ABSTRACT
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.
Subject(s)
Glycoside Hydrolases/genetics , Thermotoga maritima/enzymology , Catalysis , Cloning, Molecular , DNA, Bacterial/genetics , Enzyme Stability , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/isolation & purification , Hydrogen-Ion Concentration , Hydrolysis , Molecular Weight , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Temperature , Thermotoga maritima/genetics , Time FactorsABSTRACT
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.
Subject(s)
Polysaccharide-Lyases/chemistry , Polysaccharide-Lyases/genetics , Sugar Acids/chemical synthesis , Thermotoga maritima/enzymology , Trisaccharides/chemical synthesis , Carbohydrate Sequence , Catalysis , Cloning, Molecular , Enzyme Stability , Escherichia coli/enzymology , Escherichia coli/genetics , Hot Temperature , Hydrogen-Ion Concentration , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Polysaccharide-Lyases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Thermotoga maritima/geneticsABSTRACT
We show that the yields in saccharide synthesis by tranglycosylation with alpha-galactosidase from green coffee beans can be greatly enhanced when working in ice. Thus, methyl alpha-D-galactopyranosyl-(1-->3)-alpha-D-galactopyranoside (3a) produced by reaction of alpha-D-galactopyranosyl fluoride 1 with methyl alpha-D-galactopyranoside (2) is obtained with 51% yield in ice while only 29% is synthesized at 37 degrees C. This result, already previously found by others with proteases and by us with a beta-galactosidase appears to be a general property of hydrolases.
