APPLICATION OF SYNTHESISED GLUCOSE-6-ESTERS FOR DETERMINATION OF ESTERASE ACTIVITY

Esterases are enzymes that hydrolyses esters into acid and alcohol. Since esterases are involved in the regulation of many physiological processes such as immune system and inflammatory reactions, they are often associated with neurodegenerative and digestive diseases and inflammatory processes. During inflammatory processes in the body, immune cells are activated and the enzymes they secrete, including esterases, help fight infection. Early detection of esterase activity changes in urine or blood can lead to faster identification of inflammation and more effective treatment. In this study, esterase activity was measured using synthesized glucose-6-esters to assist in identifying inflammatory processes in diagnostic medicine. Glucose-6-esters were synthesized via a transesterification reaction catalysed by Candida antarctica lipase B (CalB). In transesterification reaction, alcohol is displaced from an ester by another alcohol. [1] In this case, the synthesis involves the exchange of the primary hydroxyl group of glucose with an ester group from a short vinyl ester, which serves as the substrate for the reaction. Since glucose has only one primary hydroxyl group, it is the best choice for the synthesis of new esters due to high selectivity of CalB for primary alcohols. [2] The idea of the work is to use synthesized glucose-6-esters and amperometric glucose biosensors to determine the esterase activity. The idea is based on the fact that glucose oxidase is used to construct the amperometric glucose sensor, which is a very specific enzyme and should not react to glucose-6-esters. In the presence of esterase in the solution and catalyzing the hydrolysis of glucose-6-esters, glucose is formed, the increase in glucose concentration is recorded by an amperometric biosensor. The rate of glucose increase is equated to esterase activity. Seven different short vinyl esters were used in the study: vinyl acetate, vinyl propionate, vinyl propionate, vinyl methacrylate, vinyl pivalate, vinyl 4-tert-butylbenzoate and vinyl benzoate. A first-generation amperometric glucose biosensor and a dinitrosalicylic acid-based method for determining reducing sugars were used to assess the efficiency of glucose-6-ester synthesis. As glucose is consumed during synthesis, the reduction in glucose detected by the glucose biosensor can be correlated with the amount of product formed. The study also found an optimum synthesis time of 24 hours and tested few glucose-short vinyl ester concentration ratios. Also, in order to verify the operation of the idea, an attempt was made to determine the activity of two esterases (Carboxyl esterase from porcine liver and acetylcholinesterase from Electrophorus electricus) using the synthesized new esters. In this work, the results obtained by the different methods were compared with each other and different synthesis conditions were tested, to obtain the highest yield.