02592nas a2200277 4500008004100000022001400041245008800055210006900143260007300212300001200285490000900297520161200306653001701918653003801935653002201973653002101995653002102016653002002037100002502057700002002082700002602102700002402128700002102152700002502173856011602198 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. uhttps://library.ncl.res.in/content/polyproline-fold-imparting-kinetic-stability-alkaline-serine-endopeptidase-002351nas a2200277 4500008004100000022001400041245010100055210006900156260005700225300001000282490000700292520140100299653001901700653000801719653002201727653002101749653001301770653002201783100002101805700002301826700002101849700002601870700002101896700002501917856013101942 2015 eng d a1572-388700aAcid stability of the kinetically stable alkaline serine protease possessing polyproline II fold0 aAcid stability of the kinetically stable alkaline serine proteas a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcFEB a60-670 v343 a

The kinetically stable alkaline serine protease from Nocardiopsis sp.; NprotI, possessing polyproline II fold (PPII) was characterized for its pH stability using proteolytic assay, fluorescence and Circular Dichroism (CD) spectroscopy, and Differential Scanning Calorimetry (DSC). NprotI was found to be functionally stable when incubated at pH 1.0, even after 24 h, while after incubation at pH 10.0, drastic loss in the activity was observed. The enzyme showed enhanced activity after incubation at pH 1.0 and 3.0, at higher temperature (50-60 A degrees C). NprotI maintained the overall PPII fold in broad pH range as seen using far UV CD spectroscopy. The PPII fold of NprotI incubated at pH 1.0 remained fairly intact up to 70 A degrees C. Based on the isodichroic point and T-m values revealed by secondary structural transitions, different modes of thermal denaturation at pH 1.0, 5.0 and 10.0 were observed. DSC studies of NprotI incubated at acidic pH (pH 1.0-5.0) showed T-m values in the range of 74-76 A degrees C while significant decrease in T-m (63.8 A degrees C) was observed at pH 10.0. NprotI could be chemically denatured at pH 5.0 (stability pH) only with guanidine thiocynate. NprotI can be classified as type III protein among the three acid denatured states. Acid tolerant and thermostable NprotI can serve as a potential candidate for biotechnological applications.

10aAcid stability10aDSC10aKinetic stability10aPolyproline fold10aProtease10aThermal unfolding1 aRohamare, Sonali1 aJavdekar, Vaishali1 aDalal, Sayli, A.1 aNareddy, Pavan, Kumar1 aSwamy, Musti, J.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/acid-stability-kinetically-stable-alkaline-serine-protease-possessing-polyproline-ii-fold-002446nas a2200277 4500008004100000022001400041245012200055210006900177260009900246300001200345490000700357520142700364653001801791653002701809653002201836653001301858653002301871100002101894700002501915700001801940700002101958700001701979700001701996700002402013856013102037 2015 eng d a1359-511300aCloning, expression and in silico studies of a serine protease from a marine actinomycete (Nocardiopsis sp NCIM 5124)0 aCloning expression and in silico studies of a serine protease fr aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLANDbELSEVIER SCI LTDcMAR a378-3870 v503 a

A serine protease (N. protease), from Nocardiopsis sp., was cloned and expressed in Escherichia coli and investigated for its potential kinetic stability. Protein expression using two vectors, pET-22b (+) and pET-39b (+) was compared based on proper folding and soluble expression of the protein. pET-39b (+) was found to be a better vector for soluble expression of this protease containing disulfide bonds. In silico studies were also carried out for N. protease. Homology modeling suggested N. protease to be a member of PA clan of proteases. The phylogenetic analysis showed relatedness of N. protease to kinetically stable proteases. Molecular docking studies performed exhibited interaction of a peptide substrate with catalytic pocket of the enzyme. High temperature MD simulations were performed on N. protease to study its unfolding behavior and comparisons were made with alpha LP. A novel approach to study `cooperativity' of protein unfolding was undertaken, wherein `P' value analysis based on phi and psi values of the protein was performed. Data showed sharper P value transition for alpha LP when compared to N. protease thus indicating relatively less kinetic stability of N. protease. Present study holds significance as the non-streptomycete actinomycetes group is least explored and ensures industrially important enzymes with exceptional stabilities. (C) 2015 Elsevier Ltd. All rights reserved.

10aActinomycetes10aCloning and expression10aKinetic stability10aProtease10aThermal simulation1 aRohamare, Sonali1 aGaikwad, Sushama, M.1 aJones, Dafydd1 aBhavnani, Varsha1 aPal, Jayanta1 aSharma, Ranu1 aChatterjee, Prathit uhttps://library.ncl.res.in/content/cloning-expression-and-silico-studies-serine-protease-marine-actinomycete-nocardiopsis-sp-0