Revisiting the enzymatic kinetics of pepsin using isothermal titration calorimetry

Luo, Qi; Chen, Dongxin; Boom, R.M.; Janssen, A.E.M.

doi:10.1016/j.foodchem.2018.06.042

Cited by 52 publications

(42 citation statements)

References 31 publications

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“…Pepsin has an optimum pH of 1-2 [8]; therefore, it was inevitable to have less effective digestion in all of the proteins tested at higher pH. Even at its optimal pH, pepsin doesn't completely hydrolyze a protein due to lower efficiency of hydrolysis of peptides than that of an intact protein [26]. Each enzyme utilized has a corresponding specificity or preference for hydrolyzing specific peptide bonds based upon amino acid sequence.…”

Section: Discussionmentioning

confidence: 99%

Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

Wang

Edrington

et al. 2020

PLoS ONE

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The susceptibility of newly expressed proteins to digestion by gastrointestinal proteases (e.g., pepsin) has long been regarded as one of the important endpoints in the weight-of-evidence (WOE) approach to assess the allergenic risk of genetically modified (GM) crops. The European Food Safety Authority (EFSA) has suggested that current digestion study protocols used for this assessment should be modified to more accurately reflect the diverse physiological conditions encountered in human populations and that the post-digestion analysis should include analytical methods to detect small peptide digestion products.The susceptibility of two allergens (beta-lactoglobin (β-Lg) and alpha-lactalbumin (α-La)) and two non-allergens (hemoglobin (Hb) and phosphofructokinase (PFK)) to proteolytic degradation was investigated under two pepsin digestion conditions (optimal pepsin digestion condition: pH 1.2, 10 U pepsin/μg test protein; sub-optimal pepsin digestion condition: pH 5.0, 1 U pepsin/10 mg test protein), followed by 34.5 U trypsin/mg test protein and 0.4 U chymotrypsin/ mg test protein digestion in the absence or presence of bile salts. All samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in conjunction with Coomassie Blue staining and, in parallel, liquid chromatography tandem mass spectrometry (LC-MS) detection. The results provide following insights: 1) LC-MS methodology does provide the detection of small peptides; 2) Peptides are detected in both allergens and non-allergens from all digestion conditions; 3) No clear differences among the peptides detected from allergen and non-allergens; 4) The differences observed in SDS-PAGE between the optimal and sub-optimal pepsin digestion conditions are expected and align with kinetics and properties of the specific enzymes; 5) The new methodology with new digestion conditions and LC-MS detection does not provide any differentiating information for prediction whether a protein is an allergen. The classic pepsin resistance assay remains the most useful assessment of the potential exposure of an intact newly expressed protein as part of product safety assessment within a WOE approach.

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Section: Discussionmentioning

confidence: 99%

Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

Wang

Edrington

et al. 2020

PLoS ONE

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“…Pepsin can attack multiple peptide-bonds, with some more easily than others. Its activity is said to be inhibited by intermediate peptides ( Qi et al., 2018 ). Salivary amylase and gastric lipase can be active in the stomach ( Sarkar et al., 2015 , Lamond et al., 2019 ), but their action is irrelevant for meals of protein gels, and hence we disregard further discussion of that.…”

Section: Models With Realistic Gastric Conditionsmentioning

confidence: 99%

Physical chemistry of gastric digestion of proteins gels

Sman

Houlder

Cornet

et al. 2020

Current Research in Food Science

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In this paper, we present the rich physics and chemistry of the gastric digestion of protein gels. Knowledge of this matter is important for the development of sustainable protein foods that are based on novel proteins sources like plant proteins or insects. Their digestibility is an important question in the design of these new protein foods. As polyelectrolyte gels, they can undergo volume changes upon shifts in pH or ionic strengths, as protein gels experience when entering the gastric environment. We show that these volume changes can be modelled using the Flory-Rehner theory, combined with Gibbs-Donnan theory accounting for the distribution of electrolytes over gel and bath. In-vitro experiments of soy protein gels in simulated gastric fluid indeed show intricate swelling behaviour, at first the gels show swelling but at longer times they shrink again. Simulations performed with the Flory-Rehner/Gibbs-Donnan theory reproduce qualitatively similar behaviour. In the final part of the paper, we discuss how the model must be extended to model realistic conditions existing in the in-vivo gastric environment.

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“…A 0.3 mM CGA-N12 solution was injected into 0.05 mM rKRE9 at 25°C, and a one-site binding model was selected to fit the data. ITC can directly measure minute enthalpy changes accompanying the binding of a peptide to a membrane [49] or to a protein [50,51]. In the present study, ITC was used as the main tool to investigate the thermodynamics of the interaction between CGA-N12 and KRE9.…”

Section: Discussionmentioning

confidence: 99%

The antifungal peptide CGA-N12 inhibits cell wall synthesis of Candida tropicalis by interacting with KRE9

Liu

Dong

et al. 2020

Biochemical Journal

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CGA-N12, an antifungal peptide derived from chromogranin A, has specific antagonistic activity against Candida spp., especially against Candida tropicalis, by inducing cell apoptosis. However, the effect of CGA-N12 on the Candida cell wall is unknown. The Candida protein KRE9, which possesses β-1,6-glucanase activity, was screened by affinity chromatography after binding to CGA-N12. In this study, the effect of CGA-N12 on KRE9 and the interaction between CGA-N12 and KRE9 was studied to clarify the effect of CGA-N12 on C. tropicalis cell wall synthesis. The effect of CGA-N12 on recombinant KRE9 β-1,6-glucanase activity was investigated by analyzing the consumption of glucose. The results showed that CGA-N12 inhibited the activity of KRE9. After C. tropicalis was treated with CGA-N12, the structure of the C. tropicalis cell wall was damaged. The interaction between CGA-N12 and KRE9 was analyzed by isothermal titration calorimetry (ITC). The results showed that their interaction process was involved an endothermic reaction, and the interaction force was mainly hydrophobic with a few electrostatic forces. The results of the fluorescence resonance energy transfer (FRET) assay showed that the distance between CGA-N12 and KRE9 was 7 ∼ 10 nm during their interaction. Therefore, we concluded that the target of CGA-N12 in the C. tropicalis cell membrane is KRE9, and that CGA-N12 weakly binds to KRE9 within a 7 ∼ 10 nm distance and inhibits KRE9 activity.

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Revisiting the enzymatic kinetics of pepsin using isothermal titration calorimetry

Cited by 52 publications

References 31 publications

Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

Presence of small resistant peptides from new in vitro digestion assays detected by liquid chromatography tandem mass spectrometry: An implication of allergenicity prediction of novel proteins?

Physical chemistry of gastric digestion of proteins gels

The antifungal peptide CGA-N12 inhibits cell wall synthesis of Candida tropicalis by interacting with KRE9

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