Enzymatic Assay

Enzymatic assay, based on oxidation-reduction reaction catalyzed by alcohol dehydrogenase, is one of the methods used to determine ethanol concentration. The present study was directed to determine the exact amount of enzyme required to accomplish oxidation-reduction reaction so that the concentration of ethanol in the sample can be determined precisely and accurately. Results of the present study indicate that the lowest unit activity of the enzyme that can be used for ethanol determination is 4000 units/mL, even though longer incubation time compared to the original method was used to ensure reaction completion. Validation of the method confirmed that the assay have acceptable linearity range within 0.01 - 0.06% (v/v) of ethanol with correlation coefficient of 0.9999. Both accuracy and precision parameters fulfill the Association of Analytical Communities (AOAC) International requirement, and therefore can be accepted as a quantitative analysis method. Limit of detection and limit of quantitation for the modified method were 0.0017% (v/v) and 0.0056% (v/v), respectively.

Symbiotic fungi are known to enhance the adaptive ability of their host plants exposed to various environmental stresses. This study aimed to quantify different modes of radical scavenging abilities of RSF including production of enzymes, phenolics and other antioxidative compounds. Total antioxidant activities of RSF were quantified using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assays. The production of phenolics was measured using Folin-Ciocalteu assay. Enzyme activities were measured in catalase (CAT) and ascorbate peroxidase (APX) katals per milligram dry weight The five RSF isolates exhibited significantly higher ascorbic acid equivalent (AAE) production per milliliter of broth compared to the broth and water control (86.98+7.28 μg/ml). M. guilliermondii produced the highest AAE (182.94+0.17 μg/ml) comparable to the AAE produced by the four other RSF isolates. In the production of phenols, all RSF isolates produced significantly higher gallic acid equivalent (GAE) compared to the broth and water control. T. simmonsii (709.38+39.40 μg/ml) produced the highest GAE per milliliter of broth. This value is more than 3x the GAE produced by the negative control (240.17+91.54 μg/ml). Antioxidant enzyme activities (both CAT and APX) were also observed in all the RSF isolates. The preliminary data on this study established the potential radical scavenging and stress adaptive mechanisms of dominant RSF isolates found on D. quercifolia.

HOW TO CITE: Aban, J. L., Barcelo, R. C., Oda, E. E., Reyes, G. A., Balangcod, T. D., Gutierrez, R. M. and Hipol, R. M. (2017). Quantification of the total antioxidative-, enzymatic- and phenolic- activities of dominant root symbiotic fungi (RSF) from Drynaria quercifolia L. Journal of Applied Environmental and Biological Sciences. 7(7).