Exopolygalacturonate lyase from Thermotoga maritima: cloning, characterization and organic synthesis application.
A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.
Parisot
Judicaël
J
Unité de Recherches en Biocatalyse (FRE-CNRS 2230), Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP 92208, F-44322, Nantes, France.
Ghochikyan
Anahit
A
Langlois
Virginie
V
Sakanyan
Vehary
V
Rabiller
Claude
C
D002240:Carbohydrate Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013400:Sugar Acids / Q000138:chemical synthesis*; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D014312:Trisaccharides / Q000138:chemical synthesis*
D011994:Recombinant Proteins; D013400:Sugar Acids; D014312:Trisaccharides; C065008:O-galactopyranosyluronic acid-(1-4)-O-galactopyranosyluronic acid-(1-4)-galactopyranosyluronic acid; D011133:Polysaccharide-Lyases; C037051:pectate disaccharide-lyase