02648nas a2200241   4500008004100000022001400041245013000055210006900185260009500254300001200349490000800361520173000369653002202099653002302121653001702144653002802161653001902189653002002208653001402228100001902242700002502261856012002286       2010                        eng d  a0006-291X00aMultistate unfolding of alpha-mannosidase from Canavalia ensiformis (Jack Bean): evidence for the thermostable molten globule0 aMultistate unfolding of alphamannosidase from Canavalia ensiform  a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcDEC  a391-3970 v4033 a<p>The relevance of partially ordered states of proteins (such as the molten-globule state) in cellular processes is beginning to be understood. We examined the conformational transitions in a multimeric and high molecular weight class II alpha-mannosidase from Canavalia ensiformis (Jack Bean) (Jb alpha-man) utilizing intrinsic fluorescence, solute quenching, hydrophobic dye binding, size exclusion chromatography and circular dichroism (CD) spectroscopy for the protein in presence of Guanidine hydrochloride (GdnHCl). The decomposition analysis of the protein spectra obtained during unfolding showed progressive appearance of class S, I, II and III trp. The parameter A and spectral center of mass showed multi state unfolding of the protein and phase diagram analysis revealed formation of an intermediate of Jb alpha-man in the vicinity of 1 M GdnHCl. The intermediate exhibited compact secondary and distorted tertiary structure with exposed hydrophobic amino acids on the surface, indicating the molten-globule nature. The dissociation, partial unfolding and aggregation of Jb alpha-man occurred simultaneously during chemical denaturation. The molten-globule possessed slightly higher hydrodynamic radius, perturbance in the structure up to 60 degrees C and stability of the structure up to 80 degrees C unlike the native Jack Bean alpha-mannosidase. The modes of chemical and thermal denaturation of the native protein were different. The solute quenching parameters confirmed the altered confirmation of the intermediate. Taken together, our results constitute one of the early reports of formation of GdnHCl induced molten globule in a class II alpha-mannosidase. (C) 2010 Elsevier Inc. All rights reserved.</p>10aalpha-Mannosidase10aCircular dichroism10afluorescence10aGuanidine hydrochloride10aMolten globule10aThermostability10aUnfolding1 aKumar, Avinash1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/multistate-unfolding-alpha-mannosidase-canavalia-ensiformis-jack-bean-evidence-002160nas a2200289   4500008004100000022001400041245011900055210006900174260007500243300001200318490000700330520114000337653001201477653002601489653001801515653001901533653002401552653002001576653001401596100002001610700002501630700001901655700001801674700002001692700002701712856013101739       2011                        eng d  a1053-050900aConformational transitions in ariesaema curvatum lectin: characterization of an acid induced active molten globule0 aConformational transitions in ariesaema curvatum lectin characte  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR  a753-7630 v213 a<p>Biophysical characterization of a lectin from Ariesaema curvatum (ACL) was carried out using steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. An intermediate with altered tryptophan microenvironment was detected in the phase diagram, which exibited pronounced secondary structure and hemagglutinating activity in presence of 0.25 M Gdn-HCl. An acid induced molten- globule like structure possessing activity and higher thermostability was detected. Transition to the molten globule state was reversible in nature. The lectin retained hemagglutinating activity even after incubation at 95 A degrees C. Both chemical and thermal unfolding of the lectin were found to consist of multistate processes. Fluorescence quenching of ACL was strong with acrylamide and KI. The single tryptophan was found to be surrounded by high density of the positively charged amino acid residues as shown by a ten fold higher K(sv) for KI compared to that for CsCl. The average lifetime of tryptophan fluorescence increased from 1.24 ns in the native state to 1.72 ns in the denatured state.</p>10aAraceae10aFluorescence lifetime10aHemagglutinin10aMolten globule10aSecondary structure10aThermostability10aUnfolding1 aSharma, Urvashi1 aGaikwad, Sushama, M.1 aSuresh, C., G.1 aDhuna, Vikram1 aSingh, Jatinder1 aKamboj, Sukhdev, Singh  uhttps://library.ncl.res.in/content/conformational-transitions-ariesaema-curvatum-lectin-characterization-acid-induced-active-001476nas a2200289   4500008004100000022001400041245011400055210006900169260007600238300001200314490000700326520044800333653002000781653002200801653001900823653002200842653002000864653002000884100002900904700001800933700001900951700002100970700002000991700002401011700001901035856013201054       2012                        eng d  a1744-309100aCrystallization and X-ray structure analysis of a thermostable penicillin G acylase from alcaligenes faecalis0 aCrystallization and Xray structure analysis of a thermostable pe  aCOMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USAbWILEY-BLACKWELLcMAR  a273-2770 v683 a<p>{The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C2221, with unit-cell parameters a = 72.9</p>10acalcium binding10adisulfide bridges10aNtn hydrolases10aorthorhombic form10atetragonal form10aThermostability1 aVarshney, Nishant, Kumar1 aKumar, Suresh1 aIgnatova, Zoya1 aPrabhune, Asmita1 aPundle, Archana1 aDodson, Eleanor, J.1 aSuresh, C., G.  uhttps://library.ncl.res.in/content/crystallization-and-x-ray-structure-analysis-thermostable-penicillin-g-acylase-alcaligenes-002491nas a2200253   4500008004100000022001400041245010500055210006900160260007900229300001200308490000800320520154900328653002101877653002101898653003301919653001701952653002001969100002101989700002702010700002602037700001402063700002802077856013202105       2012                        eng d  a0273-228900aProduction, purification, and characterization of a beta-glucosidase of penicillium funiculosum NCL10 aProduction purification and characterization of a betaglucosidas  a999 RIVERVIEW DRIVE SUITE 208, TOTOWA, NJ 07512 USAbHUMANA PRESS INCcJUL  a959-9720 v1673 a<p>Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of beta-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular beta-glucosidase production was studied in submerged fermentation. At 30 A degrees C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of beta-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major beta-glucosidase (Bgl3A) with the apparent molecular weight of similar to 120 kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60 A degrees C, and exhibited a half-life of 1 h at 60 A degrees C. K (m) of Bgl3A was found to be 0.057 mM with p-nitrophenyl beta-d-glucoside and V (max) was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K (i) of 1.5 mM. Bgl3A readily hydrolyzed glucosides with beta-linkage. Bgl3A activity was enhanced (156%) by Zn2+ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.</p>10abeta-Glucosidase10aPaper mill waste10aPenicillium funiculosum NCL110aPurification10aThermostability1 aRamani, Gurusamy1 aMeera, Balasubramanian1 aVanitha, Chinnathambi1 aRao, Mala1 aGunasekaran, Paramasamy  uhttps://library.ncl.res.in/content/production-purification-and-characterization-beta-glucosidase-penicillium-funiculosum-ncl1-002160nas a2200205   4500008004100000022001400041245011400055210006900169260011100238300001200349490000700361520132800368653002001696653002001716653000801736100002801744700002501772700002501797856013201822       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<p>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.</p>10aMetalloprotease10aThermostability10aTLP1 aMajumder, Devipriya, R.1 aKanekar, Pradnya, P.1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/purification-and-characterization-thermolysin-protease-thermoactinomyces-thalpophilus-mcmb-002079nas a2200229   4500008004100000022001400041245008700055210006900142260007300211300001000284490000700294520124300301653004001544653001701584653001901601653002001620653001401640100002701654700002701681700002501708856011601733       2014                        eng d  a0141-813000aConformational transitions of cinnamoyl CoA reductase 1 from leucaena leucocephala0 aConformational transitions of cinnamoyl CoA reductase 1 from leu  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAR  a30-350 v643 a<p>Conformational transitions of cinnamoyl CoA reductase, a key regulatory enzyme in lignin biosynthesis, from Leucaena leucocephala (L1-CCRH1) were studied using fluorescence and circular dichroism spectroscopy. The native protein possesses four trp residues exposed on the surface and 66% of helical structure, undergoes rapid structural transitions at and above 45 C and starts forming aggregates at 55 C. LI-CCRH1 was transformed into acid induced (pH 2.0) molten globule like structure, exhibiting altered secondary structure, diminished tertiary structure and exposed hydrophobic residues. The molten globule like structure was examined for the thermal and chemical stability. The altered secondary structure of Ll -CCRH1 at pH 2.0 was stable up to 90 C. Also, in presence of 0.25 M guanidine hydrochloride (GdnHCI), it got transformed into different structure which was stable in the vicinity of 2 M GdnHCI (as compared to drastic loss of native structure in 2 M GdnHC1) as seen in far UV-CD spectra. The structural transition of LI-CCRH1 at pH 2.0 followed another transition after readjusting the pH to 8.0, forming a structure with hardly any similarity to that of native protein. (C) 2013 Elsevier B.V. All rights reserved.</p>10aAggregation Cinnamoyl CoA reductase10aConformation10aMolten globule10aThermostability10aUnfolding1 aSonawane, Prashant, D.1 aKhan, Bashir, Mohammad1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/conformational-transitions-cinnamoyl-coa-reductase-1-leucaena-leucocephala-003037nas a2200241   4500008004100000022001400041245015800055210006900213260005700282300001300339490000700352520210200359653002802461653003502489653001302524653003002537653002002567100001702587700001802604700002402622700002602646856012302672       2015                        eng d  a1610-294000aInsights into the unfolding pathway and identification of thermally sensitive regions of phytase from aspergillus niger by molecular dynamics simulations0 aInsights into the unfolding pathway and identification of therma  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcJUN  aUNSP 1630 v213 a<p>Thermal stability is of great importance in the application of commercial phytases. Phytase A (PhyA) is a monomeric protein comprising twelve alpha-helices and ten beta-sheets. Comparative molecular dynamics (MD) simulations (at 310, 350, 400, and 500 K) revealed that the thermal stability of PhyA from Aspergillus niger (A. niger) is associated with its conformational rigidity. The most thermally sensitive regions were identified as loops 8 (residues 83-106), 10 (161174), 14 (224-230), 17 (306-331), and 24 (442-444), which are present on the surface of the protein. It was observed that solvent-exposed loops denature before or show higher flexibility than buried residues. We observed that PhyA begins to unfold at loops 8 and 14, which further extends to loop 24 at the C-terminus. The intense movement of loop 8 causes the helix H2 and beta-sheet B3 to fluctuate at high temperature. The high flexibility of the H2, H10, and H12 helices at high temperature resulted in complete denaturation. The high mobility of loop 14 easily transfers to the adjacent helices H7, H8, and H9, which fluctuate and partially unfold at high temperature (500 K). It was also observed that the salt bridges Asp110-Lys149, Asp205-Lys277, Asp335-Arg136, Asp416-Arg420, and Glu387-Arg400 are important influences on the structural stability but not the thermostability, as the lengths of these salt bridges did not increase with rising temperature. The salt bridges Glu125-Arg163, Asp299-Arg136, Asp266-Arg219, Asp339-Lys278, Asp335-Arg136, and Asp424-Arg428 are all important for thermostability, as the lengths of these bridges increased dramatically with increasing temperature. Here, for the first time, we have computationally identified the thermolabile regions of PhyA, and this information could be used to engineer novel thermostable phytases. Numerous homologous phytases of fungal as well as bacterial origin are known, and these homologs show high sequence similarity. Our findings could prove useful in attempts to increase the thermostability of homologous phytases via protein engineering.</p>10aConformational dynamics10aMolecular dynamics simulations10aPhytaseA10aStructurally weak regions10aThermostability1 aKumar, Kapil1 aPatel, Krunal1 aAgrawal, Dinesh, C.1 aKhire, Jayant, Malhar  uhttps://library.ncl.res.in/content/insights-unfolding-pathway-and-identification-thermally-sensitive-regions-phytase-102736nas a2200277   4500008004100000022001400041245013100055210006900186260008000255300001400335490000700349520172600356653001702082653002802099653002302127653002102150653001402171653002302185653002002208100002602228700001802254700002202272700002602294700001902320856011902339       2015                        eng d  a1367-543500aSequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases0 aSequence and structurebased comparative analysis to assess ident  aTIERGARTENSTRASSE 17, D-69121 HEIDELBERG, GERMANYbSPRINGER HEIDELBERGcNOV  a1493-15060 v423 a<p>Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in beta-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.</p>10aAlkalistable10abeta-lactam antibiotics10aConsensus approach10aDisulfide bridge10aIon pairs10aPenicillin acylase10aThermostability1 aPanigrahi, Priyabrata1 aChand, Deepak1 aMukherji, Ruchira1 aRamasamy, Sureshkumar1 aSuresh, C., G.  uhttps://library.ncl.res.in/content/sequence-and-structure-based-comparative-analysis-assess-identify-and-improve-002052nas a2200241   4500008004100000245015300041210006900194260000800263300000900271490000700280520119100287653003501478653002301513653003001536653002001566100001701586700001601603700001701619700001601636700001501652700001201667856013101679       2017                        eng d00aElucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand, hemin in its catalytic kinase domain0 aElucidation of molecular mechanism of stability of the hemeregul  cSEP  a1-170 v353 aThe eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.10aHeme Regulated Inhibitor (HRI)10aHRI-eIF2α Complex10aProtein–Ligand Modeling10aThermostability1 aBhavnani, V.1 aKaviraj, S.1 aPanigrahi, P1 aSuresh, C.G1 aYapara, S.1 aPal, J.  uhttps://library.ncl.res.in/content/elucidation-molecular-mechanism-stability-heme-regulated-eif2%CE%B1-kinase-upon-binding-its02566nas a2200253   4500008004100000022001400041245011200055210006900167260000800236300001200244490000700256520172500263653002601988653001502014653001802029653001902047653002002066653002002086100001702106700001802123700002002141700002502161856012602186       2017                        eng d  a0739-110200aFunctional stability and structural transitions of kallikrein: spectroscopic and molecular dynamics studies0 aFunctional stability and structural transitions of kallikrein sp  cMAR  a330-3420 v353 a<p>Kallikrein, a physiologically vital serine protease, was investigated for its functional and conformational transitions during chemical (organic solvents, Gdn-HCl), thermal, and pH induced denaturation using biochemical and biophysical techniques and molecular dynamics (MD) simulations approach. The enzyme was exceptionally stable in isopropanol and ethanol showing 110% and 75% activity, respectively, after 96h, showed moderate tolerance in acetonitrile (45% activity after 72h) and much lower stability in methanol (40% activity after 24h) (all the solvents [90% v/v]). Far UV CD and fluorescence spectra indicated apparent reduction in compactness of KLKp structure in isopropanol system. MD simulation studies of the enzyme in isopropanol revealed (1) minimal deviation of the structure from native state (2) marginal increase in radius of gyration and solvent accessible surface area (SASA) of the protein and the active site, and (3) loss of density barrier at the active site possibly leading to increased accessibility of substrate to catalytic triad as compared to methanol and acetonitrile. Although kallikrein was structurally stable up to 90 degrees C as indicated by secondary structure monitoring, it was functionally stable only up to 45 degrees C, implicating thermolabile active site geometry. In GdnHCl [1.0M], 75% of the activity of KLKp was retained after incubation for 4h, indicating its denaturant tolerance. A molten globule-like structure of KLKp formed at pH 1.0 was more thermostable and exhibited interesting structural transitions in organic solvents. The above results provide deeper understanding of functional and structural stability of the serine proteases at molecular level.</p>10aisopropanol tolerance10aKallikrein10aMD Simulation10aMolten globule10aSerine protease10aThermostability1 aDalal, Sayli1 aMhashal, Anil1 aKadoo, Narendra1 aGaikwad, Sushama, M.  uhttps://library.ncl.res.in/content/functional-stability-and-structural-transitions-kallikrein-spectroscopic-and-molecular