01498nas a2200205 4500008004100000022001400041245013100055210006900186260007300255300001200328490000700340520067200347653003401019653002801053653002701081100002101108700002501129700001401154856012401168 2008 eng d a0141-813000apH Induced structural alterations in an aspartic protease from Vigna radiata indicating an alkali induced molten globule state0 apH Induced structural alterations in an aspartic protease from V aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcNOV a373-3760 v433 a

pH-dependent transitions in secondary and tertiary structure are described for a plant aspartic protease from Vigna radiata. The enzyme was pH stable with pH optima of 3.0. The Lineweaver Burk analysis at various pH yielded pK(a) values of 3.3 and 4.29 indicating acidic amino acids at the active site of the enzyme. The structural changes exemplified compact secondary structure collapsed tertiary structure and exposure of hydrophobic patches at pH 10. The changes at pH 10 are typical of a molten globule state. This alkali induced molten globule is novel since acid induced molten globule state is more reported. (C) 2008 Elsevier B.V. All rights reserved.

10aAlkali induced molten globule10aPlant aspartic protease10aStructural transitions1 aKulkarni, Aarohi1 aGaikwad, Sushama, M.1 aRao, Mala uhttp://library.ncl.res.in/content/ph-induced-structural-alterations-aspartic-protease-vigna-radiata-indicating-alkali-002184nas a2200265 4500008004100000022001400041245009100055210006900146260007300215300001000288490000700298520124000305653002201545653003601567653001801603653002301621100002401644700002501668700001901693700002101712700002501733700002001758700002401778856011601802 2012 eng d a0141-813000aPurification and characterization of YxeI, a penicillin acylase from Bacillus subtilis0 aPurification and characterization of YxeI a penicillin acylase f aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcJAN a25-300 v503 a

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent Km of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93 x 10(2) M-1 and 2.51 x 10(5) M-1, respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride. (C) 2011 Elsevier B.V. All rights reserved.

10aBacillus subtilis10aConjugated bile acid hydrolases10aNtn hydrolase10aPenicillin acylase1 aRathinaswamy, Priya1 aGaikwad, Sushama, M.1 aSuresh, C., G.1 aPrabhune, Asmita1 aBrannigan, James, A.1 aDodson, Guy, G.1 aPundle, Archana, V. uhttp://library.ncl.res.in/content/purification-and-characterization-yxei-penicillin-acylase-bacillus-subtilis-002591nas a2200277 4500008004100000022001400041245008800055210006900143260007300212300001200285490000900297520161200306653001701918653003801935653002201973653002101995653002102016653002002037100002502057700002002082700002602102700002402128700002102152700002502173856011502198 2013 eng d a1570-963900aPolyproline fold-In imparting kinetic stability to an alkaline serine endopeptidase0 aPolyproline foldIn imparting kinetic stability to an alkaline se aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAR a708-7160 v18343 a

Polyproline II (PPII) fold, an unusual structural element was detected in the serine protease from Nocardiopsis sp. NCIM 5124 (NprotI) based on far UV circular dichroism spectrum, structural transitions of the enzyme in presence of GdnHCl and a distinct isodichroic point in chemical and thermal denaturation. The functional activity and conformational transitions of the enzyme were studied under various denaturing conditions. Enzymatic activity of NprotI was stable in the vicinity of GdnHCl upto 6.0 M concentration, organic solvents viz, methanol, ethanol, propanol (all 90% v/v), acetonitrile (75% v/v) and proteases such as trypsin, chymottypsin and proteinase K (NprotI:protease 10:1). NprotI seems to be a kinetically stable protease with a high energy barrier between folded and unfolded states. Also, an enhancement in the activity of the enzyme was observed in 1 M GdnHCl upto 8 h, in organic solvents (75% v/v) for 72 h and in presence of proteolytic enzymes. The polyproline fold remained unaltered or became more prominent under the above mentioned conditions. However, it diminished gradually during thermal denaturation above 60 degrees C. Thermal transition studies by differential scanning calorimetry (DSC) showed scan rate dependence as well as irreversibility of denaturation, the properties characteristic of kinetically stable proteins. This is the first report of PPII helix being the global conformation of a non structural protein, an alkaline serine protease, from a microbial source, imparting kinetic stability to the protein. (C) 2013 Elsevier B.V. All rights reserved.

10aConformation10adifferential scanning calorimetry10aKinetic stability10aNocardiopsis sp.10aPolyproline fold10aSerine protease1 aRohamare, Sonali, B.1 aDixit, Vaishali1 aNareddy, Pavan, Kumar1 aSivaramakrishna, D.1 aSwamy, Musti, J.1 aGaikwad, Sushama, M. uhttp://library.ncl.res.in/content/polyproline-fold-imparting-kinetic-stability-alkaline-serine-endopeptidase-002182nas a2200289 4500008004100000022001400041245009500055210006900150260007300219300001000292490000700302520114300309653002601452653003001478653002401508653002201532653003001554653002501584100002301609700001801632700003201650700002301682700001801705700002501723700002701748856011701775 2013 eng d a0141-813000aProbing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala0 aProbing the active site of cinnamoyl CoA reductase 1 LlCCRH1 fro aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcSEP a33-380 v603 a

Lack of three dimensional crystal structure of cinnamoyl CoA reductase (CCR) limits its detailed active site characterization studies. Putative active site residues involved in the substrate/NADPH binding and catalysis for Leucaena leucocephala CCR (Ll-CCRH1; GenBank: DQ986907) were identified by amino acid sequence alignment and homology modeling. Putative active site residues and proximal H215 were subjected for site directed mutagenesis, and mutated enzymes were expressed, purified and assayed to confirm their functional roles. Mutagenesis of S136, Y170 and K174 showed complete loss of activity, indicating their pivotal roles in catalysis. Mutant S212G exhibited the catalytic efficiencies less than 10% of wild type, showing its indirect involvement in substrate binding or catalysis. R51G, D77G, F30V and I31N double mutants showed significant changes in K-m values, specifying their roles in substrate binding. Finally, chemical modification and substrate protection studies corroborated the presence Ser, Tyr, Lys, Arg and carboxylate group at the active site of Ll-CCRH1. (c) 2013 Elsevier B.V. All rights reserved.

10aChemical modification10aCinnamoyl CoA reductase 110aDocking simulations10ahomology modeling10aSite directed mutagenesis10aSubstrate protection1 aSonawane, Prashant1 aPatel, Krunal1 aVishwakarma, Rishi, Kishore1 aSrivastava, Sameer1 aSingh, Somesh1 aGaikwad, Sushama, M.1 aKhan, Bashir, Mohammad uhttp://library.ncl.res.in/content/probing-active-site-cinnamoyl-coa-reductase-1-ll-ccrh1-leucaena-leucocephala-002159nas a2200205 4500008004100000022001400041245011400055210006900169260011100238300001200349490000700361520132800368653002001696653002001716653000801736100002801744700002501772700002501797856013101822 2013 eng d a0929-866500aPurification and characterization of a thermolysin like protease from thermoactinomyces thalpophilus MCMB-3800 aPurification and characterization of a thermolysin like protease aEXECUTIVE STE Y-2, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATESbBENTHAM SCIENCE PUBL LTDcAUG a918-9250 v203 a

The extracellular thermolysin like protease (TLP) was purified and characterized from Thermoactinomyces thalpophilus MCMB-380 (Genbank Accession No. EF397000). The enzyme was purified to homogeneity by successive ultra filtration steps using 50 kDa and 10 kDa membrane filters followed by anion exchange chromatography. The molecular mass and isoelectric point of the enzyme were found to be 34.4 kDa and 9.5, respectively. The proteolytic activity was inhibited by EDTA and the enzyme required Ca2+ to show the full activity as well as thermostability. The T-50 of the enzyme at 80 degrees C was 1 h and the activation energy was estimated to be 11.02 Kcal / mol. Atomic absorption spectrophotometric analysis revealed the presence of Zn2+ ion in the protein core indicating that it is a metalloprotease. This protease has commercial potential in catalyzing the condensation reaction of two amino acids for production of the dipeptide aspartame, an artificial sweetener. The one hour time-frame is significantly faster than that of the enzyme thermolysin from Bacillus thermoproteolyticus. Moreover the TLP was stable at 80 degrees C for one hour which makes it industrially robust. The Zn2+ ion in the T. thalpophilus protease appears to be necessary for maintaining the active conformation of the enzyme molecule.

10aMetalloprotease10aThermostability10aTLP1 aMajumder, Devipriya, R.1 aKanekar, Pradnya, P.1 aGaikwad, Sushama, M. uhttp://library.ncl.res.in/content/purification-and-characterization-thermolysin-protease-thermoactinomyces-thalpophilus-mcmb-001234nas a2200145 4500008004100000245010400041210006900145260006300214520060900277100001600886700001900902700002500921700002000946856012200966 2015 eng d00aPurification and characterization of an agglutinin from datura innoxia with antineoplastic activity0 aPurification and characterization of an agglutinin from datura i aCSIR-National Chemical Laboratory, Pune 411008, IndiacFEB3 a

Lectins are carbohydrate binding proteins of non-immune origin. Due to their multivalency, lectins can be easily detected using agglutination. Lectins are reported to induce cytotoxicity or inhibition of growth in various cancer cells., hence good therapeutic startegy against cancer. Factors such as specificity, stability, assay rapidity, and costs combine to make lectins attractive diagnostic reagents. Datura innoxia belongs to family of Solanaceae.All parts of the plant are hallucinogenic, hypnotic and narcotic. Application: at low dose acts as pain killer, relieves asthma and rheumatism.

1 aSingh, Ruby1 aNawale, Laxman1 aGaikwad, Sushama, M.1 aSuresha, C., G. uhttp://library.ncl.res.in/content/purification-and-characterization-agglutinin-datura-innoxia-antineoplastic-activity02062nas a2200181 4500008004100000022001400041245014300055210006900198260000800267300001400275490000800289520135500297100002601652700002801678700002501706700002101731856012801752 2018 eng d a0141-813000aPioglitazone inhibits advanced glycation induced protein modifications and down-regulates expression of RAGE and NF-kappa B in renal cells0 aPioglitazone inhibits advanced glycation induced protein modific cNOV a1154-11630 v1193 aThe present work aims to determine the effect of pioglitazone on in-vitro albumin glycation and AGE-RAGE induced oxidative stress and inflammation. Bovine serum albumin was glycated by methylglyoxal in absence or presence of pioglitazone. Glycation markers (fructosamine, carbonyl groups, (l-amyloid aggregation, thiol groups, bilirubin binding capacity and AOPP); protein conformational changes (native-PAGE and HPLC analysis) were determined. Cellular study was done by estimating antioxidants, ROS levels, expression profile of membrane RAGE, NF-kappa B and levels of inflammatory cytokines (IL-6, TNF-alpha) using HEK-293 cell line. We observed that levels of glycation markers were reduced at higher concentration of pioglitazone as compared to glycated albumin. Structural analysis of glycated albumin showed inhibition of protein migration and structural changes when treated with pioglitazone. Pioglitazone has potentially restored cellular antioxidants and reduced levels of IL-6 and TNF-alpha by declining expression of membrane RAGE and NF-kappa B. In conclusion, pioglitazone preferentially binds to protein and alleviates protein structural changes by maintaining its integrity. Additionally, it suppresses RAGE and NF-kappa B levels hence alleviate cellular oxidative stress and inflammation. (C) 2018 Elsevier B.V. All rights reserved.1 aAdeshara, Krishna, A.1 aAgrawal, Sanskruthi, B.1 aGaikwad, Sushama, M.1 aTupe, Rashmi, S. uhttp://library.ncl.res.in/content/pioglitazone-inhibits-advanced-glycation-induced-protein-modifications-and-down-regulates02291nas a2200217 4500008004100000022001400041245014700055210006900202260000900271300001200280490000800292520149700300653001501797653001801812653002701830100002601857700002001883700002501903700002001928856012501948 2019 eng d a0141-813000aPotent chitin-hydrolyzing enzyme from Myrothecium verrucaria affects growth and development of Helicoverpa armigera and plant fungal pathogens0 aPotent chitinhydrolyzing enzyme from Myrothecium verrucaria affe cDEC  a517-5280 v1413 a

Chitin, a crucial structural and functional component of insects and fungi, serves as a target for pest management by utilizing novel chitinases. Here, we report the biocontrol potential of recombinant Myrothecium verrucaria endochitinase (rMvEChi) against insect pest and fungal pathogens. A complete ORF of MvEChi (1185 bp) was cloned and heterologously expressed in Escherichia coli. Structure based sequence alignment of MvEChi revealed the presence of conserved domains SXGG and DXXDXDXE specific for GH-18 family, involved in substrate binding and catalysis, respectively. rMvEChi (46.6 kDa) showed optimum pH and temperature as 7.0 and 30 degrees C, respectively. Furthermore, rMvEChi remained stable within the pH range of 6.0 to 8.0 and up to 40 degrees C. rMvEChi exhibited k(cat)/K-m values of 129.83 x 10(3) [(g/L)(-1) s(-1)] towards 4MU chitotrioside. Hydrolysis of chitooligosaccharides with various degrees of polymerization (DP) using rMvEChi indicated the release of DP2 as main end product with order of reaction as DP6 > DP5 > DP4 > DP3. Bioassay of rMvEChi against Helicoverpa armigera displayed potent anti-feedant activity and induced mortality. In vitro antifungal activity against plant pathogenic fungi (Ustilago maydis and Bipolaris sorokiniana) exhibited significant inhibition of mycelium growth. These results suggest that MvEChi has significant potential in enzyme-based pest and pathogen management. (C) 2019 Elsevier B.V. All rights reserved.

10aBiocontrol10aendochitinase10aMyrothecium verrucaria1 aVidhate, Ravindra, P.1 aBhide, Amey, J.1 aGaikwad, Sushama, M.1 aGiri, Ashok, P. uhttp://library.ncl.res.in/content/potent-chitin-hydrolyzing-enzyme-myrothecium-verrucaria-affects-growth-and-development