A mid-IR flow-through sensor for direct monitoring of enzyme catalysed reactions. Case study: measurement of carbohydrates in beer

Abstract

A novel mid-IR flow-through sensor for in situ monitoring of the enzymatic reaction of amyloglucosidase with carbohydrates was developed. Amyloglucosidase was immobilised on agarose beads with N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and directly placed in a conventional IR flow-through cell. The carbohydrate content of various beer samples was then determined by following the enzymatic hydrolytic cleavage of carbohydrates to glucose with Fourier-transform infrared (FTIR) spectroscopy. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating the flow-through sensor in a sequential injection (SI) manifold. As the immobilised enzyme was directly probed by the IR beam, an in situ study of the enzymatic reaction was possible enabling determination of the Michaelis–Menten constant of the immobilised enzyme. A linear calibration curve was recorded using maltose standards in the range between 0.86 and 7.13 g L⁻¹. The proposed method was successfully applied to the determination of the carbohydrate content of four different beer samples by the standard addition method. Moreover experiments showed the possibility of monitoring in situ the immobilisation of the enzyme as well as a small organic acid (malic acid) on the agarose beads using EDC.