Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MG-oligosaccharides by NMR spectroscopy and ESI-mass spectrometry.
Alginate with long strictly alternating sequences of mannuronic (M) and guluronic (G) acid residues, F(G) = 0.47 and F(GG) = 0.0, was prepared by incubating mannuronan with the recombinant C-5 epimerase AlgE4. By partial acid hydrolysis of this PolyMG alginate at pH values from 2.8 to 4.5 at 95 degrees C, alpha-L-GulpA-(1-->4)-beta-D-ManpA (G-M) linkages were hydrolyzed far faster than beta-D-ManpA-(1-->4)-alpha-L-GulpA (M-G) linkages in the polymer chain. The ratio of the rates (kG-M/kM-G) decreased with increasing pH. The dominant mechanism for hydrolysis of (1-->4)-linked PolyMG in weak acid was thus proved to be an intramolecular catalysis of glycosidic cleavage of the linkages at C-4 by the undissociated carboxyl groups at C-5 in the respective units. The higher degradation rate of G-M than M-G glycosidic linkages in the polymer chain of MG-alginate at pH 3.5 and 95 degrees C was exploited to make oligomers mainly consisting of M on the nonreducing and G on the reducing end and, thus, a majority of oligomers with an even number of residues. The ratio of the rate constants kG-M/kM-G at this pH was 10.7. The MG-hydrolysate was separated by size exclusion chromatography and the MG oligosaccharide fractions analyzed by electrospray ionization-mass spectrometry together with 1H and 13C NMR spectroscopy. Chemical shifts of MG-oligomers (DP2-DP5) were elucidated by 2D 1H and 13C NMR.
Holtan
Synnøve
S
Norwegian Biopolymer Laboratory, Department of Biotechnology, The Norwegian University of Science and Technology (NTNU),Sem Saelands vei 6/8, N-7491 Trondheim, Norway. synnove.holtan@borregaard.com
Zhang
Qiujin
Q
Strand
Wenche Iren
WI
Skjåk-Braek
Gudmund
G
D000143:Acids / Q000737:chemistry*; D000464:Alginates / Q000737:chemistry*; D002236:Carbohydrate Conformation; D020723:Glucuronic Acid / Q000737:chemistry; D006031:Glycosylation; D006603:Hexuronic Acids / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization
D000143:Acids; D000464:Alginates; D006603:Hexuronic Acids; D009844:Oligosaccharides; C007896:guluronic acid; D020723:Glucuronic Acid; C008324:mannuronic acid