Determination of Amyloglucosidase Activity Using Flow Injection Analysis With Fourier Transform Infrared Spectrometric detection

Abstract

An automated method for the determination of amyloglucosidase (glucan 1,4-α-glucosidase) activity in aqueous solution is proposed. The method is based on the direct FTIR spectrometric monitoring of starch hydrolysis catalyzed by amyloglucosidase. It is shown that spectral changes caused by the enzyme action can be directly related to the enzyme activity, hence eliminating the need for additional consecutive reaction steps which are normally required to derive a reaction product detectable by conventional techniques such as UV/VIS spectrometry or electrochemistry. Unspecific background absorption was eliminated by calculation of a difference spectrum of the FTIR spectrum of unreacted starch and the FTIR spectrum of the reaction mixture. To guarantee optimum handling of the viscous starch solution and to obtain high sensitivity, a flow injection analysis system combining the merging zone approach and the stopped flow technique was developed. Using a stopped flow time of 5 min and a 55 g l ^-1 starch substrate solution, linear calibration curves from 50 to 2000 U ^-1 (830 - 33 300 nkat l ^-1 ) were obtained. Recoveries for spiked fermentation broth samples ranged from 102 to 98% and were determined by the standard addition method.