Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris.
Conformational stability and structural characterization of an rDex (recombinant dextranase) expressed in Pichia pastoris were studied by enzymic assays, fluorescence, CD and DSC (differential scanning calorimetry). We also identified two disulphide bridges (Cys9-Cys14, Cys484-Cys488) and two free Cys residues (Cys336, Cys415) that are not conserved between bacterial and fungal dextranases of GH-49 (glycoside hydrolase family 49) by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS. Enzymic and fluorescence studies revealed that rDex is biological and conformationally stable at acidic pH, with maximum activity at pH 4.5-5.0, while CD spectra indicated a secondary structure basically composed of beta-sheets. rDex loses biological activity at neutral pH without total disruption of its conformation. In addition, rDex preserves its conformation close to 60 degrees C, but it is thermally denatured with appreciable aggregation at temperatures above 75 degrees C. DSC studies always displayed irreversible transitions and a strong dependence on the scan rate. Our combined analysis suggested that the denaturation process of rDex is under kinetic control, which is described reasonably well by the two-state kinetic scheme.
Beldarraín
Alejandro
A
Departamento de Evaluación de Procesos y Sistemas, Planta de Producción, Centro de Ingeniería Genética y Biotecnología, P.O. Box 6162, Habana 10600, Cuba. alejandro.beldarrain@cigb.edu.cu
Acosta
Niuris
N
Betancourt
Lázaro
L
González
Luis J
LJ
Pons
Tirso
T
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D003910:Dextranase / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D011994:Recombinant Proteins; D003910:Dextranase