Key features of enzymes are substrate recognition and catalytic activity, and understanding these attributes would have a broad impact on society. Directed evolution methods have emerged as a key protein engineering tool for understanding structure/function relationships and improving protein properties. The goal is to demonstrate that Bacillus subtilis spores are useful for high-throughput screening. Protein display on the surface of B. subtilis offers advantages over more commonly utilized microbe cell-surface display systems, which include gram negative bacteria, phage, and yeast. For instance, protein folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; therefore, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. Structure/function relationships will be elucidated for metalloproteins. Initially, spore display will be demonstrated using the copper containing enzyme CotA, which is present on the B. subtilis spore coat. It will be evolved for increased redox potential and substrate selectivity. The mutants will provide a green alternative for paper bleaching and bioremediation of polycyclic aromatic hydrocarbons. Finally, the CotA system can adapted for heterologous protein display on B. subtilis spores that will be valuable for screening protein libraries.
Broader Impact The research trains undergraduate and graduate students in the multidisciplinary field of molecular evolution. The Department of Chemistry and Environmental Science at the New Jersey Institute of Technology is creating a new Biochemistry program at the undergraduate and graduate level and it is being built around the principal investigator and recent new hires. An infrastructure is being created providing expertise and resources in biochemistry, molecular biology, and protein engineering. New courses have been developed to fill missing program components and to introduce new topics. Courses include Biochemistry Lab, Molecular Biotechnology, Environmental Biology and Environmental Microbiology. Low-income and first-generation or underrepresented undergraduates have an opportunity to contribute original research through the Ronald E. McNair program at NJIT. The PI has an appointment in the Graduate Faculty in the Department of Biological Science at Rutgers-Newark, which fosters multidisciplinary research between NJIT Chemistry and Rutgers-Newark Biology. The research will also be described to a non-technical audience through the Center of Pre-College Programs at NJIT. High school students have an opportunity to compete in Chemistry Olympics, which is sponsored by NJIT, and economically disadvantaged high school students can also be involved in basic biochemistry research through Project SEED. Also, the planned research enhances international partnerships. The structures of the interesting CotA variants will be solved in collaboration with a crystallographic research group at the Universidade Nova de Lisboa in Portugal.
Significant intellectual merit resulted from designing new high-throughput screening tools for protein engineering and optimization. Directed evolution has become the method of choice for engineering proteins with improved properties and investigating structure/function relationships. The bottleneck of directed evolution is oftentimes the screen or selection. Protein surface display of proteins on Bacillus subtilis spores was demonstrated as a tool for high-throughput screening. Spore display offers advantages over more commonly utilized microbe cell-surface display systems, which include gram-negative bacteria, phage, and yeast. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; hence, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. Initially, the laccase CotA, which is present on the B. subtilis spore coat was evolved. The mutants provide a green alternative for paper bleaching and bioremediation of polycyclic aromatic hydrocarbons. Furthermore, insight can be obtaining for natural protein evolution. Finally, the CotA system can be adapted for heterologous protein display on B. subtilis spores, which will be valuable for screening protein libraries. The first goal was to demonstrate proof of principle. Bacillus subtilis spores can be used as a vehicle for directed evolution. CotA was evolved for increased substrate specificity. Next, we took advantage of the inert properties of spores and evolved CotA under extreme conditions of high organic solvent concentration and acidic pH. The proposed research had a broad impact to society by promoting teaching and learning. The research trained high school, undergraduate and graduate students in the emerging field of molecular evolution resulting in new protein engineering technologies to study structure/function relationships and to improve protein properties. In addition, a high school teacher was trained and brought the experience back to the classroom. Furthermore, teaching aids were obtained to assist in high school instruction. The research is multidisciplinary overlapping many academic departments such as chemistry, biology, chemical engineering, computer science, and physics. The Department of Chemistry and Environmental Science at the New Jersey Institute of Technology created Bachelor of Science degree in Biochemistry and it was built around the principle investigator and recent new hires. An infrastructure was created providing expertise and resources in biochemistry, molecular biology, and protein engineering, which has been previously lacking in the department. This attracted new students to the department. Low-income and first-generation or underrepresented undergraduates have contributed original research through the Ronald E. McNair program at NJIT. The PI has an appointment in the Graduate Faculty in the Department of Biological Science at Rutgers-Newark, which fosters multidisciplinary research between NJIT Chemistry and Rutgers-Newark Biology. The research was also described to a non-technical audience through the Center of Pre-College Programs, Open House, and high school visits at NJIT. High school students have the opportunity to compete in Chemistry Olympics, which is sponsored by NJIT, and economically disadvantaged high school students were involved in basic biochemistry research through Project SEED.
