Browsing by Author "Reetesh Kumar"

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A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: Purification and biochemical properties
(2008) Ritu Tomar; Reetesh Kumar; M.V. Jagannadham
Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (ε1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70°C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 μM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-α-benzoyl-DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70°C after 60 min of incubation and 74% of activity after 30 min of incubation at 80°. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures. © 2008 American Chemical Society.
Biochemical and spectroscopic characterization of a novel metalloprotease, cotinifolin from an antiviral plant shrub: Euphorbia cotinifolia
(2011) Reetesh Kumar; Kunwar Awaneesh Singh; Ritu Tomar; Medicherla V. Jagannadham
A high molecular mass novel metalloprotease, cotinifolin is purified from the latex of Euphorbia cotinifolia by a combination of anion exchange and hydrophobic interaction chromatography. The nonglycosylated enzyme has a molecular mass of 79.76 kDa (ESI-MS) and the isoelectric point of the enzyme is pH 7.7. Cotinifolin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity. The Km value of the enzyme was found to be 20 μM with azocasein. The enzyme is not prone to autolysis even at very low concentrations. Polyclonal antibodies specific to enzyme was raised and immunodiffusion reveals that the enzyme has unique antigenic determinants. Maximum caseinolytic activity of cotinifolin is observed in the range of pH 7.0-8.0 and temperature of 50 °C. Using 0.2 mL of 1 mM solution of each metal ion, the purified protease was inhibited slightly by Ba2+ and Mn2+, moderately by Mg2+, Ca2+ and Cs2+ and significantly by Zn2+, Cu2+ and Co2+. On the other hand, substantial activation in caseinolytic activity was achieved by Ni2+. The enzyme activity was also inhibited by EDTA and o-phenanthroline but not by any other protease inhibitors. Perturbation studies by temperature, pH, and chaotrophs of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Spectroscopic studies reveal that cotinifolin has secondary structural features with α/β type with approximately 9% of α-helicity. Easy availability and simple purification procedure makes the enzyme a good system for biophysical study, biotechnological and industrial applications. © 2011 Elsevier Masson SAS.
Biochemical characterization of a peroxidase isolated from Caribbean plant: Euphorbia cotinifolia
(2011) Reetesh Kumar; Kunwar Awaneesh Singh; Vijay Kumar Singh; Medicherla V. Jagannadham
A Caribbean copper plant peroxidase (CCPP) is purified from the latex of Euphorbia cotinifolia, using anion exchange chromatography. The molecular mass and isoelectic point of the enzyme is 43.11 kDa and pH 8.1 respectively. The peroxidase is found to be sensitive towards general phenolic substrates like guaiacol, pyrogallol, α-aminopterin, phloroglucinol, o-phenelenediamine and dianisidine dihydrochloride. The substrate specificity of CCPP was distinct from that of other peroxidases, and the best substrate for CCPP was guaiacol at pH 6.0 and 50°C. Sucrose and Ca2+ enhance the activity whereas the activity is significantly inhibited by NaN3 and Na 2SO3. The strong absorption at 650 nm reveals the presence of Cu ions as a prosthetic group. Spectroscopic studies reveal that CCPP has high α-helicity. The enzyme was found to be very stable at room temperature and retained more than 80% activity even after a period of 2 months and was stable for more than 6 months at 4°C without any additive or preservative. Adequate amount of latex, easy purification method, broad substrate specificity, and high stability against pH, temperature, chaotrophs and organic solvents makes this enzyme a potential candidate in biotechnological and industrial applications. © 2011 Elsevier Ltd. All rights reserved.
Crinumin, a chymotrypsin-like but glycosylated serine protease from Crinum asiaticum: Purification and physicochemical characterisation
(2010) Kunwar Awaneesh Singh; Reetesh Kumar; G.R.K. Rao; Medicherla V. Jagannadham
Plant latex could be a potential source of novel proteases usable in the food and feed industries because of broad substrate specificity with high stability in extreme conditions. Crinumin, a glycosylated serine protease with chymotrypsin-like activity was purified from the latex of Crinum asiaticum using cation-exchange column chromatography. Crinumin shows activity over a wide range of pH (4.5-11.5 and optimum at 8.5), temperature (75 °C and optimum at 70 °C) and is also functional against chaotrophs, organic solvents, and detergents, even after prolonged exposure. The molecular mass (67.7 kDa), extinction coefficient (17.7), isoelectric point (6.9), and numbers of tryptophan (13), tyrosine (24) and cysteine (15 with 7 disulphide bridges) residues were estimated. K m of the enzyme was 31.7 μM with casein and 5 × 10 4 μM with N-succinyl-l-phenylalanine-p-nitroanilide. Easy availability of the aqueous latex, simple purification procedure, high yield (33%), stability and activity in adverse conditions makes it applicable for the pharmaceutical and food industries. © 2009 Elsevier Ltd. All rights reserved.
Evidence for a molten globule state in Cicer α-galactosidase induced by pH, temperature, and guanidine hydrochloride
(2013) Neelesh Singh; Reetesh Kumar; M.V. Jagannadham; Arvind M. Kayastha
Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α+β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40°C; however, above this temperature, denaturation was observed. © 2013 Springer Science+Business Media.
Genetic diversity and population structure analyses using hypervariable microsatellite markers in foxtail millets (Setaria species): Future smart nutri-cereal crop
(Elsevier B.V., 2025) Mula Pratapa Reddy; Ved Prakash Rai; Chagam Venkata Chandra Mohan Reddy; Reetesh Kumar; Rajneesh Paliwal; B. Sinha
Knowledge of genetic diversity in germplasm resources is a prerequisite for crop improvement and successful breeding programs. Foxtail millet (Setaria species), a staple food in certain regions, stands out for its rich content of carbohydrates, proteins, dietary fiber, and essential minerals, contributing to a well-balanced diet. The genetic diversity and population structure of a foxtail millet collection consisting of 107 accessions were measured using 31 class I hypervariable SSR markers (>24 bp). 106 polymorphic alleles were detected, ranging from 2 to 8 alleles per locus, across the entire collection with an average of 3.42 alleles per locus. The average polymorphism information content (PIC), gene diversity (GD) and major allelic frequency (MAF) were 0.49, 0.56 and 0.53, respectively. Unweighted neighbor-joining based cluster analysis classified all the genotypes into four distinct major clusters I, II, III and IV, with 25, 40, 21 and 21 genotypes, respectively. Pair-wise estimates of dissimilarity values ranged from 0.094 to 0.931, with a mean of 0.719. The population structure analysis categorized the entries into four sub-populations, namely SP1, SP2, SP3 and SP4, which have 44, 26, 22, and 15 accessions, respectively. The standardized population distance (Fst) within the populations ranged from SP1 (0.604) to SP4 (0.207) with an average Fst of 0.384. The present study identified eight genotypes based on their genetic distance and favorable phenotypic traits for potential use in future breeding programs. These genotypes include four accessions of Setaria italica (ISe 1387, ISe 1234, ISe 1286, ISe 375), one accession of Setaria viridis (GS 40), one accession of Setaria faberi (SIA 2745), and two landraces (SC 13685 and SC 13653). The genetic insights presented in this study are valuable for the utilization of foxtail millet germplasm in future breeding endeavours. © 2024 SAAB
Identification of folding intermediates of streblin, the most stable serine protease: Biophysical analysis
(2014) Reetesh Kumar; Pinki Tripathi; Fabio Rogerio De Moraes; Ícaro P. Caruso; Medicherla V. Jagannadham
Streblin, a serine proteinase from plant Streblus asper, has been used to investigate the conformational changes induced by pH, temperature, and chaotropes. The near/far UV circular dichroism activities under fluorescence emission spectroscopy and 8-aniline-1-naphthalene sulfonate (ANS) binding have been carried out to understand the unfolding of the protein in the presence of denaturants. Spectroscopic studies reveal that streblin belongs to the α+β class of proteins and exhibits stability towards chemical denaturants, guanidine hydrochloride (GuHCl). The pH-induced transition of this protein is noncooperative for transition phases between pH 0.5 and 2.5 (midpoint, 1.5) and pH 2.5 and 10.0 (midpoint, 6.5). At pH 1.0 or lower, the protein unfolds to form acid-unfolded state, and for pH 7.5 and above, protein turns into an alkaline denatured state characterized by the absence of ANS binding. At pH 2.0 (1 M GuHCl), streblin exists in a partially unfolded state with characteristics of a molten globule state. The protein is found to exhibit strong and predominant ANS binding. In total, six different intermediate states has been identified to show protein folding pathways. © 2013 The Author(s).