Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger.
A method to determine the amount of galacturonic acid in blocksequence (BS) in pectin homogalacturonan (HG) is described. The method is based on a combination of endopolygalacturonase II (endo-PG II) and exopolygalacturonase (exo-PG) digestion followed by quantification of the liberated galacturonic acid monomer. The amount of monomers released is directly related to the amount of non-esterified galacturonic acid units located between two other non-esterified galacturonic acids units on the HG chain. The amount released for exo-PG digestion only corresponds to the BS located at the non-reducing end of the polymer. The difference between total- and exo-BS was calculated to be the amount of endo-BS located either within or on the reducing end of the HG. Three series of model pectins obtained by de-esterification of a high-ester pectin with either plant pectin methyl-esterase (p-PME, P-series), fungal pectin methyl-esterase (f-PME, F-series) and chemical de-esterification using base (B-series) were analysed and compared with a fully de-esterified pectic acid sample obtained from the same raw material. Clear differences for the increase of the amounts of blocksequence could be seen between de-esterification of the P- and F-series samples supporting a blockwise and a homogenous de-esterification mechanism, respectively. f-PME and base treatment showed only minor differences in the increase of galacturonic acid units in BS, despite differences seen in their methyl-esterification pattern. Differences between the amounts of galacturonic acid located in exo- and endo-BS, provided evidence for the need of a certain start side or blocklength for p-PME to de-esterify blockwise.
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; D006603:Hexuronic Acids / Q000032:analysis*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010368:Pectins / Q000737:chemistry* / Q000378:metabolism*; D011096:Polygalacturonase / Q000378:metabolism*
D006603:Hexuronic Acids; C007819:galacturonic acid; D010368:Pectins; C400187:endopolygalacturonase II; D011096:Polygalacturonase; C003181:polygalacturonic acid