Following Enzyme Activity with Infrared Spectroscopy

Abstract

Fourier transform infrared (FTIR) spectroscopy provides a direct, “on-line” monitor of enzymatic reactions. Measurement of enzymatic activity is based on the fact that the infrared spectra of reactants and products of an enzymatic reaction are usually different. Several examples are given using the enzymes pyruvate kinase, fumarase and alcohol dehydrogenase. The main advantage of the infrared method is that it observes the reaction of interest directly, i.e., no activity assay is required to convert the progress of the reaction into an observable quantity.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…Different forms of IR spectroscopy were used to measure product release rates in immobilized enzyme systems. Data were used to calculate the enzyme activity [158][159][160][161][162][163][164][165]. However, it was necessary that the reaction medium containing the solid biocatalyst was by-passed in a flow-through sensor or placed in a spectrophotometer cell.…”

“…Enzyme leaching was monitored [82] In situ reaction rates measurements Infrared spectroscopy Free enzyme Activity measurement Kinetic analysis in IR spectrophotometer [158][159][160]162,163] Infrared spectroscopy Free enzyme Reaction monitoring of synthesis and hydrolysis reactions catalyzed by an amidase Reaction media was placed in a spectrophotometer cell [155] Infrared spectroscopy Whole cell suspensions Acetonitrile was biotransformed to acetamide by a nitrile hydratase enzyme and subsequently to acetic acid (carboxylate ion) by an amidase Mid-IR spectroscopy using an immersion probe [156] Infrared spectroscopy Immobilized enzyme Kinetic analysis in a flow cell using a flow through sensor…”

Infrared Difference Spectroscopy as a Physical Tool to Study Biomolecules

“…Traditionally, the ultraviolet or visible spectral range has been used for studying enzymatic activity. Enzymatic reactions are mainly followed indirectly in this spectral range, except for cases where the substrate or product of an enzymatic reaction absorbs in the range used (115,116). Therefore, enzymatic activity can be followed either directly by colored or fluorescent analogs or indirectly by coupling to such enzymatic reactions that can be followed by ultraviolet-visible spectroscopy (115,116).…”

“…Infrared is sensitive to the structure and environment of a molecule (115,116). Therefore, one can directly follow the formation of products as well as the consumption of reactants in the infrared spectrum during the progress of a reaction.…”

A fourier transform infrared spectroscopy (FTIR) based assay for Candida parapsilosis ATCC 7330 mediated oxidation of aryl alcohols

“…Kinetics of enzymes such as lipase, ␤-fructosidase, alkaline phosphatase, ␣-amylase, urease, glucose oxidase, pyruvate kinase, fumarase, alcohol dehydrogenase, Damino acid oxidase were studied largely using FTIR. This study was performed by calibrating the spectral changes in the IR fingerprint region of 1500 cm −1 to 500 cm −1 , in aqueous media (Walde and Luisi, 1989;Cadet et al, 1995;Lendl et al, 1998;Snabe and Petersen, 2002;Karmali et al, 2004a,b;Kumar and Barth, 2010;Gardner et al, 2013). In addition to this, FTIR reportedly is an effi- * Corresponding author.…”