|Cameron, Randall - Randy|
Submitted to: Subtropical Technology Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 9/12/2005
Publication Date: 10/20/2005
Citation: Cameron, R.G., Savary, B.J. 2005. Progress in determining pectin methylesterase mode of action and structural mapping of modified pectin. Subtropical Technology Conference Proceedings. 56:14.
Technical Abstract: The functional properties of pectin are dependent on structural features of the individual pectin molecules in a solution. The primary structural components responsible for pectin functionality are the proportion of galacturonic acid (GA) residues in the homogalacturonan (HGA) region that are methyl esterified, the distribution of the esterified GA residues within the HGA (block vs. random) and the average molecular weight of the pectin molecules that make up the population. We are using a mono-component salt-independent pectin methylesterase (PME), the most abundant PME present in citrus fruit, to demethylate pectin and determine its structural affect on demethylated and methyl-protected block size. The methodology utilized involves demethylation of highly esterified pectin (DE = 94%) to various levels of deesterification. The deesterified pectins are then enzymatically digested with monocomponent preparations of endo-polygalacturonase (EPG) to release deesterified blocks from the pectin. Two monocomponent fungal EPGs (reportedly obtained from Aspergillus niger) are commercially available. It is known that EPGs from A. niger differ in their mode of action on polygalacturonic acid (PGA). In order to determine which of the commercial enzymes would be best suited for use in structural mapping of deesterified pectin it was necessary to identify the isoform they represented and to characterize their mode of action of PGA. Results from these studies indicate that based on peptide fingerprinting the commercial enzymes are most likely produced by A. aculeatus, not A. niger as listed by the manufacturer. The EPG designated M2 by the manufacturer was chosen for use in pectin structural mapping because larger GA oligomers were produced by it during digestion of PGA. This property allows for a more accurate estimate of deesterified block size in demethylated pectin.