Spontaneous, non-enzymatic breakdown of peptides during enzymatic protein hydrolysis

Butré, Claire I.; Bühler, Sofie; Sforza, Stefano; Gruppen, Harry; Wierenga, Peter A.

doi:10.1016/j.bbapap.2015.03.004

Cited by 17 publications

(19 citation statements)

References 22 publications

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“…On the other side, if postextraction protein digestion is needed, in principle, it is possible to predict at first glance which peptides are going to be generated from the known proteins and the known enzyme and pre‐select the most suitable markers. However, the not complete predictability of proteolytic enzyme behavior (missed cleavages, selectivity among different cleavage sites, impurities in the enzyme batches, and unexpected side reactions during enzymatic reactions) and the fact that some peptides might be modified by the technological processes make this theoretical approach quite unfeasible. Thus, in general, the most convenient procedure for finding marker molecules is to always carefully analyze the mixture of peptides (either already present or generated after extraction) and to identify among them the potential markers.…”

Section: Methodology Overview: Tricks and Tipsmentioning

confidence: 99%

Peptides as probes for food authentication

et al. 2018

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The aim of this review is to provide a brief summary of the recent applications of peptide molecules in the food area. Since, in the last few years, there has been growing interest in food authenticity, the role of peptides in this area has assumed greater importance. A fundamental part, as discussed in the article, was played by the growth of innovative analytical techniques, based on mass spectrometry (MS), which allows identifying and quantifying protein fragments in complex mixtures of different components. These procedures have been successfully applied for the identification of peptide markers in food authenticity. Because food‐derived proteins have high variability in chemical and physical properties, it is obvious that an exhaustive proteomic analysis cannot be applied by using a single technique for all objectives. The report shows some examples of various methodologies applied to different food matrices and some trivial aspects are also critically described.

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Section: Methodology Overview: Tricks and Tipsmentioning

confidence: 99%

Peptides as probes for food authentication

et al. 2018

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“…For β-cas, the relatively low molar sequence coverages in two parts of the sequence, i.e. and [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48], might be due to the fact that these peptides contain phosphoserines. Yet it has been reported that phosphorylated peptides are typically not well ionised 32, 33 .…”

Section: Towards Predicting Bovine Tryptic Hydrolysis 51mentioning

confidence: 99%

“…In a bovine tryptic hydrolysate of human serum albumin, the number of peptides with CSs in the AA sequences was ~60 % higher than in a porcine tryptic hydrolysate 34 . This indicated that bovine and porcine trypsins might not have the same secondary specificity.…”

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“…The a-specific cleavage was previously found to be the autolytic degradation of certain intrinsically unstable peptides bonds, referred to as the "spontaneous cleavage" 34 . It seems that the spontaneous cleavages occurred more in the hydrolysis at a higher temperature, leading to a higher DHmax,exp.…”

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“…Yet it has been reported that phosphorylated peptides are typically not well ionised 32, 33 . Based on data obtained from previous studies 3, 34,35 , the thresholds of acceptance for the average peptide and molar sequence coverages were 80 % and 70 %, respectively. From the average peptide and molar sequence coverages in this study (Figure 3.2), the quality of the peptide analysis was either better than or similar to previous studies 3, 34,35 .…”

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Towards predicting enzymatic protein hydrolysis

Deng¹

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The experimental maximum degree of hydrolysis (DHmax,exp) was typically lower than the theoretical maximum degree of hydrolysis (DHmax,theo), calculated using the number of cleavage sites (as based on protease specificity) on the protein. The aim of this thesis is, therefore, to develop a method to better define expectations of the DHmax,exp. The developed method is based on the molecular properties of the protein and the protease, and the effects of modifications on protein primary structure and changes in system conditions. Protein primary structure was modified by glycation, the initial stage of the Maillard reaction upon heating proteins in the presence of carbohydrates. The effect of glycation on DHmax,exp for five different enzymes could be predicted based on the protease primary and secondary specificity, and the sensitivity of the enzyme to modifications of the binding site. Secondary specificity was determined by linking the molecular properties of the amino acids on the binding site positions of cleavage sites with the enzyme selectivity. The enzyme selectivity, i.e. the relative hydrolysis rate constants towards cleavage sites in a protein, of bovine, porcine and human trypsins was determined after full quantitative analysis of peptides formed during hydrolysis. The prediction of DHmax,exp for bovine trypsin based on the secondary specificity was 5 times better than the prediction based on the primary specificity (DHmax,theo). The large differences found in DHmax,exp by the three trypsins could be predicted based on enzyme secondary specificity. The substrate concentration, one of the system conditions varying in the in vitro digestion models, also showed large effect on DHmax,exp. This was found to be related to changes in the mechanism of hydrolysis, illustrated by the differences in the percentage of intact protein in the hydrolysates as a function of DH. Due to the changes in mechanism, different amounts of inhibitory peptides for the protease were proposed to be present in the hydrolysates, leading to various DHmax,exp. Based on these findings, a model to predict the DHmax,exp of hydrolysis of any given protein by any specific protease was developed. The concept was shown to adequately explain the hydrolysis of proteins with various structural states and in hydrolysis by a sequential addition of enzyme. The study of in vivo protein digestibility has received a lot of attention from food and feed producers. In vitro enzymatic protein hydrolysis is often used as a screening tool to investigate the protein digestibility since the in vivo protein digestion experiments are not always feasible options. Enzymatic protein hydrolysis is also used to produce protein hydrolysates, which are used as ingredients in food products. The outcome of an enzymatic protein hydrolysis is affected by many factors, including the structure of the substrate protein, the protease used and their sensitivities towards system conditions. For example, the modifications on the protein structure during industrial process...

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Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins

Nongonierma

Fitzgerald

2017

Anal Bioanal Chem

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Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

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Spontaneous, non-enzymatic breakdown of peptides during enzymatic protein hydrolysis

Cited by 17 publications

References 22 publications

Peptides as probes for food authentication

Peptides as probes for food authentication

Towards predicting enzymatic protein hydrolysis

Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins

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