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Papers by pooja attri

Copyright © 2011 Pooja Attri et al. This is an open access article distributed under the Creative... more Copyright © 2011 Pooja Attri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dipeptidylpeptidase-III (DPP-III) from goat brain was purified and characterized using Arginyl-Arginyl-4-methoxy-βnaphthylamide (Arg-Arg-4mβNA) substrate. This enzyme retained its activity in native 10 % polyacrylamide gel when stained using Arg-Arg-4mβNA. The activity was significantly increased by 100 mM chloride. Studies for its inhibition with some peptides and chemical inhibitors revealed that Leu-Trp-Met-Arg-Phe-Ala was most potent inhibitor followed by Arg-Phe-Ala and Gly-Phe-Leu. All the studied chemical inhibitors caused 40–50 % inhibition at 1 mM. Metal ions helped to regain activity of EDTA pretreated enzyme. ZnCl2 at 50 μM almost completely restored the enzyme activity. Further ZnCl2 and CoCl2 exerted protective effects on E...

International Journal of Peptide Research and Therapeutics, 2021

An arginine aminopeptidase (EC 3.4.11.6) called aminopeptidase B was purified to apparent homogen... more An arginine aminopeptidase (EC 3.4.11.6) called aminopeptidase B was purified to apparent homogeneity from membrane extract of a potential probiotic Pediococcus acidilactici NCDC 252 using successive chromatographies on sephadex G-100 and phenylsepharose CL-4B. Purified enzyme was a heterotrimer with molecular mass of ~ 101.36 kDa. Predicted molecular weight of the enzyme from its gene (93.9 kDa) was close to the calculated molecular weight. The enzyme was optimally active at pH 7.5 and 40 °C. It was strongly inhibited by metal chelating agent and thiol protease inhibitors suggesting that enzyme is a metalloprotease with involvement of thiol. The K m and V max of enzyme for Arg-4mβNA were calculated to be 26 μM and 19.9 nmol/ml/min respectively. Its 3-D structure was modeled and validated using in-silico approach. In-silico analysis revealed Ser, His, Phe, Tyr and Thr to be present at active site of aminopeptidase B. Docking studies revealed that Arg-4mβNA binds with high affinity to the enzyme followed by Lys-4mβNA. The enzyme also hydrolyzed dipeptide-4mβNA derivatives containing hydrophobic amino acids and diaminocarboxylic acids (Arg, Lys and Asp) at the N-termini but not tripeptides, endopeptidase substrates and-βNA derivatives or peptides with proline and phenyl at their N-termini or C-termini.

Lactic acid production by three lactic acid bacteria (LAB) viz. Pediococcus acidilactici, Lactoba... more Lactic acid production by three lactic acid bacteria (LAB) viz. Pediococcus acidilactici, Lactobacillus plantarum and Lactobacillus brevis was optimized using whey. Lactic acid production by liquid and solid state fermentation was also studied with different biowastes. P. acidilactici, L. plantarum and L. brevis produced 21.3, 26.5 and 25.4 g/L of lactic acid respectively after 48 h with whey. P. acidilactici produced maximum lactic acid at 32°C, pH 6.5 and yeast extract concentration of 10 g/dm , L. plantarum at 3 32°C, pH 6.0 and yeast extract concentration of 7.5 g/dm and L. brevis at 37°C, pH 5.5 and yeast extract 3 concentration of 7.5 g/dm . Inoculum size of 2 % was optimum for all lactic acid bacteria. The results were 3 validated by employing a central composite design and response surface methodology. Fermentation resulted in significant production of lactic acid and waste disposal. Apple pomace resulted in maximum yield (>30 g/L). Practical Applications: In this study, ...

Molecular biology reports, 2018

Pediococcus acidilactici is a probiotic lactic acid bacteria possessing studied in-vitro probioti... more Pediococcus acidilactici is a probiotic lactic acid bacteria possessing studied in-vitro probiotic properties. Study of membrane proteins is crucial in developing technological and health applications of probiotic bacteria. Genome analysis of Pediococcus acidilactici revealed about more than 60 proteases/peptidases which need characterization. Dipeptidyl peptidase-III (DPP-III) is studied for first time in prokaryotes and it is a membrane protein in P. acidilactici that has been purified to apparent homogeneity. The enzyme was purified 81.66 fold with 36.75% yield. The specific activity of purified DPP-III was 202.67 U/mg. The protein moved as single band on native PAGE. The purity was also confirmed by in-situ gel assay. However SDS-PAGE analysis revealed it as high molecular weight heterotetramer with molecular weight of 108 kDa. The enzyme was maximally active at pH 8.5 and at 37 C. Purified DPP-III specifically hydrolyzed Arg-Arg-4-βNA with micromolar affinity (K = 9.0 µM) and n...

International Journal of Biological Macromolecules, 2016

Plant based diet such as vegetables, cereals, legumes and oilseeds contain 80% of total phosphoro... more Plant based diet such as vegetables, cereals, legumes and oilseeds contain 80% of total phosphorous in the form of phytic acid-cation complexes, bound phosphorous being excreted in manure due to unavailability of phytate degrader in the gastrointestinal tract of monogastric animals (Ashraf et al., 2013). The undegraded phytate leads to phosphorous deficiency in animals, elevated levels of phosphorous in soil and eutrophication of water bodies and renders phytic acid as the anti-nutritive factor by decreasing the bioavailability cations such as iron, calcium, magnesium, phosphorous, zinc, iodine, etc (Madsen, 2019; Singh et al., 2013). Most of these cations are involved in various physiological functions as their deficiency may lead to conditions such as anemia,

Annals of biology

For past 60 years, antibiotics have been critical in fighting against infectious diseases caused ... more For past 60 years, antibiotics have been critical in fighting against infectious diseases caused by bacteria and other microbes. Misuse and overuse of antibiotics leads to antibiotic resistance among bacteria and consequent treatment complications and increased healthcare costs. Vancomycin is a tricyclic glycopeptide antibiotic derived from Amycolatopsis orientalis (formerly Nocardia orientalis) that functions by inhibiting the synthesis of peptidoglycan and thus has a very broad spectrum of activity. Pediococcus acidilactici is a nonpathogenic lactic acid bacteria and is used in food and dairy industry for various purpose. It is generally regarded as safe (GRAS). But, it is an opportunistic pathogen and its vancomycin resistant strains cause blood scepticemia. Recently vancomycin resistant strains of this bacterium have been reported but no detailed study is available. We have determined the minimum inhibitory concentration (MIC) of vancomycin for Pediococcus acidilactici and vanco...

Arginyl-arginyl-4-methoxy-β-naphthylamide (Arg-Arg-4mβNA) hydrolyzing activity is known as dipept... more Arginyl-arginyl-4-methoxy-β-naphthylamide (Arg-Arg-4mβNA) hydrolyzing activity is known as dipeptidylpeptidase-III (DPP-III). DPP-III enzyme hydrolyzes various bioactive peptides, including enkephalins, angiotensins, gastric peptides and Arg-Arg-4mβNA. We report the presence of DPP-III enzyme in plants for the first time. The enzyme was noticed in mung bean [Vigna radiata] seeds germinated at 25ºC for 48 h. The crude enzyme preparation had optimum activity at alkaline pH of 8.0. Sub-cellular distribution of this enzyme revealed its cytosolic localization. Enzyme activity when investigated in different parts of mung bean plant revealed maximum activity in seeds followed by roots, stem and leaves. The ubiquitous distribution of this enzyme signifies housekeeping role of this enzyme in plant physiology. Maximum activity in seeds indicates its possible role in germination.

Applied Biochemistry and Biotechnology, 2014

DING proteins are intriguing proteins characterized by conserved N-terminal sequence. In spite of... more DING proteins are intriguing proteins characterized by conserved N-terminal sequence. In spite of unusually high sequence conservation even between distantly related species, DING proteins exhibit outstanding functional diversity. An extracellular caseinolytic alkaline enzyme was purified to homogeneity from a probiotic lactic acid bacteria Pediococcus acidilactici NCDC 252 using a simple procedure involving ammonium sulphate precipitation and gel filtration chromatography. This was purified 45.72-fold with a yield and specific activity of 43.5 % and 250 U/mg, respectively. The calculated molecular weight was 38.7 and 38.9 kDa by MALDI and SDS-PAGE, respectively, and pI was 7.77. The enzyme exhibited optimal activity at pH 8.0 and 40 °C. It was considerably stable up to pH 12. For casein, the enzyme had K m of 20 μM with V max of 26 U/ml. The enzyme was resistant to organic solvents but sensitive to DTNB and EDTA that confirmed it as thiol protein with involvement of metal ions in catalysis. Its tryptic peptide fragments showed 95 % similarity with eukaryotic DING, i.e., human phosphate binding protein (HPBP). Homology-based structure evaluation using HBPB as template revealed both to be structurally conserved and also possessing conserved phosphate binding motifs.

International Journal of Dairy Technology, 2015

ABSTRACT In vitro probiotic attributes of Pediococcus acidilactici, viz. tolerance to acidity and... more ABSTRACT In vitro probiotic attributes of Pediococcus acidilactici, viz. tolerance to acidity and bile salt, phenol resistance, lactic acid production, antioxidant activity, cell surface hydrophobicity, lysozyme resistance and the presence of β-galactosidase, proteolytic, lipase and peptidoglycan hydrolase activities, were studied. The strain was found to be rich in different proteolytic activities, β-galactosidase and antioxidant activities, and produced lactic acid. The studies conducted support that P. acidilactici national collection of dairy cultures (NCDC) 252 is a potential probiotic for humans with all the essential basic probiotic properties. This testing is useful to gain insight into this strain and its mechanism of action. All these attributes add to its therapeutic importance and industrial significance.

Enzyme Research, 2011

Dipeptidylpeptidase-III (DPP-III) from goat brain was purified and characterized using Arginyl-Ar... more Dipeptidylpeptidase-III (DPP-III) from goat brain was purified and characterized using Arginyl-Arginyl-4-methoxy-β-naphthylamide (Arg-Arg-4mβNA) substrate. This enzyme retained its activity in native 10% polyacrylamide gel when stained using Arg-Arg-4mβNA. The activity was significantly increased by 100 mM chloride. Studies for its inhibition with some peptides and chemical inhibitors revealed that Leu-Trp-Met-Arg-Phe-Ala was most potent inhibitor followed by Arg-Phe-Ala and Gly-Phe-Leu. All the studied chemical inhibitors caused 40–50% inhibition at 1 mM. Metal ions helped to regain activity of EDTA pretreated enzyme. ZnCl2at 50 μM almost completely restored the enzyme activity. Further ZnCl2and CoCl2exerted protective effects on EDTA pretreated enzyme for its susceptibility to DTNB inhibition. Therefore, DPP-III is a metalloprotease with the involvement of cysteine residues either located at the catalytic site or involved in regulation.

Current Pharmaceutical Design, 2012

Analytical Methods, 2012

ABSTRACT A simple and reproducible method for extraction of membrane enzymes from Gram positive b... more ABSTRACT A simple and reproducible method for extraction of membrane enzymes from Gram positive bacteria in active form has been developed. The method employs the incubation of cells with lysozyme and sonication in the presence of salt and detergent followed by repeated freezing and thawing to disrupt the lipid–protein interactions. Although developed for Dipeptidylpeptidase-III as a demonstration, this protocol has been used for the successful extraction of some synthetic substrate hydrolyzing activities from three different Gram positive bacteria with yield of more than 90% for most studied activities. This method can be routinely used for a wide range of applications and it can also be used to extract and solubilize other enzymes/proteins only by manipulating detergent, salt and their concentration. It is simple, straightforward, reproducible, cost effective and a less time consuming protocol for extracting and solubilizing membrane enzymes.