Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger.
A series of pectins with different distribution patterns of methyl ester groups was produced by treatment with either plant (p-PME) or fungal pectin methyl esterases (f-PME) and compared with those obtained by base catalysed de-esterification. The products generated by digestion of these pectins with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de-esterification caused by p-PME treatment gave a decrease of partly methylated oligomers in PL fingerprints, which did not effect the relative composition of partly methylated oligomers. PG II fingerprints showed a constant increase of monomers and oligomers without any methyl ester groups with decreasing degree of esterification (DE), but almost no change in the concentration of partly methylated compounds. PL fingerprints of f-PME and chemically treated pectins showed decreasing amounts of partly methyl esterified oligomers with decreasing DE, together with a relative shift towards longer oligomers. PG II fingerprints were characterised by an increase of partly methylated and not methylated oligomers with decreasing DE. But differences were also seen between these two forms of homogenous de-esterification. Introduction of a certain pattern of methyl ester distribution caused by selective removal of certain methyl ester groups by f-PME is the most reasonable explanation for the detected differences.
Limberg
G
G
Danisco Biotechnology, Copenhagen K, Denmark. gl@avh.de
Körner
R
R
Buchholt
H C
HC
Christensen
T M
TM
Roepstorff
P
P
Mikkelsen
J D
JD
D001234:Aspergillus niger / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010368:Pectins / Q000737:chemistry* / Q000378:metabolism*; D011096:Polygalacturonase / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010368:Pectins; C400187:endopolygalacturonase II; D011096:Polygalacturonase; D011133:Polysaccharide-Lyases; C027349:pectin lyase