Optimization of Elicitation Conditions to Enhance the Production of Potent Metabolite Withanolide from Withania somnifera (L.).

This study aimed at optimizing conditions for increased withanolide production in Withania somnifera. The elicitors used for the foliar spray on the aerial parts of the plant were salicylic acid, jasmonic acid, and chitosan for the enhancement of withanolides in Withania somnifera under different environmental regimes. Three different elicitors, i.e., chitosan, jasmonic acid and salicylic acid, were applied on the plants through foliar route every 15th day for 6 months, and later plants were used for sample preparation. Further, the elicitors were used in different concentration, i.e., jasmonic acid (50, 200 and 400 ppm), chitosan (10, 50 and 100 ppm) and salicylic acid (0.5, 1 and 2 ppm). The elicitors were sprayed on the foliar parts of the plant between 10:00-11:00 a.m. on application days. For elicitor spray, a calibrated sprayer was used. The withanolide A/withaferin A was quantified through HPLC. It was found that in an open environment, maximum withaferin A content, i.e., 0.570 mg/g (DW), was recorded with jasmonic acid (50 ppm) treatment in comparison to control (0.067 mg/g DW). Thus, there was an 8.5-fold increase in the withaferin A content. Maximum withanolide A content of 0.352 mg/g (DW) was recorded when chitosan (50 ppm) was sprayed, while in the control, withanolide A content was recorded to be 0.031 mg/g (DW); thus, chitosan application increased the production of withanolide A by 11.3-fold. Under controlled conditions, maximum withaferin A content of 1.659 mg/g (DW) was recorded when plants were sprayed with chitosan (100 ppm), which was 8.1 times greater than the control content of 0.203 mg/g (DW). Maximum withanolide A content of 0.460 mg/g (DW) was recorded when chitosan (100 ppm) was applied, whereas in the control, withanolide A content was found to be 0.061 mg/g (DW). Thus, foliar spraying of elicitors in very low concentrations can serve as a low-cost, eco-friendly, labor-intensive and elegant alternative approach that can be practiced by farmers for the enhancement, consistent production and improved yield of withanolide A/withaferin A. This can be a suitable way to enhance plant productivity, thus increasing the availability of withanolide A and withaferin A for the health and pharma industry.

Composition, Antioxidant and Anti-inflammatory activities of Hexane and Methanol extracts of Acmella uliginosa from Terai region of Uttarakhand

Abstract Acmella uliginosa, an edible herb belonging to Asteraceae family, was collected from the Terai region of Uttarakhand, India. Methanol and hexane extracts of the whole plant were prepared using soxhlet apparatus. The GC-MS analysis of plant extracts identifies 22 and 35 major compounds of methanol and hexane extracts which comprises of 74.21% and 73.20% of the total composition of extracts, respectively. The major compound in hexane was 2, 4-heptadienal (7.99%) whereas trans, trans-9, 12-octadecadienoic acid propyl ester (16.96%) was major compound in methanol extract. The extracts were evaluated for antioxidant and anti-inflammatory properties. Methanol extract showed higher free radical scavenging and reducing power activities with IC50 value 153.82±1.69 µg/mL and RP50 value of 152.28±0.41 µg/mL, respectively. The metal chelating activity was higher in hexane extract as compared to methanol extract i.e., 62.08±0.25 µg/mL. The anti-inflammatory activity assessed by its ability to inhibit denaturation was higher in methanol having IB50 value 87.33±0.15 µg/mL. The total phenolic content (TPC), total flavonoid content (TFC) and ortho-dihydric phenol content (ODP) of methanol and hexane extracts were also evaluated. TPC, TFC and ODP was higher in methanol extract having value of 122.23±0.22, 35.01±0.29 and 8±0.86 mg/mL, respectively. Acmella uliginosa, might be considered as a natural source for antioxidant and anti-inflammatory properties

Foliar application of elicitors enhanced the yield of withanolide contents in Withania somnifera (L.) Dunal (variety, Poshita).

This study involves a detailed investigation about the effect of three elicitors, such as chitosan, jasmonic acid and salicylic acid (SA) on withaferin A and withanolide A contents of Withania somnifera (L.) Dunal (Poshita variety). Moreover, the different environmental regimes were also studied to assess and optimise the accumulation of withaferin A and Withanolide A contents. In an open environment, the total withaferin A content was found to be increased 6.3 and 5.8 times when sprayed with chitosan, 10 ppm and 50 ppm, respectively, as compared to control. Similarly, the total withanolide A content was found to be increased 4.5 and 3.6 times when sprayed with jasmonic acid (400 ppm and 200 ppm, respectively) with respect to control. In a controlled condition, the total withaferin A content was found to be increased 6 and 4.5 times when sprayed with jasmonic acid (400 ppm and 200 ppm, respectively) as compared to control. On the other hand, the total withanolide A content was found to be enhanced by 7 and 4.3 times when sprayed with jasmonic acid (400 ppm) and SA (1 ppm), respectively, as compared to control. Therefore, this study was focussed on the optimisation of enhanced accumulation of withaferin A and withanolide A contents in the aerial parts of the plant in open and controlled environment by foliar application of elicitors in minimal concentrations.

Production of Thermostable Organic Solvent Tolerant Keratinolytic Protease from Thermoactinomyces sp. RM4: IAA Production and Plant Growth Promotion.

There are several reports about the optimization of protease production, but only few have optimized the production of organic solvent tolerant keratinolytic proteases that show remarkable exploitation in the development of the non-polluting processes in biotechnological industries. The present study was carried with aim to optimize the production of a thermostable organic solvent tolerant keratinolytic protease Thermoactinomyces sp. RM4 utilizing chicken feathers. Thermoactinomyces sp. RM4 isolated from the soil sample collected from a rice mill wasteyard site near Kashipur, Uttrakhand was identified on the basis of 16S rDNA analysis. The production of organic solvent tolerant keratinolytic protease enzyme by Thermoactinomyces sp. RM4 was optimized by varying physical culture conditions such as pH (10.0), temperature (60°C), inoculum percentage (2%), feather concentration (2%) and agitation rate (2 g) for feather degradation. The result showed that Thermoactinomyces sp. RM4 potentially produces extra-cellular thermostable organic solvent tolerant keratinolytic protease in the culture medium. Further, the feather hydrolysate from keratinase production media showed plant growth promoting activity by producing indole-3-acetic acid itself. The present findings suggest that keratinolytic protease from Thermoactinomyces sp. RM4 offers enormous industrial applications due to its organic solvent tolerant property in peptide synthesis, practical role in feather degradation and potential function in plant growth promoting activity, which might be a superior candidate to keep ecosystem healthy and functional.

Physicochemical and antioxidant properties of kiwifruit as a function of cultivar and fruit harvested month

The present study was carried out to find the effect of fruit harvesting stage (October, November and December) on the physicochemical and antioxidant properties in five kiwi cultivars (Abbot, Bruno, Allison, Hayward, Monty). Results showed that soluble solid content (SSC) and pH increased while ascorbic acid (Vit C), titrated acidity (TAD) and SSC/TAD decreased in all the cultivars with delay in harvesting. Total polyphenols (TP) were decreased while total flavonoids (TF) increased in all tested cultivars with delay in harvesting. The highest concentration of TP (2.02 mg gallic acid equivalent/g fresh weight) and TF (51.12 mg catechin equivalent/100g FW) were found in cultivar 'Allison' in the month of October and December, respectively. Antioxidant activities (AA) were genotype depended and no trend was observed with month of harvesting. Principal component analysis (PCA) showed strong correlation between Vit C, TP and antioxidant activities. Two major clusters were computed using agglomerative hierarchical clustering (AHC). All the studied important traits may be used in the breeding programmes to increase the variability for different physiochemical and antioxidative characteristics and to make suitable selections that could be acceptable to consumers.

Mannitol-induced drought stress on calli of Trigonellafoenum-graecum L. Var. RMt-303.

Different explants of fenugreek, T. foenum-graecum L. (Var. RMt-303), were compared for their callus induction and subsequent shoot regeneration capabilities on Murashige and Skoog media supplemented with different phytohormones in varying concentration. The highest percentage of callus induction frequency was observed in 1 ppm benzylaminopurine (BAP). Maximum shoots were induced on media supplemented with 0.5 ppm BAP using leaf and stem tissues as explants. However, root tissues showed only callusing with no subsequent shooting. Cotyledonary node responded better than hypocotyls in terms of shoot induction on media supplemented with thidiazuron (0.1 ppm). The callus was subjected to drought stress as simulated by reduced water potential of growth media due to addition of mannitol. Calli could withstand -2 MPa water potential till 30 days indicating that the drought stress tolerance mechanisms are functional in this variety. Chlorophyll a and b and total chlorophyll, proline and total phenolic contents, total peroxidase and catalase activities increased under stress conditions suggesting the tolerance of callus to drought stress. However, ascorbate peroxidase, guaiacol peroxidase activities were found to decrease slightly. Malondialdehyde and H2O2 contents were found to decrease while only a slight disturbance was found in membrane stability index. These results underline the mechanisms that are crucial for drought stress tolerance in fenugreek.

Cloning, Sequencing, and In Silico Analysis of ß-Propeller Phytase Bacillus licheniformis Strain PB-13.

ß -Propeller phytases (BPPhy) are widely distributed in nature and play a major role in phytate-phosphorus cycling. In the present study, a BPPhy gene from Bacillus licheniformis strain was expressed in E. coli with a phytase activity of 1.15 U/mL and specific activity of 0.92 U/mg proteins. The expressed enzyme represented a full length ORF "PhyPB13" of 381 amino acid residues and differs by 3 residues from the closest similar existing BPPhy sequences. The PhyPB13 sequence was characterized in silico using various bioinformatic tools to better understand structural, functional, and evolutionary aspects of BPPhy class by multiple sequence alignment and homology search, phylogenetic tree construction, variation in biochemical features, and distribution of motifs and superfamilies. In all sequences, conserved sites were observed toward their N-terminus and C-terminus. Cysteine was not present in the sequence. Overall, three major clusters were observed in phylogenetic tree with variation in biophysical characteristics. A total of 10 motifs were reported with motif "1" observed in all 44 protein sequences and might be used for diversity and expression analysis of BPPhy enzymes. This study revealed important sequence features of BPPhy and pave a way for determining catalytic mechanism and selection of phytase with desirable characteristics.

Molecular and biochemical characteristics of recombinant ß-propeller phytase from Bacillus licheniformis strain PB-13 with potential application in aquafeed.

Phytic acid is the major storage form of organic phosphorus in nature- and plant-based animal feed. It forms insoluble complexes with nutritionally important metals and proteins that are unavailable for monogastric or agastric animals. Phytases initiate the stepwise hydrolysis of phytic acid and release inorganic orthophosphate. In the present investigation, the phytase gene from a phytase producing Bacillus licheniformis strain PB-13 was successfully expressed in Escherichia coli BL21. Recombinant phytase 'rPhyPB13' was found to be catalytically active, with an activity of 0.97 U/mL and specific activity of 0.77 U/mg. The rPhyPB13 was purified to 14.10-fold using affinity chromatography. Similar to other ß-propeller phytases, purified rPhyPB13 exhibited maximal activity at pH 6.0-6.5 and 60 °C in the presence of 1 mM Ca(2+) and was highly active over a wider pH range (pH 4.0-8.0) and high temperature (80 °C). It has shown maximum activity towards Na-phytate as substrate. The observed K m , V max and k cat of purified rPhyPB13 were 1.064 mM, 1.32 µmol/min/mg and 27.46 s(-1), respectively. PhyPB13 was resistant to trypsin inactivation, activated in presence of Ca(2+) and inhibited in presence of EDTA. Crude rPhyPB13 has good digestion efficiency for commercial feed and soybean meal. These results indicate that PhyPB13 is a ß-propeller phytase that has application potential in aquaculture feed.

Evaluation of phytase producing bacteria for their plant growth promoting activities.

Bacterial inoculants are known to possess plant growth promoting abilities and have potential as liquid biofertilizer application. Four phytase producing bacterial isolates (phytase activity in the range of 0.076-0.174 U/mL), identified as Advenella species (PB-05, PB-06, and PB-10) and Cellulosimicrobium sp. PB-09, were analyzed for their plant growth promoting activities like siderophore production, IAA production, HCN production, ammonia production, phosphate solubilization, and antifungal activity. All isolates were positive for the above characteristics except for HCN production. The solubilization index for phosphorus on Pikovskaya agar plates was in the range of 2-4. Significant amount of IAA (7.19 to 35.03 µ g/mL) production and solubilized phosphate (189.53 to 746.84 µ g/mL) was noticed by these isolates at different time intervals. Besides that, a greenhouse study was also conducted with Indian mustard to evaluate the potential of these isolates to promote plant growth. Effect of seed bacterization on various plant growth parameters and P uptake by plant were used as indicators. The plant growth promoting ability of bacterial isolates in pot experiments was correlated to IAA production, phosphate solubilization, and other in vitro tests. On the basis of present findings, isolate PB-06 was most promising in plant growth promotion with multiple growth promoting characteristics.

Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films.

Isolation of phytase-producing bacteria from Himalayan soils and their effect on growth and phosphorus uptake of Indian mustard (Brassica juncea).

Phytase-producing bacteria (PPB) is being investigated as plant growth promoting rhizobacteria (PGPR) to improve the phosphorus (P) nutrition and growth of plants grown in soil with high phytate content. Phytate is dominant organic P forms in many soils and must be hydrolyzed to be available for plants. Indian mustard (Brassica juncea) is a plant with economic importance in agriculture and phytoremediation, therefore biotechnological tools to improve growth and environmental stress tolerance are needed. In this study, we isolated and characterized PPB from Himalayan soils and evaluated their effect on growth and P uptake by B. juncea under greenhouse conditions. Sixty five PPB were isolated and based on phytate hydrolysis, three efficient PPB were chosen and identified as Acromobacter sp. PB-01, Tetrathiobacter sp. PB-03 and Bacillus sp. PB-13. Selected PPB showed ability to grow at wide range of pH, temperature and salt concentrations as well as to harbour diverse PGPR activities, such as: solubilization of insoluble Ca-phosphate (193-642 µg ml(-1)), production of phytohormone indole acetic acid (5-39 µg ml(-1)) and siderophore. Tetrathiobacter sp. PB-03 and Bacillus sp. PB-13 showed 50 and 70 % inhibition of phytopathogen Rhizoctonia solani, respectively. Greenhouse potting assay also showed that the bacterization of B. juncea seeds with Tetrathiobacter sp. PB-03 and Bacillus sp. PB-13 significantly increased the biomass and P content in 30 days old seedlings. This study reveals the potential of PPB as PGPR to improve the growth of B. juncea.

Enhanced phytase production from Achromobacter sp. PB-01 using wheat bran as substrate: prospective application for animal feed.

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB-01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X-100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB-01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non-starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro-industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium.

In silico characterization of histidine Acid phytase sequences.

Histidine acid phytases (HAPhy) are widely distributed enzymes among bacteria, fungi, plants, and some animal tissues. They have a significant role as an animal feed enzyme and in the solubilization of insoluble phosphates and minerals present in the form of phytic acid complex. A set of 50 reference protein sequences representing HAPhy were retrieved from NCBI protein database and characterized for various biochemical properties, multiple sequence alignment (MSA), homology search, phylogenetic analysis, motifs, and superfamily search. MSA using MEGA5 revealed the presence of conserved sequences at N-terminal "RHGXRXP" and C-terminal "HD." Phylogenetic tree analysis indicates the presence of three clusters representing different HAPhy, that is, PhyA, PhyB, and AppA. Analysis of 10 commonly distributed motifs in the sequences indicates the presence of signature sequence for each class. Motif 1 "SPFCDLFTHEEWIQYDYLQSLGKYYGYGAGNPLGPAQGIGF" was present in 38 protein sequences representing clusters 1 (PhyA) and 2 (PhyB). Cluster 3 (AppA) contains motif 9 "KKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDP" as a signature sequence. All sequences belong to histidine acid phosphatase family as resulted from superfamily search. No conserved sequence representing 3- or 6-phytase could be identified using multiple sequence alignment. This in silico analysis might contribute in the classification and future genetic engineering of this most diverse class of phytase.