A thermogravimetric method for studying the kinetics of enzyme catalysed reactions that produce a volatile co-product

A general method, using a thermogravimetric balance, has been developed for determining the rate of enzyme reactions that involve loss of a volatile co-product in the presence of an involatile product, the method requiring only small amounts of both substrate and enzyme. Following our interest in enzyme mediated polyester formation, the method was developed using the Candida antarctica lipase B catalysed reaction of various diols with adipic acid or monoethyl adipate (MEAA) to form polyesters and water or ethanol, respectively. The reactions were shown to be first order in enzyme concentration. 1,4-Butanediol was found to be too volatile for accurate rate determination, but the volatilities of 1,6-hexanediol and polytetramethylene diol (PTMEG 650) were sufficiently low for reaction rates to be determined below 80 °C for 1,6-hexanediol and 140 °C with PTMEG 650. Although Candida antarctica lipase B is a thermozyme it does show significant degradation of reaction rate above 130 °C. Arrhenius parameters for the reaction of 1,6-hexanediol with adipic acid and MEAA confirm that the enzyme has a distinct preference for reaction with ester rather than carboxylic acid groups.