Effect of enzymatic cross-linking of β-casein on proteolysis by pepsin

Monogioudi, Evanthia; Faccio, Greta; Lille, Martina; Poutanen, Kaisa; Büchert, Johanna; Mattinen, Maija‐Liisa

doi:10.1016/j.foodhyd.2010.05.007

Cited by 94 publications

(62 citation statements)

References 46 publications

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“…Monogioudi et al [153] observed for β-casein cross-linked by TGase that very large fragments (>20 kg/mol) remain after hydrolysis with pepsin, and that low molecular weight fractions (<4 kg/mol) exhibit a different amino acid composition as determined by mass spectrometry. Hellwig et al [154,155] performed simulated gastrointestinal digestion and used SEC to isolate fractions below and above 0.2-0.5 kg/mol for further analysis.…”

Section: Literature Review Of Studies On Cross-linked Caseinmentioning

confidence: 99%

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

et al. 2018

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Abstract:Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size separation techniques were applied for characterisation of these reactions: gel electrophoresis and size exclusion chromatography. This review summarises their separation principles and discusses the outcome of studies on cross-linked casein from the last~20 years. Both methods, however, show limitations concerning separation range and are applied mainly under denaturing and reducing conditions. In contrast, field flow fractionation has a broad separation range and can be easily applied under native conditions. Although this method has become a powerful tool in polymer and nanoparticle analysis and was used in few studies on casein micelles, it has not yet been applied to investigate cross-linked casein. Finally, the principles and requirements for absolute molar mass determination are reviewed, which will be of increased interest in the future since suitable calibration substances for casein polymers are scarce.

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Section: Literature Review Of Studies On Cross-linked Caseinmentioning

confidence: 99%

Size Separation Techniques for the Characterisation of Cross-Linked Casein: A Review of Methods and Their Applications

et al. 2018

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“…In the stomach, the stability of protein polymers depends on their particular chemical structures (19). For example, enzymatic cross-linking modification of ␤-casein results in the formation of high-molecular-mass polymers with a compact structure and enhanced resistance to in vitro pepsin digestion (20).N-Glycosylation modification at the N in the N-X-S/T sequon commonly occurs in eukaryotic expression systems (21). Previous studies indicate that N-glycosylation confers resistance to proteolysis.…”

mentioning

confidence: 99%

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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“…Such controlled aggregation allows the management of molecular interactions to achieve the desired protein structures. Enzymatically cross-linked -casein decreased their digestibility in comparison with native -casein (Monogioudi et al, 2011). Heat treatment of milk (sterilization) increases protein resistance to the in vitro digestion in comparison with unheated milk, probably because of the structural changes caused by denaturation and aggregation of whey proteins and/or casein with whey proteins through disulfide bounds (Almaas et al, 2006;Dupont et al, 2010).…”

Section: Proteinsmentioning

confidence: 99%