Characterization of digestive proteases in the gut of a basal deuterostome

Abstract

Digestive systems are complex organs that allow organisms to absorb energy from their environment to fuel vital processes like growth, development and the maintenance of homeostasis. A comprehensive understanding of digestive physiology is therefore essential to fully understand the energetics of an organism. The digestion of proteins is of particular importance because most heterotrophic organisms are not able to synthesize all essential amino acids. While Echinoderms are basal deuterostomes that share a large genetic similarity with vertebrates their digestion physiology remains largely unexplored. Using a genetic approach, this work demonstrated that several protease genes including an enteropeptidase, aminopeptidase, carboxypeptidase and trypsin involved in mammalian digestive networks are also found in sea urchin larvae. Through characterization including perturbation experiments with different food treatments and pharmacological inhibition of proteases using specific inhibitors, as well as transcriptomic analysis we conclude that the trypsin-2 gene codes for a crucial enzyme for the protein digestion in Strongylocentrotus purpuratus. Measurements of in vivo digestion rates in the transparent sea urchin larva were not altered by pharmacological inhibition of trypsin (Soybean Trypsin Inhibitor) or serine proteases (Aprotinin) suggesting that proteases are not critically involved in the initial step of microalgal breakdown. This work provides new insights into the digestive physiology of a basal deuterostome and allows comparisons from the molecular to the functional level in the digestive systems of vertebrates and mammals. This knowledge will contribute to a better understanding for conserved digestive mechanisms that evolved in close interaction with their biotic and abiotic environment.

Electrochemical Impedance Spectroscopy for the Sensing of the Kinetic Parameters of Engineered Enzymes

“…A detailed kinetic analysis of these enzymes, particularly when genetically modified to enhance their properties, is vital for both understanding their biological functions and their application in therapeutic and industrial contexts [ 2 ]. Despite advances in enzymatic studies, accurately determining the kinetic parameters of modified enteropeptidases remains a challenge, impeding the full exploitation of their potential [ 3 ]. According to the MEROPS classification, enterokinase (enteropeptidase) EC 3.4.21.9 falls under the clan of PA proteases, subclan PA(S), family S1 , and subfamily S1A peptidases.…”

“…Traditional methods for enzyme kinetics, such as fluorescence spectroscopy, or other, usually indirect experimental methods, face limitations regarding environmental versatility, cost, and operational complexity. These limitations are particularly pronounced when analyzing samples with varying optical properties or in complex matrices, restricting the comprehensive assessment and utilization of engineered enzymes [ 3 , 9 , 10 , 11 , 12 ]…”

“…Traditional methods for enzyme kinetics, such as fluorescence spectroscopy, or other, usually indirect experimental methods, face limitations regarding environmental versatility, cost, and operational complexity. These limitations are particularly pronounced when analyzing samples with varying optical properties or in complex matrices, restricting the comprehensive assessment and utilization of engineered enzymes [3,[9][10][11][12] Innovative methodologies are necessary to determine critical kinetics parameters, such as the maximum reaction rate (v max ), Michaelis constant (K m ), and turnover number (k cat ), due to discrepancies in their values [6,[13][14][15][16][17][18]. This study aimed to address the aforementioned limitations by utilizing electrochemical impedance spectroscopy (EIS) to determine the kinetic parameters of genetically engineered enteropeptidases.…”