Engineering of Aspergillus oryzae cutinase to improve its stability and activity on synthetic polyester substrates

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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.