Humicola insolens cutinase; a novel catalyst for polymer synthesis reactions

The cutinase class of enzymes, specifically Humicola insolens cutinase (HiC), was methodically studied for its use as a potential biocatalyst for various polymer synthesis reactions; as a substitute or possibly a better alternative to other commercially available biocatalysts.;Biocatalysis is an emerging field of research that has gained prominence in the past decade with greater emphasis on development of environmentally friendly processes and/or resources for development of materials with unique physical and chemical properties. This work focused on the development and optimization of immobilized HiC as it would allow for the optimal use of this enzyme in organic media, at substantially higher reaction temperatures and to facilitate removal of enzyme from the product at the termination of the reaction so that it may be re-used over multiple reaction cycles. This involved understanding how enzyme activity is affected by different methods of enzyme immobilization such as chemical verse physical immobilization; effects of the immobilization support such as porosity, surface chemistry and bead size; and the efficiency of enzyme loading onto immobilization supports. Furthermore, work was carried out to determine effects of various organic solvents on immobilized HiC enzyme activity, optimal reaction temperature and catalyst concentration. Working at the highest possible reaction temperature where the enzyme remains active is crucial in order to limit diffusion constraints in reactions of polymer synthesis. In the first phase of this work, an HiC immobilized on Amberzyme oxirane resin (HiC-AO) system is optimized for use in polyester condensation reactions. In the second phase, HiC-AO was used to catalyze polycondensation reactions. The emphasis of this work was to gain an understanding of enzyme selectivity for different chain length diacid, diol and &omega hydroxyacid substrates. These reactions were also performed in parallel with CALB (N435) catalysis, and the results of the HiC-AO and CALB catalyzed reactions are compared. Moreover, a similar series of small molecule esterification experiments were carried out with both acids and alcohols of various chain lengths using both N435 (CALB) and HiC-AO. These products were analyzed and conclusions were drawn as to the chain length selectivity of the two enzymes in small molecule esterification reactions. Furthermore, comparisons were drawn between similar and dissimilar trends found in the two series of experiment.;In the third phase of this work, the HiC-AO was used to catalyze the synthesis of various polyol polyesters. Reactions were performed by changing the molar ratio of the alditol added to each reaction. Many of these reactions were repeated with N435 catalysis and enzyme activities as well as in-depth structural analysis of the products are compared. This work also includes an extensive thermostability study of 4 different wild type cutinase enzymes and preliminary data comparing the chain length selectivity of these enzymes in small molecule esterification reactions between octanol and acids of varying chain lengths.