1a. Objectives (from AD-416)
Purify and characterize pectin methylesterase isoforms from papaya. Use them to modify a model homogalacturonan, introducing demethylated block structure which will be characterized for average block size, number of blocks per molecule and their intermolecular distribution within the population of molecules. These data will be used to mathematically model enzyme mode of action. Introduced structural features will be correlated to measured functional properties for predictive modeling of food product quality.
1b. Approach (from AD-416)
Pectin methylesterases (PME) present in commercial papaya fruit (Carica papaya) extracts, used industrially in the formulation of food ingredients for their inherent PME activity, will be purified with a multi-dimensional strategy incorporating affinity chromatography. The hypothesis that these enzymes can be used to rationally design functional polysaccharide food ingredients will be tested. They will be characterized for biochemical, physical and kinetic properties, and their ability to manipulate pectin nano-structure. Mass spectrometry and N-terminal amino acid sequencing will be used to establish identity tags and relatedness of individual PME forms, establishing a basis for subsequent cloning. A recently defined model homogalacturonan will be demethylated with purified papaya PME(s) to pectins with predetermined degrees of methylation. Introduced demethylated block sequence will be characterized by limited digestion with endo polygalacturonase to release demethylated fragments. Released oligomers of galacturonic acid will be visualized and quantified using HPAEC coupled to an evaporative light scattering detector, allowing for the estimation of average demethylated block size and number per pectin molecule. Capillary electrophoresis will be used to probe the intermolecular distribution of introduced demethylated blocks. These data will be used to mathematically model the enzyme mode of action/degree of processivity. Modified homogalacturonans will be submitted to functional testing (rheology, calcium sensitivity and suspension properties) to enable comparison of nano-structure features to functional properties. The relationship between block size/number and influence of reaction conditions on enzyme mode of action and pectin functionality will allow for predictive modeling of food product quality related to the listed variables.
3. Progress Report
This project is related to Objective 3 of this in-house project: Enzymatic conversion of citrus processing waste and its separation into phenolic and polysaccharide streams; and enzymatic release of furanocoumarins from grapefruit processing retentate. A Post Doctoral Research Associate has been selected and is in the process of obtaining a visa. Several commercial papaya extracts have been evaluated as a source of the enzyme (pectin methylesterase) that is the focus of the study. One of these extracts has been selected and work has been initiated on preparing it for chromatographic isolation of the pectin modifying enzyme. MONITORING: Progress was monitored by meetings, email and phone conversations.