Mechanistic studies of the enzymatic synthesis of polyesters
A thermogravimetric technique for following lipase reaction kinetics has been developed. The relationships between the reaction rate and substrate have been determined for Candida Antarctica lipase B. At higher concentrations, evidence of substrate inhibition was found, due to the pH of the system decreasing as the concentration of acid increased. This has a dramatic adverse effect on the enzyme activity as it moves away from the optimum pH 7.0. Buffering the system closer to optimum pH increased the reaction rate. Also, it was found that as the hydrophobicity of the medium, expressed as C logP, increased the reaction rate became slower. The effect of substitution on the acid substrates was studied; it was found that the enzyme accepted substrates with substitution in the 3-position but not in the 2-position. As a result of modelling, this effect was explained by the disruption of hydrogen bonding, which stabilised the acyl enzyme tetrahedral intermediate. The thermodynamic reaction parameters were determined using Isothermal Titration Calorimetry. The difference in reactivity of ester and acid carbonyls was determined together with the entropy and enthalpy of formation of the acyl intermediates. In the solvent free reaction low dispersity and high molecular weight polyesters are formed due to the limited solubility of the polyester in the diol, only limited transesterification occurring at the ends of the chain. Molecular modelling was used to map the surface hydrophobicity around the enzyme active site in an attempt to explain the observed hydrophobic effects. Modelling around and within the active site was carried out in order to explain the activity of different substrates. The information gained from these studies led to the synthesis of several novel polyesters and polyurethanes, which may have commercial utility in coatings and adhesives. We have investigated the secondary structure of the Candida antarctica lipase B enzyme using conventional CD and synchrotron radiation CD in aqueous buffers and solvents. The secondary structure was determined under different conditions, using the CDSSTR and Selcon programs. Little difference was found between the structure in aqueous buffers and solvents such as hexane, however, really polar solvents like dioxane and THF unfolded the protein. Novel Near Infrared (NIR) spectroscopic methods were developed for the determination of the acid number and the hydroxyl number of polyesters. The effect of changes in the backbone structure of the polyester on the NIR spectrum of the polyester was determined and calibration curves developed for all the common types of linear aliphatic polyesters. The importance of the intra-molecular hydrogen bonding between the acid carbonyl and the hydroxyl groups has been established and the effect of temperature on the degree of association determined. It was found that even at a temperature of 120°C there was still substantial association between the two groups. Partial least squares analysis was developed for the simultaneous determination of both acid number and hydroxyl number. It has been shown that the principal difference between the conventionally synthesised polyesters and those synthesised using enzymatic catalysis is that the latter have little or no carboxyl termination at the ends of the polymer chain. This effect has been explained by the mechanism of the enzymatic polymerisation.