Evaluation of Kluyveromyces marxianus endo-polygalacturonase activity through ATR-FTIR

The endo-polygalacturonase enzyme (endoPG: EC 3.2.1.15) plays an important role in the fruit juice and wine industries, so the development of new tools for the quantitative and qualitative analysis of its enzymatic action is necessary. In this work, we report the development of a simple, fast and practical method that did not use any chemical reagent to identify and evaluate the action of the endoPG enzyme, produced by the yeast Kluyveromyces marxianus CCT3172, using attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy combined with principal component analysis-linear discriminant analysis (PCA-LDA). This method evaluated the action of the endoPG enzyme on the polygalacturonic acid (PGA) substrate at 5 different times (0, 10, 15, 20 and 30 minutes), and at each time interval the samples were analyzed by ATR-FTIR. It was demonstrated that there was clear segregation between the samples that were and that were not subjected to the action of the endoPG enzyme, and it was also possible to distinguish the samples that were subjected to different incubation times with the enzyme. Through PCA-LDA it was possible to obtain wavelengths that are biomarkers for this enzymatic reaction and the observed changes as a function of hydrolysis duration were found to be in agreement with the breakdown of the glycosidic chain (1011 cm^-1-CH-O- CH stretching) of PGA and release of oligosaccharides (1078 cm^-1 C-OH elongation). The activity of the endoPG enzyme and the release of galacturonic acid were verified by the dinitrosalicylic acid (DNS) method in all samples. The efficacy of an automatic classifier using a principal component analysis-linear discriminant classifier (PCA-LDC) was evaluated to diagnose the action of the endoPG enzyme. The results showed an accuracy of 100% for the identification of the endoPG enzyme action and from 91.67% to 100% for classification according to the hydrolysis duration in which PGA was exposed to endoPG. The present study indicates that this methodology may be a new approach for the qualitative evaluation of the endoPG enzyme with the potential to be used in laboratories and industries.