Assessment of α-Amylase Activity in a Microanalysis System: Experimental Optimization and Evaluation of Type of Inhibition

Abstract

Enzymes, enzymatic kinetics, and enzyme inhibition are topics covered in undergraduate biochemistry textbooks, having perceptible relevance to everyday life. The experimental use of enzymes involved in basic physiologic processes may be of utmost pedagogic interest because they bring evidence on how a significant number of drugs are used to control illnesses. Salivary and pancreatic α-amylases are essential digestive enzymes responsible for the partial hydrolysis of starch, the primary source of calories in human diet. As the structures of α-amylase from human pancreas and porcine pancreas are highly similar, the latter is an enzyme that can be easily used in laboratory classes at an accessible price. The activity of this enzyme can also be easily and affordably evaluated by resorting to the substrate 2-chloro-4-nitrophenyl-α-d-maltotrioside (CNPG3). Here, we propose an active-learning environment activity where students use a physiologically relevant enzyme (α-amylase) to develop an experimental protocol, explore their own experimental results, and discriminate between different kinetic models. For this purpose, students are invited to select the best experimental protocol (optimizing the activity conditions/concentration of the enzyme and substrate, pH and temperature parameters), to discriminate different enzymatic models and determine the kinetic parameters through both linear transformations of the Michaelis–Menten equations (Lineweaver–Burk double-reciprocal transformation) and nonlinear regression using the Solver tool of Microsoft Office Excel.

Untargeted Metabolomic Analysis of Randia echinocarpa Cell Cultures Treated with L-Tyrosine.

“…These results were in accordance with estimated free binding energies predicted among α-amylase and small molecules such as avonoid or pirazole derivatives (Rydberg et al, 2002;Mai et al, 2023). The enzyme α-amylase exists in two isoforms, present in both saliva and pancreatic secretions (Freitas et al, 2023). Both isoforms are composed of 496 amino acids and could be divided into three different domains (A, B, and C), with the catalytic triad (Asp197, Glu233, and Asp300) responsible for the cleaving function of the enzymes located in the A domain.…”

Paper-based chemometer device for the estimation of α-amylase—a biomarker for pancreatitis

“…The CNPG3 method is a good assessment for microanalysis system. 50 This CNPG3 method works with UV-visible spectroscopy, which has linearity up to 2000 U L −1 . The accuracy of the PCD was determined by estimating the concentrations, i.e., 70, 140, 210, 280, 350, and 420 U L −1 of a-amylase in ABS.…”

Analysis of the Effects of Windthrows on the Microbiological Properties of the Forest Soils and Their Natural Regeneration

“…To assess the intensity of microbial activity in soil, the activity of specific enzymes [23] such as sucrase [24], amylase [25], glucosidase [26], urease [27], dehydrogenase [28], catalase [29], phosphatase [30], etc., are determined. Among all soil enzymes, dehydrogenases are one of the most important and are used as an indicator of the overall microbial activity of the soil, because they are produced by soil microorganisms intracellularly and capable of multiplication.…”