2016-2017 Funded Proposal
Description of Proposed Research Area
We have entered what some are calling the “age of plastics.” The vast majority of plastics that are produced are non- or poorly biodegradable, and rely on non-renewable petrochemicals for production. Thus, there is considerable interest in identifying bio-based and biodegradable alternatives to traditional plastics. Polylactic acid (or polylactide) (PLA) is a bio-based biodegradable polymer derived from renewable plant feedstocks (typically corn or sugarcane), and has attracted much attention for its good optical properties, strength, and compostability. PLA is commonly used in the production of compostable food service items, packaging and agricultural biodegradable plastic mulch films. While this polymer has demonstrated degradability under industrial compost conditions, municipal composting programs are still very limited in the U.S., and much of this PLA is expected to end up in other environments (e.g. soils, landfills) where it’s fate is unknown (Brodhagen et al. 2015). Thus, as production of PLA is anticipated to continue to increase, there is a need to reveal the microbial mechanism of breakdown of these polymers.
Several isolates of fungi and bacteria have been identified that can degrade PLA. It has been suggested that protease-type enzymes are responsible for the initial depolymerization of PLA, however the mechanism is not well characterized (Shah et al. 2008). Thus, the specific objective for this proposed project is to identify microbial (fungal and/or bacterial) enzymes involved in the degradation of PLA. Our central hypothesis is that fungi involved in the initial degradation of PLA will secrete enzymes (possibly proteases) that can be isolated, and then their cognate genes identified through reverse genetics.
This is a new collaboration between Dr. Reynolds, who brings expertise in secreted proteins and enzymology, and Dr. DeBruyn, who has isolated several strains of bacteria and fungi which appear to degrade biodegradable polymers. Characterization of the degradation pathways used by these microbes will provide critical preliminary data which will position us to apply for extramural funding.
Aim 1: Development of an assay to detect lactic acid released from degradation of PLA. The degradation of PLA by microbes presumably must occur initially through breakdown of the polymer by secreted enzymes from soil microbes such as fungi or bacteria (Tokiwa et al, 2009). The commercially available protease, Proteinase K has been experimentally shown to break down PLA, as have some lipases (Fukuzaki, et al, 1989;Lim, et al 2005; Williams, 1981). Thus, we have some idea of what types of enzymes may be involved. In this aim, we intend to develop a quantitative colorimetric assay for detecting the release of lactic acid (LA) from PLA in the laboratory.
For the purposes of assay development, the release of LA will initially be facilitated using commercially available proteases such as Proteinase K and some commercially available lipases as well. Detection of released LA will be assessed using several commercially available kits marketed for detecting either L-LA or D-LA, which are both used to make different types of PLA. We will test these kits for the ability to detect L- or D-LA released from PLA over time in different conditions with a variety of proteases and lipases. This will be correlated with other assays that include loss of mass from PLA and clearing of regions within PLA. We will test colorimetric kits for lactic acid detection from Megazyme and MyBioSource.
Alternatively, if these kits are not sensitive enough, we will use liquid chromatography and mass spectrometry to detect LA released from PLA. This is available through the mass spectrometry core facility at UTK.
Aim 2. Identification of enzymes from microbes that can break down PLA. Using microbes isolated in association with PLA from soil, we will identify natural enzymatic activities that release LA from PLA. Several candidate isolates of fungi or bacteria have been isolated by the DeBruyn lab using enrichment on minimal media with PLA-containing plastic films as the sole carbon source. We will culture microbes with PLA (Tokiawa, et al 2009), and then test culture filtrates for the presence of LA using the assay(s) developed in aim 1. This will be accomplished by growing these organisms in liquid culture with PLA: both small squares of PLA-based plastic films and pure powdered PLA will be used. Subsamples from the cultures will be taken at regular time intervals and filtered. Filtrate will be tested for released LA using the methods developed in aim 1.
Alternatively, if we do not detect sufficient LA release, since protease activity is associated with PLA breakdown, we will test for protease activity using a quantitative, colorimetric protease assay (Pierce).
Once activities are detected using either approach above, we will fractionate the culture proteins from the microbes on a polyacrylamide gel by electrophoresis (PAGE) and then use an in-gel assay to detect protease activities. This will allow us to determine which protein band(s) is(are) associated with the activity.