Submitted to: Pectins and Pectinases 2001
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 27, 2002
Publication Date: March 27, 2002
Citation: F. Voragen et al. (eds.), Advances in Pectin and Pectinase Research. p.345-361. 2003. Kluwer Academic Publishers. Interpretive Summary: We are interested in developing enzyme technologies for the preparation of novel specialty pectins and pectic materials from U.S. agricultural processing residues for new uses in food and non-food applications. Enzymes can be used to selectively modify pectin structure and thereby improve performance and increase functionality. Recent work in our laboratories has focused on plant pectin methylesterases (PMEs). We have determined the major PME purified from Valencia orange peel to be a salt-independent isoenzyme, which has properties that may be advantageous in enzymatic modification of pectin and pectic materials. Furthermore, the orange peel PME available in a commercial enzyme preparation was demonstrated to be this same isoenzyme, providing a convenient and standardized source of the enzyme for other investigators. Details of the purification and characterization of the salt-independent PME are presented as well as results demonstrating a block-wise mode of action. W have now used the salt-independent PME to prepare a series of enzymatically de-esterfied pectins to study pectin structure-functional property relationships. Analysis by HPSEC (with RI, viscometry, and MALLS detectors in series) revealed no changes in molecular properties, except for reduced intrinsic vicosity at low levels of methylesterification. From these studies, we show the potential industrial utility of this unique plant PME isoenzyme by demonstrating it can be used to prepare modified pectins with increased calcium sensitivity while preserving the molecular weight.
Technical Abstract: The two major PME isoenzymes occurring in Valencia orange peel have now been purified and further characterized to establish unequivocal identity. Using chromatography and electrophoretic elution profiles, and biochemical activity patterns, we have distinguished a fruit-specific PME-I from the vegetative-type PME-IIs previously examined in detail by other research groups. Identity of the Valencia PME-I can be correlated to enzymatic activity in the absence of supplemented salts. We have now obtained partial amino acid sequences for the salt-independent PME-I. These sequences were used to design a molecular probe for screening an extensive citrus EST database. A total of 15 different PME-like clones were identified in 5 of 8 tissue libraries. We previously confirmed the salt-independent PME-I acts on pectin in a block-wise mode of action by 1H-NMR and a calcium-sensitive pectin precipitation assay, and we now show it can be used to selectively modify pectin methyester patterns. Enzymatically de-esterfied pectins were prepared and examined with a HPSEC system fitted with RI, viscometry, and MALLS detectors in series. We observed no reduction in molecular weight associated with pectin depolymerization or changes in the radius of gyration and Mark-Houwink exponent of de-esterified pectin. Below DE 40%, treated pectins did show significant reduction in their intrinsic viscosity. This may be due to increased cross-link density of pectin networks by intramolecular hydrogen bonding. The potential industrial utility of the salt-independent PME-I is demonstrated by the ability to prepare calcium-sensitive pectins, with a wide range of degree of esterification, without degradation of molecular weight.