Submitted to: Proceedings of Florida State Horticultural Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2009
Publication Date: 3/19/2010
Citation: Cameron, R.G., Luzio, G.A., Savary, B., Nunez, A., Goodner, K.L. 2010. Digestion patterns of two commercial endopolygalacturonases on polygalacturonate oligomers with a degree of polymerization from 7 – 21. Proceedings of Florida State Horticultural Society. 122:295-302. Interpretive Summary: Fungal endo polygalacturonases (EPGs) are important enzymes due to their industrial significance, biological role in pathogenesis and experimental utility. They are significant components of many commercial pectinase preparations used for fruit juice clarification, pulp treatment, fruit and vegetable maceration/liquefaction and fruit juice clarification and have been purified from commercial preparations. Often these enzymes are added during the maceration step to improve juice extraction, filtration and concentration. Depectinization to produce a sparkling clear juice is common with many fruits including various berries, grapes, pears and apples. Viscosity reduction for pulp wash concentration is the major application of pectinases in the citrus industry although it also can be used to stabilize juice cloud by reducing the molecular weight of pectin fragments, preventing their precipitation. The experimental uses of EPG largely have relied on material purified in the laboratory. The results of studies using EPG to probe pectin structure and pectin methylesterase (PME) mode of action do suggest that the enzymes have utility for characterizing pectin nanostructure. However the need to first purify the enzymes from fungal cultures places an added burden on the experimental design and limits their potential utility. In this study we report on the fragmentation of galacturonic acid (GalA) oligomers present in a narrow-range size-class of DP 7 – 21 resulting from digestion with two different commercially available EPGs and compare the results using a computer simulation model to describe the fragmentation process. We also provide data on the fungal source of the EPGs based on matrix assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) peptide mass fingerprinting to correlate identity to previously described Aspergillus niger EPGs.
Technical Abstract: Many fruits and fruit juices are enzymatically treated to aid the process of liquefaction, juice extraction, viscosity reduction and juice clarification. Polygalacturonases are a major component of enzyme mixtures used for these industrial applications as well as an enzymatic tool for elucidating pectin nano structure. Endopolygalacturonases (EPG) fragment the pectin homogalacturonan chain, cutting the molecule within a contiguous stretch of demethylated galacturonic acid (GalA) residues. To increase our understanding of this process, GalA oligomers with a degree of polymerization (DP) ranging from 7 – 21 were digested with two commercial endopolygalacturonase preparations (EPG M1 and EPG M2). Identity of the EPGs as EPG II from Aspergillus niger (EPG M1) and endo-PG I from A. aculeatus (EPG M2) was suggested from peptide mass fingerprinting with matrix assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) Aliquots were collected at various time points during the digestion and the resulting fragmentation patterns were determined. Individual oligomer masses were estimated and converted to molar concentrations. The distribution of cleavage products produced by the EPGs differed. Smaller fragments, especially GalA monomer, were produced earlier with EPG M1. Rate constants for each of 87 possible reactions were estimated by a computer simulation model. When compared to optimized data for individual reactions, the experimental data for EPG M1 provided a good fit, thus indicating the model was a good approximation of the fragmentation process.