Cloning expression and characterization of a thermostable exopolygalacturonase from Thermotoga maritima.
A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 degrees C, pH 6.0 and retains 90% of activity after heating at 90 degrees C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of 1H NMR spectroscopy revealed an inversion of configuration during hydrolysis of alpha-(1-->4)-galacturonic linkage.
Parisot
Judicaël
J
Unité de Recherches en Biocatalyse (unité CNRS 2230), Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP, 92208 F-44322, Nantes, France.
Langlois
Virginie
V
Sakanyan
Vehary
V
Rabiller
Claude
C
D002384:Catalysis; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D013997:Time Factors
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C020136:exopolygalacturonase