Enzymatic hydrolysis coupled to hot water extraction for determination of flavonoids in plants

Abstract: The word sustainability is more and more frequently used within the field of chemistry and in many other fields. Green or sustainable chemistry was introduced during the 90 ́s as a field of chemistry where the environmental and health impacts of processes and products are evaluated and improved. Within analytical chemistry the awareness of sustainability has also increased, which has resulted in optimisation and development of new techniques and methods. The easiest way to improve the sustainability of an analytical method is to decrease and/or replace the use of hazardous chemicals with ones known to have less negative impact on the environment. This thesis reports on a study of the extraction and hydrolysis of quercetin glucosides from onion using sustainable solvents and catalysts. Water at elevated temperatures has been used as a green extraction solvent and, to catalyse the hydrolysis reaction, a thermostable beta-glucosidase expressed from Thermotoga neapolitana, TnBgl1A has been used. It is shown that the use of water as extraction solvent and enzyme as catalyst have less environmental impact than the conventional method where aqueous methanol and a high concentration of HCl are used as extraction solvent and catalyst, respectively. In studying the rate of hydrolysis, it was found that glucose bound at the 3-position of quercetin was slower to hydrolyse than glucose bound at 4 ́-position when TnBgl1A was used as catalyst. Different amino acids in the active site of TnBgl1A were mutated and one mutant, TnBgl1A_N221S/P342L, showed an increased hydrolysis rate of glucose at 4 ́-position, but also of glucose at 3-position, compared to the wild type enzyme. TnBgl1A_N221S/P342L was then immobilised to support materials to allow reuse and also to be used in a continuous flow system. The combination of extraction and hydrolysis of quercetin glucosides from onion in a continuous flow system was not as trivial as originally expected. The total time was quite long, 2 hours, due to slow extraction at optimal hydrolysis conditions. However, by using an on-line method, manual work was reduced and no extra separation step of extract and immobilised enzyme was needed. Molecularly imprinted polymers were developed to accomplish the step after extraction and hydrolysis, i.e. isolation of quercetin from the onion extract. The imprinted polymers were tested for isolation of quercetin from aqueous yellow onion extract, and it was found that the binding capacity of quercetin to MIP increased significantly with increasing temperature.