Nature is a repository of potential solutions to some difficult problems. So believe the team of Investigators of Richard Gross and Jin Montclare of the Polytechnic University of New York and Richard Bonneau and Glenn Butterfoss of New York University. The problem is to determine a method for decomposing plastic materials, in particular PET plastics. The potential solution is sought in enzyme catalysts that attack similar natural polymers. Cutin is a biopolyester built from a complex array of C16 and C18 omega-hydroxyfatty acids which functions to protect plant surfaces from invasion by pathogenic organisms. Cutinase is natures equalizing response and is an enzyme present in various pathogens which will attack the natural biopolyesters. However, cutinases are an enzyme family that, thus far, has received disproportionally little attention relative to other ester hydrolase enzyme families. This is changing as cutinases are emerging as one of the primary benchmark hydrolase enzymes for synthetic polymer modification as they exhibit the extraordinary ability to catalyze a number of important polymer biotransformations on poly(ethyleneterephthalate) (PET), Nylon 6,6, polyvinylacetate, polyacrylonitrile and others. This is remarkable as these polymer substrates deviate dramatically in structure from the natural substrate for these enzymes.
The PIs have put together a well-planned program to provide a thorough study leading to a deep understanding of structural features that lead to high thermal stability and enhanced activity of cutinases. The program includes kinetics and mechanistic studies for cutinase-catalyzed hydrolysis of PET and other specific polymeric materials. A comprehensive study of this type is thus far lacking in published literature. In order to do this, plans include engineering AoC variants (Aspergillus oryzae cutinases) that will be synthesized and tested using the above substrates with the goal of achieving both high stability (temperature, pH) and catalytic activity. Studies will include modeling efforts and analysis of degradation products to further build understanding.
Cutinase activity for polymer degradation is only one feature of this work. Numerous polymer applications require tailoring of surface properties to enhance biocompatibility, chemical resistance, hydrophobicity, adhesion and wettability. Current methodologies to modify polymer surfaces include wet chemical modification, plasma treatments, and application of polymeric surface coatings. These methodologies exhibit negative features including generation of large volumes of solvent waste, limitation to batch processing, and safety hazards. Furthermore, there is an increased demand for materials with surfaces that can function to self-clean, repel and/or kill microbes, and have advanced biological properties. The PIs will be able to consider an engineered cutinase with sufficient stability and activity to be immobilized on such surfaces and function to modify or degrade the surface layer of a material, thereby engineering in various of these surface properties.
Funding will provide important research opportunities to the NYU-POLY student body which is diverse demographically, socio-economically and includes many children of first generation immigrants, eager to reach the next step of the economic ladder through education. The PIs participate in NYUPOLYs institutionalized UG summer research program, have a very active high school mentoring program (6-10 students per year) and work with the Kids Science Challenge team to create new modules aimed at 3rd to 6th graders to teach, for example, about magic microbes.
Project Summary: Increasingly, scientists are being challenged to solve environmental problems due to chemical processes that generate toxic by-products and require high energy consumption. This program addressed these concerns by focusing on a promising but understudied family of enzymes known as cutinases. This enzyme family has its active site exposed to solvent that allows it to access rigid substrates that are generally not accessible to other esterase families such as lipases. The natural function of cutinases is the hydrolysis of cutin (a high-molecular weight polyester) that protects plants from attack by plant pathogenic fungi. In this program we focused on developing cutinases with enhanced activity for hydrolyzing non-natural polymers such as: i) polyethylene terephthalate (PET), a polyester used in synthetic fibers (e.g. in clothes), beverage containers (e.g. water bottles) and ii) cellulose acetate (CA) used in synthetic fibers, cigarette filters and coatings. Research focused on developing a deep understanding of cutinase structure-thermostability-catalytic activity relationships. Applications of cutinases such as in PET recycling and surface modification are discussed in greater detail in the Broader Impacts section. For these applications, increasing the thermostability (resistance to heat that causes protein unfolding) of cutinases is critically important so that they can be used above the polymers glass transition temperatures (Tg) where chains have increased mobility and, therefore, are more easily accessed and hydrolyzed by cutinase active sites. Intellectual Merit: By combining computational protein design methods with intuitive rationalizations, variants (changes in the structure of the naturally occurring protein) of the cutinase from Aspergillus oryzae (AoC) were developed with up to a 7 oC increase in thermostability. The best variants were those where salt bridges were created at the enzymes surface. Furthermore, the kinetic stability (how quickly a protein unfolds) increased by 10-fold at 60 oC. However, the increase in thermostability did not result in a corresponding increase in the optimum activity temperature of AoC (Fig 1). Hence, we learned that while these thermostable variants increased the temperature of protein unfolding, changes in structure still occurred at the proteins active site that decrease AoC’s activity. Future work will address better stabilization of the active site region. Study of the cutinase produced by the thermophilic fungi Thiellevia terestris (TtC) revealed that the pH optimum was found to be acidic and basic for poly(e-caprolactone), PCL, hydrolysis and cellulose acetate deacetylation, respectively (Fig 2). The disparity in pH optimum was explained based on differences in TtC binding to these substrates at varying pH values. The binding constant was determined using enzyme kinetic analysis and data fitting. This work highlighted the importance of amino acids that closely neighbor the active site that are critical to productive interactions of proteins with heterogenous substrate surfaces. The effect of glycosylation (attachment of sugars) on TtC properties was also studied. Glycosylation improved TtC’s thermostability (Fig 3). Intriguingly, the extent of kinetic stabilization was higher than the thermodynamic stabilization which was understood by analysis of TtC’s thermal deactivation pathway (Fig 4). Hence, introduction of glycosylation site(s) is a powerful strategy to improve the thermostability of the cutinases. Future work will address the identification of locations where new glycosylation sites will be of greatest benefit to cutinase functional properties. Metagenomic analyses of leaf branch compost by Sulaiman et al. lead to the identification of a novel cutinase (LCC). The unfolding temperature (Tm) of LCC is 86 oC, thus it is highly thermostable. Similar to the above observation with TtC, thermostability analysis revealed that glycosylated LCC has a higher thermostability than non-glycosylated LCC. Furthermore, unlike AoC, the global stabilization of LCC by glycosylation also resulted in stabilization of the active region resulting in a higher optimum temperature for LCC activity. The comparative activity analysis of different cutinase homologues towards PET hydrolysis revealed that LCC is most active. This in part is due to the high thermal stability of LCC that allows the protein to function at temperatures above PET’s glass transition temperature (Tg). Interestingly, LCC also showed better activity than the other cutinases at temperatures below PET’s Tg. Broader Impacts: This research program resulted in important new knowledge in cutinase structure-activity relationships. Insights gained will be used in future work to develop cutinases with industrially relevant performance. Cutinases will enable the performance of chemical transformations such as recycling of PET and CA (in combination with cellulases) to its monomeric units, surface modification, and other chemical reactions that have not yet been identified. Replacing traditional chemical catalysts by enzymes will have important benefits such as reduction in energy consumption, toxic catalyst use and generation of toxic co-products. In addition, there use will result in processes that operate under mild conditions instead of at high temperatures-pressures that compromise worker safety. Developing robust economic enzyme-catalysts represents an inevitable paradigm shift in the chemical industry.
