Sivashanmugam Karthikeyan

OsPRP3, a flower specific proline-rich protein of rice, determines extracellular matrix structure of floral organs and its overexpression confers cold-tolerance

Proline-rich protein (PRP), a cell wall protein of plant, has been studied in many plant species. Yet, none of the PRPs has been functionally elucidated. Here we report a novel flower-specific PRP designated OsPRP3 from rice. Expression analysis showed that the OsPRP3 transcript was mainly present in rice flower and accumulated abundantly during the late stage of the flower development. To study the function of OsPRP3, we constructed and transformed a binary vector containing a full clone of OsPRP3 in sense orientation and also an RNAi vector to achieve overexpression and knockout of the gene, respectively. Our overexpression plants showed a significant increase in cold tolerance than the WT plants which is conferred by the accumulation of OsPRP3 protein during cold treatment. Further the microscopic analysis revealed that OsPRP3 enhances the cell wall integrity in the cold tolerant plant and confers cold-tolerance in rice. Microscopic analysis of the RNAi mutant flower revealed that blocking OsPRP3 function caused significant defects in floral organogenesis. Taken together, the results suggested that OsPRP3 is a cell wall protein, playing a crucial role in determining extracellular matrix structure of floral organs.

Purification, characterization and functional analysis of the immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus and their antibiofilm properties

ABSTRACT The present study reveals purification and characterization of immune molecule lectin from the haemolymph of blue swimmer crab Portunus pelagicus (Pp‐Lec). The Pp‐Lec was purified by affinity chromatography with mannose coupled sepharose CL‐4B column and it exhibits single band with a molecular weight of 155 kDa in SDS‐PAGE. The surface morphology of purified Pp‐Lec displays the homogeneous nature of protein. A distinct peak with a retention time of 3.3 min was appeared in high performance liquid chromatography (HPLC) and X‐ray diffraction (XRD) analysis expresses a single peak at 31.5° which shows the purity and crystalline nature of the protein respectively. Functional analysis of purified Pp‐Lec exhibits encapsulation activity against sepharose beads and yeast agglutination activity against Saccharomyces cerevisiae. Moreover, the purified Pp‐Lec has the ability to agglutinates with the human erythrocytes among tested and which was observed by light microscopy. In addition, purified Pp‐Lec showed the broad spectrum of antibacterial activity against Gram‐positive Bacillus pumulis, Bacillus thuringiensis, Enterococcus faecalis and Gram negative Citrobacter amalonaticus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Proteus vulgaris, Citrobacter murliniae, Citrobacter freundii, Morganella morganii. Antibiofilm potential of purified Pp‐Lec against selective Gram‐negative bacteria showed the disruption of biofilm architecture at the concentration of 50 &mgr;g ml−1. HighlightsImmune molecule lectin was successfully purified from the blue swimmer crab Portunus pelagicus using mannose coupled sepharose CL‐4B column.Functional role of lectin in the immune system of P. pelagicus was noticed.Antibiofilm activity of lectin in destroying biofilm architecture of Gram negative bacteria was discussed.

Systematic functional analysis and application of a cold-active serine protease from a novel Chryseobacterium sp.

Psychrotolerant bacteria isolated from natural and artificially cold environments were screened for synthesis of cold-active protease. The strain IMDY showing the highest protease production at 5°C was selected and phylogenetic analysis revealed that IMDY as novel bacterium with Chryseobacterium soli(T) as its nearest neighbor. Classical optimization enhanced the protease production from 18U/mg to 26U/mg and the enzyme was found to be active at low temperature, activity enhanced by CaCl2, inhibited by PMSF, stable against NaCl, and its activity retained in the presence of surfactants, organic solvents and detergents. On testing, the meat tenderization, myofibril fragmentation, pH, and TBA values were favorable in IMDY-protease treated meat compared to control. SDS profiling and SEM analysis also showed tenderization in meat samples. Hence, this study proposes to consider the cold-active protease from Chryseobacterium sp. IMDY as a pertinent candidate to develop potential applications in food processing industry.

Astaxanthin from psychrotrophic Sphingomonas faeni exhibits antagonism against food-spoilage bacteria at low temperatures.

Food production and processing industry holds a perpetual relationship with microorganisms and their by-products. In the present study, we aimed to identify beneficial cold-adapted bacteria devoid of any food spoilage properties and study their antagonism against common food-borne pathogens at low temperature conditions. Ten isolates were obtained on selective isolation at 5 °C, which were spread across genera Pseudomonas, Sphingomonas, Psychrobacter, Leuconostoc, Rhodococcus, and Arthrobacter. Methanol extracts of strains were found to contain several bioactive metabolites. Among the studied isolates, methanol extracts of S. faeni ISY and Rhodococcus fascians CS4 were found to show antagonism against growth of Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, Listeria monocytogenes and Vibrio fischeri at refrigeration temperatures. Characterization of the abundant yellow pigment in methanol extracts of S. faeni ISY through UV-Vis spectrophotometry, high performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) revealed the presence of astaxanthin, which, owing to its presence in very large amounts and evidenced to be responsible for antagonistic activity of the solvent extract.

β-Keto esters from ketones and ethyl chloroformate: a rapid, general, efficient synthesis of pyrazolones and their antimicrobial, in silico and in vitro cytotoxicity studies.

Background Pyrazolones are traditionally synthesized by the reaction of β-keto esters with hydrazine and its derivatives. There are methods to synthesize β-keto esters from esters and aldehydes, but these methods have main limitation in varying the substituents. Often, there are a number of methods such as acylation of enolates in which a chelating effect has been employed to lock the enolate anion using lithium and magnesium salts; however, these methods suffer from inconsistent yields in the case of aliphatic acylation. There are methods to synthesize β-keto esters from ketones like caboxylation of ketone enolates using carbon dioxide and carbon monoxide sources in the presence of palladium or transition metal catalysts. Currently, the most general and simple method to synthesize β-keto ester is the reaction of dimethyl or ethyl carbonate with ketone in the presence of strong bases which also requires long reaction time, use of excessive amount of reagent and inconsistent yield. These factors lead us to develop a simple method to synthesize β-keto esters by changing the base and reagent. Results A series of β-keto esters were synthesized from ketones and ethyl chloroformate in the presence of base which in turn are converted to pyrazolones and then subjected to cytotoxicity studies towards various cancer cell lines and antimicrobial activity studies towards various bacterial and fungal strains. Conclusion The β-keto esters from ethyl chloroformate was successfully attempted, and the developed method is simple, fast and applicable to the ketones having the alkyl halogens, protecting groups like Boc and Cbz that were tolerated and proved to be useful in the synthesis of fused bicyclic and tricyclic pyrazolones efficiently using cyclic ketones. Since this method is successful for different ketones, it can be useful for the synthesis of pharmaceutically important pyrazolones also. The synthesized pyrazolones were subjected to antimicrobial, docking and cytotoxicity assay against ACHN (human renal cell carcinoma), Panc-1 (human pancreatic adenocarcinoma) and HCT-116 (human colon cancer) cell line, and lead molecules have been identified. Some of the compounds are found to have promising activity against different bacterial and fungal strains tested.

Identification and analysis of the salt tolerant property of AHL lactonase (AiiATSAWB) of Bacillus species

Bacterial biofilms communicate by a process called Quorum Sensing. Gram negative bacterial pathogens specifically talk through the production, detection, and response to the signal or autoinducer called Acyl Homoserine Lactones. Bacterial lactonases are important AHL hydrolysing or quorum quenching enzymes. The present study deals with ten endospore forming gram positive isolates of the saltern soil. Preliminary screening for Quorum Quenching activity with the QS Inhibition indicator strain Chromobacterium violaceum ATCC 12472, showed positive activity in four isolates namely TS2, TS16, TSAWB, and TS53B. AHL lactonase (AiiA) specific primers amplified Acyl Homoserine Lactone lactonase gene in the TSAWB genome alone. Phylogenetic relationship of the identified AiiATSAWB confirmed its evolutionary relationship with bacterial AiiA like AHL lactonase of the metallo‐beta‐lactamase super family. Our in vitro AHL hydrolysis assay under wide percentage (0–5) of salt solutions with TSAWB isolate and also its intracellular soluble protein fraction showed halotolerant AHL hydrolysis ability of the AiiATSAWB enzyme. In silico determination of putative tertiary structure, the ESBRI derived conserved salt bridges, aminoacid residue characterization with high mole percent of acidic and hydrophobic residues reaffirmed the halotolerant ability of the enzyme. So we propound the future use of purified AiiATSAWB, as hypertonic suspension for inhalation to substitute the action of inactivated hosts paraoxonase in treating Pseudomonas aeruginosa infection in cystic fibrosis patients.

Insight on biochemical characteristics of thermotolerant amylase isolated from extremophile bacteria Bacillus vallismortis TD6 (HQ992818)

Halotolerant bacterium Bacillus vallismortis (HQ992818) was isolated from saltern sediments in India, and produced significantly high levels extracellular amylase. A detailed investigation on the culture conditions including period of incubation, media pH, and inoculum size in addition to different sources of carbon and nitrogen, metal ions, NaCl, and amino acids was carried out for optimized production. Maximum amylase production (62 U/mL) was attained after 26 h of incubation. The optimized conditions for maximal production of amylase were found to be 1% NaCl, pH 8, temp 37°C, 1% starch, 1% sodium nitrate, phenyl alanine (0.01%) and calcium chloride (10 mM). The biochemical characteristics of the extracellular amylase were studied with respect to change in temperature, pH and metal ions. The enzyme was found to be optimally active in the temperature range of 40–70°C and pH 8. Activation of the enzyme by Ca2+ (135%), Fe2+ (113%) and Mg2+ (109%) occurred at 5 mM concentration and strongly inhibited by Hg2+, Zn2+ and Mn2+ occurred at 10 mM. Significant compatibility of the enzyme with the commercial laundry detergents and the results of washing performance test confirmed its effectiveness. Available data on the optimized culture conditions enables for easily adaptable setup of large scale production of the enzyme for use in detergent formulations.

Identification, characterization and immune response of prophenoloxidase from the blue swimmer crab Portunus pelagicus and its antibiofilm activity

Prophenoloxidase is a conserved Cu-containing enzyme acting as a major defense molecule in the immune response of crustaceans. In the present research, we purified prophenoloxidase from the haemolymph of Portunus pelagicus (Pp-proPO) by Blue Sepharose CL-6B chromatography. Pp-proPO exhibited only one band with molecular weight of 75kDa on SDS-PAGE. The purified Pp-proPO was characterized through X-ray diffraction (XRD) and high-performance liquid chromatography (HPLC). Pp-proPO showed phagocytic activity on the yeast Saccharomyces cerevisiae as well as encapsulation on sepharose CL-6B beads associated with CM sepharose and beads of sodium alginate. Pp-proPO also led to strong agglutination on human erythrocytes. Furthermore, Pp-proPO showed magnified PO activity when altered with activated particles acting as pathogen combined molecular patterns (PAMPs), metal ions or other chemicals. Pp-proPO showed relevant antibiofilm activity on Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. Overall, the above results allowed us to claim that Pp-proPO play a key role in immune defense mechanisms of P. pelagicus crabs, in particular towards microbial pathogens; notably we added basic information to the functional characterization of Pp-proPO, as well as to understand its immunological role in crustaceans defense systems.

Phenoloxidase activation, antimicrobial, and antibiofilm properties of β-glucan binding protein from Scylla serrata crab hemolymph

In this study, we purified β-GBP from hemolymph of Scylla serrata crabs using affinity chromatography. The purified S. serrata β-GBP (Ss-β-GBP) had 100kDa molecular mass in the SDS-PAGE. MALDI-TOF/TOF analysis was conducted, revealing that the purified 100kDa protein had 96% similarity with β-GBP of Astacus leptodactylus. Ss-β-GBP was characterized using high-performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, which confirmed the structure of the Ss-β-GBP. The purified Ss-β-GBP was functionally analyzed by yeast agglutination and phagocytic reaction assays. Moreover, the PO enhancing ability of Ss-β-GBP was evidenced through PO activity. Specifically, the antibacterial activity of the Ss-β-GBP against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was evaluated by determining its minimum inhibitory concentration (MIC)<60μg/ml for all tested species. Furthermore, the antibiofilm efficacy of Ss-β-GBP at 50 and 100μg/ml was outlined using light microscopy and confocal laser scanning microscopy (CLSM). Bacterial viability assays also outlined the dose-dependent activity of Ss-β-GBP based on the ratio of live/dead bacterial cells. The results of this study revealed that crab-borne Ss-β-GBP might be widely used to suppress the growth of pathogenic bacteria.

Simple, fast and efficient synthesis of β-keto esters from the esters of heteroaryl compounds, its antimicrobial study and cytotoxicity towards various cancer cell lines

A series of β-keto esters were synthesized from heteroaryl esters and ethyl acetate using LiHMDS as base at -50 to -30 °C. The increase in yields of cross condensed product were observed and the percentage of self condensed product was reduced drastically by applying the suitable base (LiHMDS), solvent and the minimum amount of ethyl acetate. All these β-keto esters were characterized using (1)H NMR, (13)C NMR and mass spectral data. A plausible mechanism is also depicted to prove the formation of trans-esterified products. All the synthesized compounds were subjected to test for their cytotoxicity towards various cancer cell lines and also tested for their antimicrobial activity towards various bacterial and fungal strains and some of them were found to have promising activity.