Browsing by Author "Arvind M. Kayastha"

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A comparison of proline, thiol levels and GAPDH activity in cyanobacteria of different origins facing temperature-stress
(2005) A.P. Singh; R.K. Asthana; Arvind M. Kayastha; S.P. Singh
Three cyanobacterial strains originating from different habitats were subjected to temperature shift exposures and monitored for levels of proline, thiol and activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Thermophile Mastigocladus laminosus (growth optimum, 40°C), raised the proline level 4.2-fold at low temperature (20deg;C), for the psychrophile Nostoc 593 (growth optimum, 20°C), it was raised 8-fold at 40°C while in the mesophile Nostoc muscorum (growth optimum, 30°C), the imino acid level increased 2.3-fold during temperature 'shiftdown' to 20°C or 3.5-fold in sets facing 'shiftup' (40°C). Alterations in thiol levels in the above strains were in line with proline. It is suggested that such fluctuations reflect metabolic shifts as a response to stress. Interestingly, GAPDH activity was maximum at the respective growth temperature optimum of M. laminosus (122 nmol NADPH oxidized min-1 mg-1 protein) and Nostoc 593 (141 nmol NADPH oxidized min-1 mg-1 protein) while in N. muscorum, it increased at 40°C (101 nmol NADPH oxidized min-1 mg-1 protein) and to 93.3 nmol NADPH oxidized min-1 mg-1 protein (20°C) relative to 86 nmol NADPH oxidized min -1 mg-1 protein at 30°C. It seems that extremophiles maintain the GAPDH activity/level during growth at their respective temperatures optimal while the mesophile increases it in order to cope up with temperature-stress.
A novel application of Cicer α-galactosidase in reduction of raffinose family oligosaccharides in soybean flour
(Springer, 2013) Neelesh Singh; Arvind M. Kayastha
Raffinose family oligosaccharide (RFOs) especially raffinose and stachyose are responsible for flatulence during intake of soybean derived products. This gives negative impression on the acceptance of soy-based foods. The effect of soaking, cooking and Cicer α-galactosidase treatment for reducing stachyose and raffinose content from the soybean flour was investigated. Cicer α-galactosidase treatment was found to be most effective in comparison to traditional methods, soaking and cooking. Enzymatic treatment for 6 h reduces raffinose and stachyose component of soybean flour by 80.0 % and 85.0 %, respectively. This can be utilized effectively for improving the nutritional quality of soy-based foods on large scale, which could be one of the best alternatives for lactose intolerant population for fulfilling their protein requirement. © 2012 Society for Plant Biochemistry and Biotechnology.
A simple laboratory experiment for teaching enzyme immobilization with urease and its application in blood urea estimation
(Elsevier Ltd, 1999) Arvind M. Kayastha; Nilanjana Das
An experiment is described in which students carry out urease purification, immobilization and its application in blood urea estimation. Urease from pigeonpea is partially purified using acetone fractionation and then immobilized on calcium alginate in the form of beads. The immobilized enzyme has a better shelf-life at 4°C than soluble enzyme. Various aspects of enzyme immobilization are discussed. Blood urea estimation is carried out with immobilized enzyme beads and the beads can be used repeatedly for this purpose making it an economical procedure compared to commercial kits.
A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification, biochemical properties and industrial applications
(2012) Devesh Kishore; Arvind M. Kayastha
A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg-1, with approximately 1840 fold purification. Analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC-MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a Km value of 1.73 mM at 37°C. The energy of activation (Ea) calculated in the range of 35 to 60°C, using Arrhenius equation, was determined to be 11.32 kcal mol-1. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40°C. Km and Ea for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol-1, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60°C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology. © 2012 Elsevier Ltd. All rights reserved.
A β-galactosidase from pea seeds (PsBGAL): purification, stabilization, catalytic energetics, conformational heterogeneity, and its significance
(2009) Alka Dwevedi; Arvind M. Kayastha
A basic glycosylated β-galactosidase (PsBGAL) has been purified from pea seeds by 910-fold with a specific activity of 77.33 μmol min-1 mg-1 protein. The purified enzyme is an electrophoretically homogeneous protein consisting of a single protein band with an apparent Mr of 55 kDa, while the deglycosylated enzyme has a Mr of 54.2 kDa on SDS-PAGE under reducing conditions. According to MALDI-TOF measurements of the 55 kDa band, the enzyme showed a homology with BGAL from other sources present in the SWISS-PROT database, while it showed no resemblance to any lectin. The N-terminal sequence of PsBGAL was determined as TIECK and showed a resemblance to BGAL from Arabidopsis thaliana (Q93Z24). The enzyme showed an unique property of multiple banding patterns on SDS-PAGE at 20 mA current, with tryptic digests of all bands having similar m/z values (using MALDI-TOF) while it showed only a single band at 10 mA current. PsBGAL is effectively compartmentalized during seed maturation inside vacuoles (pH ∼ 5). The enzyme is capable of hydrolyzing pea seed xyloglucan, and it may be involved in modifying the cell wall architecture during seedling growth and development. The enzyme has a protonated carboxyl group at its active site as observed by ionization constant, thermodynamics, and chemical modification studies. © 2009 American Chemical Society.
Advances in the molecular recognition of DNA - A step towards therapeutic drug design
(1995) Arvind M. Kayastha; Prakash Prabhakar; O.P. Malhotra
[No abstract available]
An antioxidant rich novel β-amylase from peanuts (Arachis hypogaea): Its purification, biochemical characterization and potential applications
(Elsevier B.V., 2018) Ranjana Das; Arvind M. Kayastha
β-Amylase from un-germinated seeds of peanut (Arachis hypogaea) was purified to apparent electrophoretic homogeneity with final purification fold of 205 and specific activity of 361 μmol/min/mg protein. The enzyme was purified employing simple purification techniques for biochemical characterization. The purified enzyme was identified as β-amylase with Mr of 31 kDa. The enzyme displayed its optimum catalytic activity at pH 5.0 and 60 °C with activation energy of 4.5 kcal/mol and Q10 1.2. The enzyme displayed Km and Vmax values, for soluble potato starch of 1.28 mg/mL and 363.63 μmol/min/mg, respectively. Thermal inactivation of β-amylase at 65 °C resulted into first-order kinetics with rate constant 0.0126 min− 1 and t½ 55 min. The enzyme was observed to act on native granular potato starch, which could minimize the high cost occurring from solubilization of starch in industries. Enzyme fractions scavenge 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical, indicating its antioxidative nature. In addition, the purified β-amylase was successfully utilized for the improvement of antioxidant potential of wheat. These findings suggest that β-amylase from peanuts have potential application in food and pharmaceutical industries. © 2018 Elsevier B.V.
An Assay Procedure for Determining the Rate of an Enzyme Reaction Lacking an Optical Signal: Validity of Coupled Enzyme Assays
(Elsevier Ltd, 1996) O.P. Malhotra; P.K. Ambasht; Prakash Prabhakar; Ashish K. Lal; Arvind M. Kayastha
[No abstract available]
An NMR based panorama of the heterogeneous biology of acute respiratory distress syndrome (ARDS) from the standpoint of metabolic biomarkers
(John Wiley and Sons Ltd, 2020) Akhila Viswan; Chandan Singh; Arvind M. Kayastha; Afzal Azim; Neeraj Sinha
Acute respiratory distress syndrome (ARDS), manifested by intricate etiology and pathophysiology, demands careful clinical surveillance due to its high mortality and imminent life support measures. NMR based metabolomics provides an approach for ARDS which culminates from a wide spectrum of illness thereby confounding early manifestation and prognosis predictors. 1H NMR with its manifold applications in critical disease settings can unravel the biomarker of ARDS thus holding potent implications by providing surrogate endpoints of clinical utility. NMR metabolomics which is the current apogee platform of omics trilogy is contributing towards the possible panacea of ARDS by subsequent validation of biomarker credential on larger datasets. In the present review, the physiological derangements that jeopardize the whole metabolic functioning in ARDS are exploited and the biomarkers involved in progression are addressed and substantiated. The following sections of the review also outline the clinical spectrum of ARDS from the standpoint of NMR based metabolomics which is an emerging element of systems biology. ARDS is the main premise of intensivists textbook, which has been thoroughly reviewed along with its incidence, progressive stages of severity, new proposed diagnostic definition, and the preventive measures and the current pitfalls of clinical management. The advent of new therapies, the need for biomarkers, the methodology and the contemporary promising approaches needed to improve survival and address heterogeneity have also been evaluated. The review has been stepwise illustrated with potent biometrics employed to selectively pool out differential metabolites as diagnostic markers and outcome predictors. The following sections have been drafted with an objective to better understand ARDS mechanisms with predictive and precise biomarkers detected so far on the basis of underlying physiological parameters having close proximity to diseased phenotype. The aim of this review is to stimulate interest in conducting more studies to help resolve the complex heterogeneity of ARDS with biomarkers of clinical utility and relevance. © 2019 John Wiley & Sons, Ltd.
An Overview on Starch Processing and Key Enzymes
(Springer Nature, 2023) Ranjana Das; Arvind M. Kayastha
Starch makes an important component of human and cattle diets. A large portion of our food and fodder originates from them. In addition, chemically or enzymatically processed starch is used as a variety of products such as starch hydrolysates, glucose syrups, maltose, fructose, starch or maltodextrin derivatives or cyclodextrins. These enzymes for starch processing exist in natural resources and are categorized as (i) endoamylases, (ii) exoamylases, (iii) debranching enzymes, and (iv) transferases; on the type of reaction, they catalyze. The individual category and their corresponding enzymes are discussed in detail in this chapter. The aim of this chapter is a general study of these enzymes’ operation in various industries for the production of newly refined starch. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023.
Antibacterial potential of γ-linolenic acid from Fischerella sp. colonizing Neem tree bark
(2006) Ravi K. Asthana; Arunima Srivastava; Arvind M. Kayastha; Gopal Nath; Sureshwar P. Singh
Pharmaceutically important γ-linolenic acid (GLA) was produced (4.1 mg g-1 dry wt) by laboratory grown cyanobacterium Fischerella sp. colonizing Neem (Azadirachta indica) tree bark. GLA isolated from the test cyanobacterium was active against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25992, Salmonella typhi (local strain), Pseudomonas aeruginosa ATCC 27853 and Enterobacter aerogenes MTCC 2822. The overproduction of GLA was also monitored by altering phosphate and nitrate levels in the nutrient medium. A doubling in phosphate concentration (58 μM) increased GLA level up to 12% over that of control cells while half of this phosphate level reduced GLA synthesis by 8%. In contrast, elevated nitrate concentrations (5 and 10 mM) stimulated biomass yield but not GLA, as the levels approximated to the nitrate-lacking control. The antibacterial potential of GLA from Fischerella sp. grown at varying P or N levels was at variance as evidenced by the diameter of inhibition zones against S. aureus. This variation in inhibition zones reflected differing levels of GLA as ascertained quantitatively by HPLC. © Springer 2005.
Application of short column gel permeation in the study of protein - protein interactions
(Elsevier, 1996) O.P. Malhotra; Prakash Prabhakar; Arvind M. Kayastha
A simple and rapid procedure based on the gel filtration principle is described together with its applicability to the study of protein - protein interactions including subunit - subunit and enzyme - enzyme interactions. Using this procedure, it is shown that phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GPDH) interact with a stoichiometry of one PGK molecule combining with one monomeric subunit of GPDH. This interaction has been observed with both enzymes being from the same, as well as from different, species. The Kd values for rabbit muscle PGK and porcine muscle GPDH complex and that for the rabbit muscle PGK and yeast GPDH complex are found to be (4.5 ± 2.0) × 10-7 M and (6.5 ± 1.7) × 10-7 M, respectively. The specificity of bienzyme association is stronger when enzymes are from the same species than when they are from different species.
B-amylase from starchless seeds of Trigonella foenum-graecum and its localization in germinating seeds
(Public Library of Science, 2014) Garima Srivastava; Arvind M. Kayastha
Fenugreek (Trigonella foenum-graecum) seeds do not contain starch as carbohydrate reserve. Synthesis of starch is initiated after germination. A β-amylase from ungerminated fenugreek seeds was purified to apparent electrophoretic homogeneity. The enzyme was purified 210 fold with specific activity of 732.59 units/mg. Mr of the denatured enzyme as determined from SDS-PAGE was 58 kD while that of native enzyme calculated from size exclusion chromatography was 56 kD. Furthermore, its identity was confirmed to be β-amylase from MALDI-TOF analysis. The optimum pH and temperature was found to be 5.0 and 50°C, respectively. Starch was hydrolyzed at highest rate and enzyme showed a Km of 1.58 mg/mL with it. Antibodies against purified Fenugreek β-amylase were generated in rabbits. These antibodies were used for localization of enzyme in the cotyledon during different stages of germination using fluorescence and confocal microscopy. Fenugreek β-amylase was found to be the major starch degrading enzyme depending on the high amount of enzyme present as compared to α-amylase and also its localization at the periphery of amyloplasts. A new finding in terms of its association with protophloem was observed. Thus, this enzyme appears to be important for germination of seeds. © 2014 Srivastava, Kayastha.
Biochemical and thermodynamic characterization of de novo synthesized β-amylase from fenugreek
(Elsevier B.V., 2019) Dinesh Chand Agrawal; Alka Dwevedi; Arvind M. Kayastha
β-Amylase has been de novo synthesized from germinating fenugreek seeds. Enzyme has been isolated and purified from 36 h germinated seeds with 226-fold purification and specific activity of 763 U/mg. Homogeneity of the purified β-amylase has been confirmed with size-exclusion chromatography, SDS-PAGE and MALDI MS/MS analysis. The isoelectric point, optimum pH and temperature of the enzyme were found to be pH 5.2, 5.7 and 57 °C, respectively. The enzyme was specific for soluble starch with Km and Vmax of 2.4 mg/mL and 833.3 U/mg, respectively. Maltose was found to be competitive inhibitor of the enzyme with inhibition constant (Ki) of 14 mM. However, metallic ions like Ag+ and Hg2+ were found to be non-competitive inhibitors of the enzyme. Thermodynamic parameters like Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes have further revealed that thermal denaturation of the enzyme has followed first-order with the enzyme unfolding rather an aggregation with the process being irreversible. The activation energy of β-amylase during thermal activation and denaturation were 27.5 kJ/mol and 145.23 kJ/mol, respectively at R2 > 0.92. Thus, the enzyme was stable even at higher temperature with ability of undergoing catalysis making it commercially exploitable, particularly in food and pharmaceutical industries. © 2019 Elsevier B.V.
Bioinformatics for legume genomics research
(Springer New York, 2014) Vinay Kumar Singh; A.K. Singh; Arvind M. Kayastha; B.D. Singh
Enormous legume genome sequence data are becoming available at a rapid rate through the Next-Gen Sequencing platforms. One of the biggest problems relates to management and analysis of the huge data derived from whole genome sequencing projects. To resolve this problem, researchers index their data in major biological depository systems and availability of algorithms, tools, softwares and databases and provide opportunities for analysis, annotation, and visualization of sequence data at the computational level. Different types of tools and softwares are available for the interpretation of genomes, proteomes and genes. Now researchers are using various in-silico techniques in Bio-omics (genomics, proteomics, metabolomics and transcriptomics) era for management, planning and prediction of data in cost effective and less time consuming manner. Bio-omics plays an important role in comparative, structural and functional biology at computational level and will play major role in different biological investigations. Identification of signal transduction pathway-associated members and gene family members will help in functional elucidation and relationship among them. In this context identification of potential candidate genes will provide an opportunity to researchers for improvement and nutritional quality enhancement of crop genomes. Based on genome blue-prints (plants, animals, fungus, microbes) one can develop potential applications to understand systems biology of legumes in fullness. © Springer Science+Business Media New York 2014. All rights are reserved.
Biophysical Investigation of the Interplay between the Conformational Species of Domain-Swapped GB1 Amyloid Mutant through Real–Time Monitoring of Amyloid Fibrillation
(American Chemical Society, 2021) Renuka Ranjan; Nidhi Tiwari; Arvind M. Kayastha; Neeraj Sinha
Mutant polypeptide GB1HS#124F26A, which is known to aggregate into amyloid-like fibrils, has been utilized as a model in this study for gaining insights into the mechanism of domain-swapped aggregation through real-time monitoring. Size exclusion with UV monitoring at 280 nm and dynamic light scattering (DLS) profiles through different time points of fibrillation reveal that the dimer transitions into monomeric intermediates during the aggregation, which could further facilitate domain swapping to form amyloid fibrils. The 1D 1H and 2D 1H–13C HSQC nuclear magnetic resonance (NMR) spectra profiling through different time points of fibrillation reveal that there may be some other species present along with the dimer during aggregation which contribute to different trends for the intensity of protons in the spectral peaks. Diffusion NMR reveals changes in the mobility of the dimeric species during the process of aggregation, indicating that the dimer gives rise to other lower molecular weight species midway during aggregation, which further add up to form the oligomers and amyloid fibrils successively. The present work is a preliminary study which explores the possibility of utilizing biophysical methods to gain atomistic level insights into the different stages of aggregation. © 2021 The Authors. Published by American Chemical Society
Biotechnology programme at Banaras Hindu University, India
(1992) Arvind M. Kayastha; B.D. Singh
[No abstract available]
Boric acid and boronic acids inhibition of pigeonpea urease
(2006) K. Ravi Charan Reddy; Arvind M. Kayastha
Urease from the seeds of pigeonpea was competitively inhibited by boric acid, butylboronic acid, phenylboronic acid, and 4-bromophenylboronic acid; 4-bromophenylboronic acid being the strongest inhibitor, followed by boric acid > butylboronic acid > phenylboronic acid, respectively. Urease inhibition by boric acid is maximal at acidic pH (5.0) and minimal at alkaline pH (10.0), i.e., the trigonal planar B(OH)3 form is a more effective inhibitor than the tetrahedral anionic form. Similarly, the anionic form of phenylboronic acid was least inhibiting in nature.
Carbon nanotubes molybdenum disulfide 3D nanocomposite as novel nanoscaffolds to immobilize Lens culinaris β-galactosidase (Lsbgal): Robust stability, reusability, and effective bioconversion of lactose in whey
(Elsevier Ltd, 2019) Anjali Yadav; Sumit Kumar Pandey; Dinesh Chand Agrawal; Himanshu Mishra; Anchal Srivastava; Arvind M. Kayastha
Multiwalled carbon nanotubes molybdenum disulfide 3D nanocomposite (MWCNT-MoS2 NC) was successfully synthesized via eco-friendly hydrothermal method. The microstructural characterization of synthesized nanocomposite was carried out using different spectroscopic and microscopic techniques. Nanocomposite was activated using glutaraldehyde chemistry and used as a platform to immobilize Lens culinaris β-galactosidase (Lsbgal) which resulted in 93% of immobilization efficiency. Attachment of Lsbgal onto nanocomposite was confirmed by AFM, FE-SEM, FTIR, and CLSM. The nanobiocatalyst showed broadening in operational pH and temperature working range. Remarkable increase in thermal stability was observed as compared to soluble enzyme. Nanobiocatalyst showed outstanding increase in storage stability, retained 92% of residual activity over a period of 8 months. This offers good reusability as it retained ∼50% residual activity up to 21 reuses and exhibited higher rate of lactose hydrolysis in whey. MWCNT-MoS2 NC conjugated to biomolecules can serve as a potential platform for fabrication of lactose biosensor. © 2019 Elsevier Ltd
Cicer a-galactosidase immobilization onto functionalized graphene nanosheets using response surface method and its applications
(Elsevier Ltd, 2014) Neelesh Singh; Garima Srivastava; Mahe Talat; Himanshu Raghubanshi; Onkar Nath Srivastava; Arvind M. Kayastha
Cicer a-galactosidase was immobilized onto functionalized graphene with immobilization efficiency of 84% using response surface methodology (Box-Behnken design). The immobilized enzyme had higher thermal stability than the soluble one, attractive for industrial applications. Immobilization of the enzyme lowered the Km to 1/3rd compared to the soluble enzyme. Raffinose family oligosaccharides (RFOs) are mainly responsible for flatulence by taking soybean derived food products. The immobilized enzyme can be used effectively for the hydrolysis of RFOs. After ten successive runs, the immobilized enzyme still retained approximately 60% activity, with soybean RFOs. The easy availability of enzyme source, ease of its immobilization on matrices, non-toxicity, increased stability of immobilized enzyme and effective hydrolysis of RFOs increase the Cicer a-galactosidase application in food processing industries. © 2013 Elsevier Ltd.