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Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris
Author(s) -
Beldarraín Alejandro,
Acosta Niuris,
Betancourt Lázaro,
González Luis J.,
Pons Tirso
Publication year - 2003
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20030063
Subject(s) - pichia pastoris , chemistry , differential scanning calorimetry , denaturation (fissile materials) , recombinant dna , kinetics , fluorescence , glycoside hydrolase , circular dichroism , hydrolase , enzyme , hydrolysis , stereochemistry , biochemistry , nuclear chemistry , physics , quantum mechanics , gene , thermodynamics
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 (Cys 9 –Cys 14 , Cys 484 –Cys 488 ) and two free Cys residues (Cys 336 , Cys 415 ) 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 β‐sheets. rDex loses biological activity at neutral pH without total disruption of its conformation. In addition, rDex preserves its conformation close to 60 °C, but it is thermally denatured with appreciable aggregation at temperatures above 75 °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.