Application of infrared spectroscopy (attenuated total reflection) for monitoring enzymatic activity on substrate films

Infrared film analysis, a method based on infrared spectroscopy in the mode of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), is demonstrated as a novel analytical method for monitoring enzymatic activity on surface-attached substrate films in the mid infrared range (400-4000 cm(-1)). The ATR-FTIR technique is sensitive to molecules within a distance of approximately 1 microm from the ATR sampling unit surface (a 7 cm(2) hydrophobic ZnSe crystal). Applying a 0.2-0.3 microm thick film on the ATR unit surface, any chemical changes within this film as well as at the interface can be continuously monitored, even having an aqueous phase on top of the film. Infrared film analysis is considered especially useful for studying detergent enzymes, which act on surface bound films consisting of food component like vegetable oils (triacylglycerols) and carbohydrates (e.g. starch). Experimental data are presented for hydrolysis of a triacylglycerol film (triolein) by use of a triacylglycerol lipase (cutinase), and starch film degradation by use of an alpha-amylase.