Endophytes isolated from Ecuadorian Amazonian plant samples were screened for their ability to degrade polyester polyurethane (PUR). Almost half of the organisms displayed some activity in the initial plate clearance assay. Eighteen active and two inactive endophytes were further characterized. Eight of the most active organisms belonged to the Pestalotiopsis
genus. The current literature reports some fungi with the ability to degrade PUR (4
), although these studies have focused primarily on organisms isolated from soil samples. This is the first study that demonstrates PUR degradation by endophytic fungi. The broad distribution of activity suggests that endophytes might be a promising source of biodiversity in which to test for activities important for bioremediation.
All active fungi were identified as Ascomycota, with a cluster of organisms belonging to the class Dothidiomycetes and the order Pleosporales. A large portion of the active fungi belonged to the class Sordariomycetes, including those identified as Pestalotiopsis sp. strains.
Although robust activity was observed among several Pestalotiopsis sp. isolates, not all of them demonstrated equivalent levels of activity. Of the nine isolates tested, three were highly active (E2712A, E3317B, and E2711A), five were moderately active (E3314A, E2520A, E2911H, E3412F, and E2708A), and one was inactive (E4112A). This variability in activity among distinct Pestalotiopsis microspora isolates suggests that there are genetic differences among the organisms.
The genus Pestalotiopsis
is grouped in the Xylariales
order and comprises several known plant pathogens. The fungus is not host specific and causes rot and disease in a wide variety of plant species (29
), although these isolates were all endophytic and the plants showed no pathogenic symptoms. Pestalotiopsis microspora
isolates have previously been shown to have a propensity for horizontal gene transfer. In one notable case, a Pestalotiopsis microspora
strain isolated as a fungal endophyte from the taxol-producing plant Taxus wallachiana
had acquired the ability to synthesize taxol (27
). Such a propensity for horizontal gene transfer may have contributed to the ability of a subset of these isolates to degrade polyester polyurethane as a sole carbon substrate, or it may reflect a significant level of phenotypic diversity among the genus.
There are no previous reports of members of the genus Pestalotiopsis having biodegradation activity. We found that two isolates of Pestalotiopsis microspora (E2712A and E3317B) were able to degrade PUR when grown anaerobically with Impranil DLN serving as the sole carbon source. For these two organisms, the level of activity was the same when grown under either aerobic or anaerobic conditions. This is in contrast to the control fungus Aspergillus niger, which showed substantially less activity when grown anaerobically. This observation may have practical significance in that fungal growth on and metabolism of PUR by Pestalotiopsis microspora could be used in anaerobic fermentation systems.
The enzyme produced by Pestalotiopsis microspora that is responsible for PUR degradation appears to be a member of the serine hydrolase family. Furthermore, activity extended throughout the medium at a distance well removed from the areas of fungal growth. This suggests that the enzyme responsible for degradation is extracellular, secreted, and diffusible. In comparison to an inactive cell-free filtrate from a fungal culture grown in rich medium, we found that the polyurethanase is inducible when Pestalotiopsis microspora E2712A is grown in minimal PUR-Lmin medium containing a suspension of Impranil DLN. By using activity-based probes, the active enzyme was identified as a serine hydrolase with an approximate molecular mass of 21 kDa. The protein was shown to be able to degrade PUR after subsequent purification, showing that activity is independent of other components of the culture filtrate.
Polyurethanases have previously been isolated and characterized from protein extracts of several organisms, including the bacteria Pseudomonas chlororaphis
) and Comamonas acidovorans
), as well as the fungus Candida rugosa
). The active enzymes have been classified (17
) as esterases (5
), lipases (24
), and proteases and ureases (19
), suggesting degradation of the PUR substrate by cleavage of the ester bond. The IR analysis and molecular inhibition of PUR degradation by Pestalotiopsis microspora
suggest that ester hydrolysis by a serine hydrolase is responsible for PUR biodegradation.
This investigation established the robust polyurethane degradation activity under anaerobic conditions in which the synthetic polymer served as the only carbon source for the fungus. A cell extract of the active culture containing a critical serine hydrolase is able to clear the polymer in under 1 h using the PUR concentrations reported here. This work establishes that endophytes are a useful source of biodiversity with potential application for bioremediation. The relative ease with which organisms can be isolated and screened makes this a highly accessible and environmentally relevant project for engaging undergraduate students in scientific research. It is possible that activities against other, more recalcitrant polymers could be discovered using this abundant source of biodiversity.