Biocatalysis plays a very important role in numerous natural and technological processes. One of the major deficiencies of many native enzymes, however, is their inability to transform water-insoluble substrates, which often necessitates the use of harsh reaction conditions and polluting technologies based on toxic organic and inorganic catalysts, solvents and their mixtures.
The main objective of this proposal is to develop a family of new highly efficient, recyclable and environmentally benign biocatalytic structures for the synthesis of hydrophobic substrates of biological, pharmaceutical or biotechnological importance, and test the ability of these nanodevices to facilitate biotransformations in water and at ambient temperature. The novelty in the design of these nano-constructs is that individual or multiple glycoproteins will be positioned in the core of a micelle or supramolecular hydrogel, constructed of linear-dendritic copolymers. This macromolecular architectural principle is clearly distinguishable from previous and current research on dendrimer- and polymer-supported enzymes. In this project the dendritic fragments do not serve as crosslinking agents and are not covalently attached to the enzymes. They bind the glycoproteins by soft regioselective nanocontacts. Thus these perfectly branched fragments will serve not only as supports of the enzymatic functions, but will also act as a cooperative micro-environment to provide transition state stabilization, enhancement of enzymatic activity, and selectively promote the uptake and internal transport of substrates and release of products. This protective nanoporous shell will also facilitate the recovery and recycling of the enzymatic assemblies by simple filtration or centrifugation.
Three specific aims will be pursued to produce the targeted biocatalysts and demonstrate their advantages and applicability for sustained, "green" chemistry processes: 1) Construct series of recyclable linear-dendritic enzyme complexes in micellar- or physical hydrogel form without chemical bond formation and explore the influence of chemical, structural and environmental factors on the mechanism and kinetics of the catalyzed reactions; 2) Test single-enzyme constructs with the highest catalytic activity in selected tandem processes, which will involve formation of novel biologically active dimers, oligomers and polymers for multi-purpose applications; 3) Evaluate single- and dual-enzyme complexes for "green chemistry" cascade reactions, where the products, formed in the dendritic shell or through the first enzyme will serve as exclusive substrates of the second enzyme. The project will integrate in a unique way elements from the kinetics of enzymatic biocatalysis and fundamental self-assembly processes with the synthesis of chemically functional organic and polymeric materials.
The impact of this research will result in the development of novel environmentally friendly synthetic strategies for important fine chemicals and pharmaceutical intermediates using water, low energy consumption and renewable natural resources. Broader impacts of the proposed activities will be manifested by the creation of new procedures for in situ drug synthesis and delivery using unique biocompatible nanodevices. The proposed work will also pave the way to a better understanding of vital natural biosynthetic processes involving multiple enzymes. The investigations in this project also provide excellent educational and training opportunities for undergraduate and graduate students in a truly interdisciplinary research at the intersection of polymer materials and supramolecular chemistry, biotechnology and enzymatic catalysis, organic synthesis and macromolecular nanoscience.
One of the many important functions of enzymes in Nature is to induce the formation of polymers. These natural catalysts are characterized with high efficiency, selectivity and functionality. An important advantage of the enzymatic catalysis is that it proceeds under mild reaction conditions – usually in water and at ambient temperature. These features make enzymes very promising substitutes for the typical industrial catalysts, which often require the use of elevated temperatures and toxic solvents. Due to their natural functions many enzymes could not transform water-insoluble substances. The strategic goal of this project was to design, prepare and test an enzymatic complex, able to catalyze in water reactions with hydrophobic compounds. This was achieved without any genetic engineering or any chemical intervention on the enzyme, but with the use of linear-dendritic copolymers. These macromolecules were able to spontaneously adhere to specific domains in the enzymes (polysaccharide residues) and serve as binding sites for water-insoluble reagents. The complex formed with the enzyme laccase was more active than the native natural form and was applied in several diverse areas: The potentially toxic chemical Bisphenol-A was removed from aqueous waste by incorporating it in a water-insoluble copolymer with possible applications in biotechnology; New polymers were prepared from natural steroids with promising biological activity as poly-drugs; The first ever fullerene (buckyball) oxidation in water and at temperatures close to ambient was achieved thus eliminating the use of concentrated sulfuric acid, nitric acid and sodium hydroxide and temperatures above 100°C. The resulting fullerenols have been shown by others to be effective agents in cancer radiotherapy; Novel hydrogels were constructed with the aim of binding enzymes, drugs or fluorescent markers; A new class of water-soluble perfectly branched macromolecules (dendrimers) was created as environmentally friendly catalysts for challenging organic reactions. Findings from the studies within the project were summarized in six articles, published in leading polymer science and biotechnology journals and were presented as invited lectures at three international conferences and at University of Cambridge (Cambridge, England), Swiss Federal Institute of Technology (Zurich, Switzerland), Johannes Gutenberg University (Mainz, Germany) and several other institutions in Bulgaria, Germany, Spain and Switzerland. Most importantly two graduate and two undergraduate students worked on the project and were trained to perform interdisciplinary research at the cross-section of organic and polymer synthesis, "green" and supramolecular chemistry, enzymatic biocatalysis and nano-science. All of them are currently employed by well-established chemical companies in fields related to their academic research.
