Simultaneous determination of α-amylase and amyloglucosidase activities using flow injection analysis with fourier transform infrared spectroscopic detection and partial least-squares data treatment Original Research Article

A flow injection analysis (FIA) system with fourier transform infrared (FTIR) spectroscopic detection for the simultaneous determination of amyloglucosidase (exo-glucan 1,4-α-glucohydrolase, EC 3.2.1.3) and α-amylase (endo-glucan 1,4-α-glucanohydrolase, EC 3.2.1.1) activities in aqueous solution is proposed. Starch hydrolysis catalyzed by both enzymes was monitored in the mid-IR range by recording IR-spectra of the assay solutions before and after hydrolysis. The intensities of the resulting difference spectra were directly related to the enzyme activities. Furthermore, the different reaction pathways of the two enzymes under investigation produced distinct spectral changes resulting in difference spectra characteristic for each enzyme. Using partial least squares (PLS) regression it was possible to simultaneously determine both enzyme activities covering a range of 38–150 U/l (630–2500 nkat/l) for amyloglucosidase and a range of 500–3750 U/l (8300–62 000 nkat/l) for α-amylase. Absorption due to the sample matrix was successfully eliminated by calculation of the difference spectra. The developed method was also successfully applied to the analysis of spiked fermentation broth samples yielding mean deviations of 2.6% and 10.8% for amyloglucosidase and α-amylase, respectively.