Study on extraction, purification and characterization of a novel peroxidase from white Spanish broom (Cytisus multiflorus)

Peroxidases (EC 1.11.1.7) are a large group of enzymes widely distributed in the plant kingdom. The present work describes a study on the isolation, purification and some features of a novel peroxidase from white Spanish broom (Cytisus multiflorus), a tree legume very abundant in the northern half of Spain and Portugal. Optimal conditions are proposed for enzyme extraction, removal of phenolic compounds and enzyme purification by consecutive hydrophobic, ion-exchange and size-exclusion chromatographies. Peroxidase from Cytisus multiflorus (CMP) was found to have a molecular weight of 49 kDa. The spectrum of CMP showed a Soret band at 403 nm with a Rz factor of 3.3. Substrate specificity and the effect of some variables on the activity of CMP with guaiacol as cosubstrate have also been investigated.This work was partially supported by project SA052A10-2, funded by Consejería de Educación de la Junta de Castilla y León (Spain).Peer reviewe

Structural stability of adenylate kinase from the sulfate-reducing bacteria Desulfovibrio gigas

Biophysical Chemistry 110 (2004) 83–92A novel adenylate kinase (AK) has recently been purified from Desulfovibrio gigas and characterized as a Co2+/Zn2+-containing enzyme: this is an unusual characteristic for AKs from Gram-negative bacteria, in which these enzymes are normally devoid of metals. Here, we studied the conformational stability of holo- and apo-AK as a function of temperature by differential scanning calorimetry (DSC), circular dichroism (CD), and intrinsic fluorescence spectroscopy. The thermal unfolding of AK is a cooperative two-state process, and is sufficiently reversible in the 9–11 pH range, that can be correctly interpreted in terms of a simple two-state thermodynamic model. The spectral parameters as monitored by ellipticity changes in the CD spectra of the enzyme as well as the decrease in tryptophan intensity emission upon heating were seen to be good complements to the highly sensitive but integral DSC-method.supported in part by the Fundação para a Ciência e a Tecnologia, Portugal, fellowships BD/13775/97 to OYG, BPD/3518/00 to SAB, SFRH/BD/1067/2000 to DGP, and NATO Scientific Programme Fellowships for Spain, call 2002 to GGZ

Thermostability of cardosin A from Cynara cardunculus L.

The structural stability of cardosin A, a plant aspartic proteinase (AP) from Cynara cardunculus L., has been investigated by high-sensitivity differential scanning calorimetry, intrinsic fluorescence and circular dichroism spectroscopy, and enzymatic activity assays. Even though the thermal denaturation of cardosin A is partially irreversible, valid thermodynamic data can be obtained within a wide pH region. Also, although cardosin A is a heterodimeric enzyme its thermal denaturation occurs without simultaneous dissociation to unfolded monomers. Moreover, in the 3-7 pH region the excess heat capacity can be deconvoluted into two components corresponding to two elementary two-state transitions, suggesting that the two polypeptide chains of cardosin A unfold independently. Detailed thermodynamic and structural investigations of cardosin A at pH=5.0, at which value the enzyme demonstrates maximum stability and enzymatic activity, revealed that after thermal denaturation the polypeptide chains of this protein retain most of their secondary structure motifs and are not completely hydrated.http://www.sciencedirect.com/science/article/B6THV-47P1SF6-4/1/edc14f851e47459fcd87b748d068439

Steady-state kinetics of Roystonea regia palm tree peroxidase

ABSTRAC

Thermal stability of peroxidase from Chamaerops excelsa palm tree at pH 3

7 pages, 5 figures, 4 tables.-- PMID: 19428462 [PubMed].-- Printed version published May 1, 2009.The structural stability of a peroxidase, a dimeric protein from palm tree Chamaerops excelsa leaves (CEP), has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism and steady-state tryptophan fluorescence at pH 3. The thermally induced denaturation of CEP at this pH value is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, leading to the conclusion that in solution CEP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of CEP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N -(k)-> D [View source], where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate.This work was partially supported by projects SA-06-00-0 ITACYL-Universidad de Salamanca and SA 129A07 (Junta de Castilla y León) and BFU2004-01432 and BFU2007-68107-C02-02/BMC (Ministerio de Educación y Ciencia) Spain. L.S.Z was fellowship holders from the Junta de Castilla y León, Spain (Ref. EDU/1490/2003).Peer reviewe