, induction of multiple catalase isozymes was observed in all the screened susceptible genotypes and their activities were not inhibited upon Foc or SA treatments. The above results support the possible role of these isozymes as a marker to identify which genotype of chickpea is expressing systemic acquired resistance.

Interaction of plant cell signaling molecules, salicylic acid and jasmonic acid, with the mitochondria of Helicoverpa armigera.

The cotton bollworm, Helicoverpa armigera is a polyphagous pest in Asia, Africa, and the Mediterranean Europe. Salicylic acid (SA) and jasmonic acid (JA) are the cell signaling molecules produced in response to insect attack in plants. The effect of these signaling molecules was investigated on the oxidative phosphorylation and oxidative stress of H. armigera. SA significantly inhibited the state III and state IV respiration, respiratory control index (RCI), respiratory complexes I and II, induced mitochondrial swelling, and cytochrome c release in vitro. Under in vivo conditions, SA induced state IV respiration as well as oxidative stress in time- and dose-dependent manner, and also inhibited the larval growth. In contrast, JA did not affect the mitochondrial respiration and oxidative stress. SA affected the growth and development of H. armigera, in addition to its function as signaling molecules involved in both local defense reactions at feeding sites and the induction of systemic acquired resistance in plants.

Production of alkaliphilic, halotolerent, thermostable cellulase free xylanase by Bacillus halodurans PPKS-2 using agro waste: single step purification and characterization.

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular alkaliphilic, thermostable and halotolerent xylanase. The culture conditions for xylanase production were optimized with respect to pH, temperature, NaCl and inexpensive agro waste as substrates. Xylanase yield was enhanced more than four fold in the presence of 1% corn husk and 0.5% peptone or feather hydrolysate at pH 11 and 37°C. Xylanase was purified to 11.8-fold with 8.7% yield by using traditional chromatographic methods whereas the same enzyme purified to 20-fold with 72% yield by using corn husk as ligand. Its molecular mass was estimated to be 24 kDa by SDS-PAGE. The xylanase had maximal activity at pH 11 and 70°C. The enzyme was active over broad range, 0-20% sodium chloride. The enzyme was thermostable retaining 100% of the original activity at 70°C for 3 h. The apparent K (m) values for oat spelt xylan and brichwood xylan were 4.1 and 4.4 mg/ml respectively. The deduced internal amino acid sequence of PPKS-2 xylanase resembled the sequence of ß-1,4-endoxylanase, which is member of glycoside hydrolase family 11.

Super Annigeri 1 and improved JG 74: two Fusarium wilt-resistant introgression lines developed using marker-assisted backcrossing approach in chickpea (Cicer arietinum L.).

Annigeri 1 and JG 74 are elite high yielding desi cultivars of chickpea with medium maturity duration and extensively cultivated in Karnataka and Madhya Pradesh, respectively. Both cultivars, in recent years, have become susceptible to race 4 of Fusarium wilt (FW). To improve Annigeri 1 and JG 74, we introgressed a genomic region conferring resistance against FW race 4 (foc4) through marker-assisted backcrossing using WR 315 as the donor parent. For foreground selection, TA59, TA96, TR19 and TA27 markers were used at Agricultural Research Station, Kalaburagi, while GA16 and TA96 markers were used at Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur. Background selection using simple sequence repreats (SSRs) for the cross Annigeri 1 × WR 315 in BC1F1 and BC2F1 lines resulted in 76-87% and 90-95% recurrent parent genome recovery, respectively. On the other hand, 90-97% genome was recovered in BC3F1 lines in the case of cross JG 74 × WR 315. Multilocation evaluation of 10 BC2F5 lines derived from Annigeri 1 provided one superior line referred to as Super Annigeri 1 with 8% increase in yield and enhanced disease resistance over Annigeri 1. JG 74315-14, the superior line in JG 74 background, had a yield advantage of 53.5% and 25.6% over the location trial means in Pantnagar and Durgapura locations, respectively, under Initial Varietal Trial of All India Coordinated Research Project on Chickpea. These lines with enhanced resistance and high yield performance are demonstration of successful deployment of molecular breeding to develop superior lines for FW resistance in chickpea.

Purification and characterization of an esterase isozyme involved in hydrolysis of organophosphorus compounds from an insecticide resistant pest, Helicoverpa armigera (Lepidoptera: Noctüidae).

An esterase isozyme was purified from the insecticide resistant pest, Helicoverpa armigera collected from field crops. Purification involved ammonium sulfate precipitation, hydrophobic interaction and ion exchange chromatography followed by gel filtration chromatography. The purification was 212-fold with 1% yield of the enzyme. The optimum pH of the isozyme was found to be 10.5 and 8.5 for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme was unstable at temperature >50 degrees C. The molecular mass determined by SDS-PAGE was 66 kDa. Cations such as Hg(+2), Ag(+2), Cd(+2) inhibited the activity while Zn(+2) stimulated it. Kinetic studies indicated that the enzyme had low K(m) values of 0.238 and 0.348 mM for p-nitrophenyl phosphate and paraoxon, respectively. The enzyme had broad substrate specificity with high K(m) values for ATP, ADP and beta-glycerophosphate. This enzyme was partially sequenced and identified as an alkaline phosphatase.

Hydrolysis of organophosphorus compounds by an esterase isozyme from insecticide resistant pest Helicoverpa armigera.

Esterase activity of resistant and susceptible H. armigera were compared in gels with different substrate such as naphthyl acetate, naphthyl phosphate, paraoxon and monocrotophos. Whole body extract of resistant H. armigera hydrolyzed paraoxon, monocrotophos and naphthyl phosphate in gels. Resistant H. armigera showed high esterase, phosphatase and paraoxon hydrolase activity compared to susceptible ones.

Production of extremely alkaliphilic, halotolerent, detergent, and thermostable mannanase by the free and immobilized cells of Bacillus halodurans PPKS-2. Purification and characterization.

The alkaliphilic Bacillus halodurans strain PPKS-2 was shown to produce extracellular extreme alkaliphilic, halotolerent, detergent, and thermostable mannanase activity. The cultural conditions for the maximum enzyme production were optimized with respect to pH, temperature, NaCl, and inexpensive agro wastes as substrates. Mannanase production was enhanced more than 4-fold in the presence of 1 % defatted copra meal and 0.5 % peptone or feather hydrolysate at pH 11 and 40 °C. Mannanase was purified to 10.3-fold with 34.6 % yield by ion exchange and gel filtration chromatography methods. Its molecular mass was estimated to be 22 kDa by SDS-PAGE. The mannanase had maximal activity at pH 11 and 70 °C. This enzyme was active over a broad range of NaCl (0-16 %) and thermostable retaining 100 % of the original activity at 70 °C for 3 h. Immobilization of whole cells proved to be effective for continuous production of mannanase. Since the strain PPKS-2 grows on cheaper agro wastes such as defatted copra meal, corn husk, jowar bagasse, and wheat bran, these can be exploited for mannanase production on an industrial scale.

Production of bioethanol from fermented sugars of sugarcane bagasse produced by lignocellulolytic enzymes of Exiguobacterium sp. VSG-1.

Exiguobacterium sp. VSG-1 was isolated from the soil sample and characterized for the production of lignocellulolytic enzymes. Production of these enzymes by the strain VSG-1 was carried out using steam-exploded sugarcane bagasse (SCB) and found to secrete cellulase, pectinase, mannanase, xylanase, and tannase. The growth and enzyme production were found to be optimum at pH 9.0 and 37 °C. Upon steam explosion of SCB, the cellulose increased by 42 %, whereas hemicelluloses and lignin decreased by 40 and 62 %, respectively. Enzymatic hydrolysis of steam-exploded SCB yielded 640 g/l of total sugars. Fermentation of sugars produced from pretreated SCB was carried out by using Saccharomyces cerevisiae at pH 5.0 and 30 °C. The alcohol produced was calculated and found to be 62.24 g/l corresponding to 78 % of the theoretical yield of ethanol. Hence, the strain VSG-1 has an industrial importance for the production of fermentable sugars for biofuels.

Salicylic acid is a modulator of catalase isozymes in chickpea plants infected with Fusarium oxysporum f. sp. ciceri.

The relationship between salicylic acid level catalases isoforms chickpea cv. ICCV-10 infected with Fusarium oxysporum f. sp. ciceri was investigated. Pathogen-treated chickpea plants showed high levels of SA compared with the control. Two isoforms of catalases in shoot extract (CAT-IS and CAT-IIS) and single isoform in root extract (CAT-R) were detected in chickpea. CAT-IS and CAT-R activities were inhibited in respective extracts treated with pathogen whereas, CAT-IIS activity was not inhibited. These isoforms were purified and their kinetic properties studied in the presence or absence of SA. The molecular mass determined by SDS-PAGE of CAT-IS, CAT-IIS and CAT-R was found to be 97, 40 and 66 kDa respectively. Kinetic studies indicated that Km and V(max) of CAT-IS were 0.2 mM and 300 U/mg, 0.53 mM and 180 U/mg for CAT-IIS and 0.25 mM and 280 U/mg for CAT-R, respectively. CAT-IS and CAT-R were found to be more sensitive to SA and 50% of their activities were inhibited at 6 and 4 µM respectively, whereas CAT-IIS was insensitive to SA up to 100 µM. Quenching of the intrinsic tryptophan fluorescence of purified catalases were used to quantitate SA binding; the estimated K(d) value for CAT-IS, CAT-IIS and CAT-R found to be 2.3 µM, 3.1 mM and 2.8 µM respectively. SA is a modulator of catalase isozymes activity, supports its role in establishment of SAR in chickpea plants infected with the pathogen.

Identification of factors responsible for insecticide resistance in Helicoverpa armigera.

Moth larvae (Helicoverpa armigera Hübner) collected from field crops were tested for resistance to cypermethrin, fenvalerate, endosulfan, monocrotophos and quinolphos. Larvae were treated with a dose of the pesticide that would kill 99% of the susceptible insects. The percent survival of the resistant strains was determined. Highest seasonal average percentage survival was recorded by fenvalerate (65.0%) followed by cypermethrin (62.4%). Acetylcholinesterase of resistant larvae was less sensitive to monocrotophos and methyl paraoxon. Resistant larvae showed higher activities of esterases, phosphatases and methyl paraoxon hydrolase compared with susceptible larvae. The presence of high activity of esterases was attributed to appearance of extra bands of esterases in native PAGE. The presence of P-glycoprotein expression was detected in resistant larvae using P-gp antibodies; this was not detected in the susceptible larvae. Our results indicate that the high level of resistance detected in the field pests could be because of a combined effect of decreased sensitivity to AChE, higher levels of esterases, phosphatases and the expression of P-gp.