With the potential for each droplet to act as a unique reaction vessel, droplet microfluidics is a powerful tool for high-throughput discovery. Any attempt at compound screening miniaturization must address the significant scaling inefficiencies associated with library handling and distribution. Eschewing microplate-based compound collections for one-bead-one-compound (OBOC) combinatorial libraries, we have developed hνSABR (Light-Induced and -Graduated High-Throughput Screening After Bead Release), a microfluidic architecture that integrates a suspension hopper for sedimentation-mediated compound library bead introduction, droplet generation, microfabricated waveguides that precisely irradiate (365 nm) the droplet flow and photochemically cleave the compound from the bead to dose the droplet, incubation, and laser-induced fluorescence for assay readout. Avobenzone-doped PDMS (0.6% w/w) patterning confines UV exposure to the desired illumination region, generating in-droplet compound concentrations (> 10 µM) that are reproducible between devices. Beads displaying photocleavable pepstatin A were distributed into droplets and exposed with 5 different UV intensities to demonstrate dose-response screening in an HIV-1 protease activity assay. This microfluidic architecture introduces a new analytical approach for OBOC library screening, and represents a key component of a next-generation distributed small molecule discovery platform.
During the last 50 years human anisakiasis has been rising while parasites have increased their prevalence at determined fisheries becoming an emergent major public health problem. Although artificial enzymatic digestion procedure by CODEX (STAN 244-2004: standard for salted Atlantic herring and salted sprat) is the recommended protocol for anisakids inspection, no international agreement has been achieved in veterinary and scientific digestion protocols to regulate this growing source of biological hazard in fish products. The aim of this work was to optimize the current artificial digestion protocol by CODEX with the purpose of offering a faster, more useful and safer procedure for factories workers, than the current one for anisakids detection. To achieve these objectives, the existing pepsin chemicals and the conditions of the digestion method were evaluated and assayed in fresh and frozen samples, both in lean and fatty fish species. Results showed that the new digestion procedure considerably reduces the assay time, and it is more handy and efficient (the quantity of the resulting residue was considerably lower after less time) than the widely used CODEX procedure. In conclusion, the new digestion method herein proposed based on liquid pepsin format is an accurate reproducible and user-friendly off-site tool, that can be useful in the implementation of screening programs for the prevention of human anisakiasis (and associated gastroallergic disorders) due to the consumption of raw or undercooked contaminated seafood products.
Pepsin is formed as the zymogen, pepsinogen, which includes an additional 44 residue prosegment (PS) on the N-terminus. Upon acidification (pH <3) the PS is removed, yielding active pepsin. The PS is critical to such processes as the initiation of correct folding and protein stability. In the present study, the NMR assignments of the 34.6 kDa native porcine pepsin and porcine pepsin complexed with pepstatin are reported in order to obtain structural information regarding PS-catalyzed protein folding. Such information would contribute to a better understanding of the nature of folding/unfolding energy barrier of pepsin and other aspartic proteases.
Glycopeptides are generated from the enzymatic digestion of glycoproteins with a specific or nonspecific protease. Whether this enzymatic conversion of glycoproteins into glycopeptides and peptides is done in-solution or in-gel, an efficient digestion protocol is one of the key components of a successful outcome in a mass spectrometry-based experimental workflow. This chapter outlines an optimized in-solution digestion protocol to prepare samples for glycopeptide-based mass analysis.
The ingestion of nucleic acids (NAs) as a nutritional supplement or in genetically modified food has attracted the attention of researchers in recent years. Discussions over the fate of NAs led us to study their digestion in the stomach. Interestingly, we found that NAs are digested efficiently by human gastric juice. By performing digests with commercial, recombinant and mutant pepsin, a protein-specific enzyme, we learned that the digestion of NAs could be attributed to pepsin rather than to the acidity of the stomach. Further study showed that pepsin cleaved NAs in a moderately site-specific manner to yield 3'-phosphorylated fragments and the active site to digest NAs is probably the same as that used to digest protein. Our results rectify the misunderstandings that the digestion of NAs in the gastric tract begins in the intestine and that pepsin can only digest protein, shedding new light on NA metabolism and pepsin enzymology.
Celiac disease is characterized by intestinal inflammation triggered by gliadin, a component of dietary gluten. Oral administration of proteases that can rapidly degrade gliadin in the gastric compartment has been proposed as a treatment for celiac disease; however, no protease has been shown to specifically reduce the immunogenic gliadin content, in gastric conditions, to below the threshold shown to be toxic for celiac patients. Here, we used the Rosetta Molecular Modeling Suite to redesign the active site of the acid-active gliadin endopeptidase KumaMax. The resulting protease, Kuma030, specifically recognizes tripeptide sequences that are found throughout the immunogenic regions of gliadin, as well as in homologous proteins in barley and rye. Indeed, treatment of gliadin with Kuma030 eliminates the ability of gliadin to stimulate a T cell response. Kuma030 is capable of degrading >99% of the immunogenic gliadin fraction in laboratory-simulated gastric digestions with minutes, to a level below the toxic threshold for celiac patients, suggesting great potential for this enzyme as an oral therapeutic for celiac disease.
Modifying the protein conformation appears to improve the digestibility of proteins in the battle against allergies. However, it is important not to lose the protein functionality in the process. Light pulse technology has been recently tested as an efficient non-thermal process which alters the conformation of proteins while improving their functionality as stabilizers. Also, in order to rationally design emulsion based food products with specific digestion profiles, we need to understand how interfacial composition influences the digestion of coated interfaces. This study has been designed to investigate the effects of pulsed light (PL) treatment on the gastrointestinal digestion of protein covered interfaces. We have used a combination of dilatational and shear rheology which highlights inter and intra-molecular interactions providing new molecular details on protein digestibility. The in vitro digestion model analyses sequentially pepsinolysis, trypsinolysis and lipolysis of β-lactoglobulin (BLG) and pulsed light treated β-lactoglobulin (PL-BLG). The results show that the PL-treatment seems to facilitate digestibility of the protein network, especially regarding trypsinolysis. Firstly, PL treatment just barely enhances the enzymatic degradation of BLG by pepsin, which dilutes and weakens the interfacial layer, due to increased hydrophobicity of the protein owing to PL-treatment. Secondly, PL treatment importantly modifies the susceptibility of BLG to trypsin hydrolysis. While it dilutes the interfacial layer in all cases, it strengthens the BLG and weakens the PL-BLG interfacial layer. Finally, this weakening appears to slightly facilitate lipolysis as evidenced by the results obtained upon addition of lipase and bile salts (BS). This research allows identification of the interfacial mechanisms affecting enzymatic hydrolysis of proteins and lipolysis, which demonstrates an improved digestibility of PL-BLG. The fact that PL treatment did not affect the functionality of the protein makes it a valuable alternative for tailoring novel food matrices with improved functional properties such as decreased digestibility, controlled energy intake and low allergenicity.
AS-48 is a bacteriocin with potential application as food biopreservative. In order to optimize its use for oral consumption, we assess the impact of gastrointestinal digestion, both in bulk and adsorbed at the air-water interface. Analysis of AS-48 digestion fragments in bulk by SDS-PAGE, RP-HPLC, and MALDI-TOF proves that the previous pepsin exposition promotes digestion by trypsin/chymotrypsin by exposing new cleavage sites. Regarding adsorbed AS-48, the in vitro digestion profile shows that the conformational change undergone by AS-48 upon adsorption affects its digestibility. Gastrointestinal enzymes cleave only susceptible residues, which are oriented into the aqueous phase, while hydrophobic susceptible residues remain undigested. Evaluation of the elasticity of the adsorbed layer confirms also the presence of undigested AS-48. These results are important towards the use of AS-48 in food formulations; assuring that some intact AS-48 resists digestion guarantees its antibacterial activity throughout the gastrointestinal tract.
Oral delivery of biopharmaceuticals is limited by the absorption barriers in the gastrointestinal tract (GIT) such as low permeability across the biological membranes and the enzymatic degradation by proteases. In this study, lipid based drug delivery systems were proposed to overcome these obstacles.
The digestibility of fucosylated glycosaminoglycan (FG) and its effects on digestive enzymes were investigated using an in vitro digestion model. Results showed that the molecular weight and the reducing sugar content of FG were not significantly changed, and no free monosaccharides released from FG were detected after the salivary, gastric and intestinal digestion, indicating that both the backbone and the sulfated fucose branches of FG are resistant to be cleaved in the saliva and gastrointestinal tract environments. Furthermore, FG can dose-dependently inhibit digestive enzymes such as α-amylase, pepsin and pancreatic lipase in different degrees under the simulated digestion conditions due to the sulfate and carboxyl groups. These physiological effects of FG may help control the postprandial glucose concentration and have the potential in the prevention or treatment of reflux disease and obesity. The findings may provide information on the digestibility and beneficial physiological effects of FG as a potential natural product to promote human health.