The soluble recombinant N-terminal domain of HMW 1Dx5 and its aggregation behavior

Wang, Jing Jing; Liu, Guang; Qing-jun, Zeng; Shen, Xing; Hou, Yi; Li, Lin; Hu, Song-Qing

doi:10.1016/j.foodres.2015.10.010

Cited by 25 publications

(18 citation statements)

References 28 publications

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“…Despite the above, cereal scientists have gradually realized the importance of terminal domains in dough quality . The structure model shows that nonrepetitive N-terminal domain and C-terminal domain appear to be more similar to globular proteins, containing α-helix and aperiodic structure .…”

Section: Introductionmentioning

confidence: 99%

Dissecting the Disulfide Linkage of the N-Terminal Domain of HMW 1Dx5 and Its Contributions to Dough Functionality

Wang

Liu

Huang

et al. 2017

J. Agric. Food Chem.

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The N-terminal domain of HMW-GS 1Dx5 (1Dx5-N) contains three cysteine residues (Cys10, Cys25, Cys40), which are the basis of gluten network formation through disulfide bonds. Disulfide linkage in 1Dx5-N was dissected by site-directed mutagenesis and LC-MS/MS, and its contributions to structural and conformational stability of 1Dx5-N and dough functionality were investigated by circular dichroism, intrinsic fluorescence, surface hydrophobicity determination, size exclusion chromatography, nonreducing/reducing SDS-PAGE, atomic force microscopy, and farinographic analysis. Results showed that Cys10 and Cys40 of 1Dx5-N were the active sites for intermolecular linkage. Meanwhile, Cys40 also exhibited the ability to form intrachain disulfide linkage with Cys25. Moreover, Cys10 and Cys40 played a functionally important role in maintaining the structural and conformational stability and high surface hydrophobicity of the N-terminal domain of HMW-GS, which in turn facilitated the formation of HMW polymers and massive disulfide linkage of HMW-GS through hydrophobic interaction. Additionally, the 1Dx5-N mutants in which Cys were replaced by serine (Ser) presented different effects on dough functionality, while only the C25S mutant produced positive effects compared with wild type 1Dx5-N. NaCO-induced β-elimination of cystine might occur in glutenin without heating, which would make it much easier to reduce the nutritional quality of flour products by the cost of lysine. Therefore, these results give a deep understanding of the disulfide linkage of the N-terminal domain of HMW-GS and its functional importance, which will provide a practical guide to effectively generate a superior HMW-GS allele by artificial mutagenesis.

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Section: Introductionmentioning

confidence: 99%

Dissecting the Disulfide Linkage of the N-Terminal Domain of HMW 1Dx5 and Its Contributions to Dough Functionality

Wang

Liu

Huang

et al. 2017

J. Agric. Food Chem.

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“…Nevertheless, the mechanism of such aggregate formation for soluble MnP still needs further study. Chihi et al [54] demonstrated that the hydrophobic interactions and the formation of new disulfide bridges resulted in soluble protein aggregates; while, Wang et al [55] emphasized the importance of the hydrophobic interaction in promoting the protein aggregate, as compared with disulfide bonds. Therefore, we will shed light on the intriguing mechanism of polymerization for soluble MnP in the future.…”

Section: Discussionmentioning

confidence: 99%

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes

et al. 2016

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BackgroundManganese peroxidase (MnP) from Irpex lacteus F17 has been shown to have a strong ability to degrade recalcitrant aromatic pollutants. In this study, a recombinant MnP from I. lacteus F17 was expressed in Escherichia coli Rosetta (DE3) in the form of inclusion bodies, which were refolded to achieve an active enzyme. Further, we optimized the in vitro refolding conditions to increase the recovery yield of the recombinant protein production. Additionally, we attempted to express recombinant MnP in soluble form in E. coli, and compared its activity with that of refolded MnP.ResultsRefolded MnP was obtained by optimizing the in vitro refolding conditions, and soluble MnP was produced in the presence of four additives, TritonX-100, Tween-80, ethanol, and glycerol, through incubation at 16 °C. Hemin and Ca2+ supplementation was crucial for the activity of the recombinant protein. Compared with refolded MnP, soluble MnP showed low catalytic efficiencies for Mn2+ and H2O2 substrates, but the two enzymes had an identical, broad range substrate specificity, and the ability to decolorize azo dyes. Furthermore, their enzymatic spectral characteristics were analysed by circular dichroism (CD), electronic absorption spectrum (UV-VIS), fluorescence and Raman spectra, indicating the differences in protein conformation between soluble and refolded MnP. Subsequently, size exclusion chromatography (SEC) and dynamic light scattering (DLS) analyses demonstrated that refolded MnP was a good monomer in solution, while soluble MnP predominantly existed in the oligomeric status.ConclusionsOur results showed that two forms of recombinant MnP could be expressed in E. coli by varying the culture conditions during protein expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0317-2) contains supplementary material, which is available to authorized users.

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“…A mobile phase consisting of 0.1 M NaCl (pH 12.0) was used to elute the sample at an equidistant flow rate of 0.5 ml/min, and the absorbance was determined at 280 nm. SDS (15 mM) and DTT (25 mM) were added to the protein samples to analyze the protein aggregation (16).…”

Section: Size Exclusion High Performance Liquid Chromatographymentioning

confidence: 99%

“…In Figure 1C, the role of hydrophobic interaction and disulfide bond in proteins aggregation was determined after treatment with hydrophobic interaction denaturant SDS and disulfide bond reducing agent DTT (16). The elution profile consisted of four components, including high molecular weight protein (HMW, I), large molecular weight protein (LAMW, II), medium molecular weight protein (MMW, III) and low molecular weight protein (LMW, IV).…”

Section: Aggregation Behaviormentioning

confidence: 99%

“…When the pH value was 3, 7, and 9, the ζpotential values were >30 mV, and when the pH value was 5, a minimum 20.2 mV was reached. Previous studies have shown that pH 5 is close to the isoelectric point of giant shrimp protein, which reduces the surface charge of protein and forms aggregates (16). However, the proteins had a high positive charge at pH 3, and a high negative charge at pH 7 and 9, thus inhibiting the aggregation of proteins and maintaining the stability of emulsions.…”

Section: Effect Of Phmentioning

confidence: 99%

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Characterization of a Novel Food Grade Emulsion Stabilized by the By- Product Proteins Extracted From the Head of Giant Freshwater Prawn (Macrobrachium rosenbergii)

Wang

et al. 2021

Front. Nutr.

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The aim of this work was to develop a food-grade emulsion that stabilized by the by-product proteins in the head of giant freshwater prawn. The physicochemical properties of protein particles were characterized, and the stability of proteins-stabilized emulsions under different environmental stresses was evaluated. Results showed that the proteins were relatively hydrophilic and preferentially resided in the aqueous phase to form oil/water emulsions. On this basis, the proteins exhibited superior ability to stabilize the emulsions, and remarkably, the emulsions stabilized by 2% proteins and 3:7 oil/water ratio efficiently resisted the freeze-thaw treatment and the change of pH (3–9), salt addition (NaCl, 50–400 mM), and storage temperatures (4–60°C). Furthermore, the emulsions showed a matched long-term stability with the existing biopolymers-stabilized emulsions. Consequently, this is the first finding of the by-product proteins in the head of giant freshwater prawn as an excellent emulsifier to stabilize emulsions, which help to improve the stability of food-grade emulsions and the added value of aquatic products.

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The soluble recombinant N-terminal domain of HMW 1Dx5 and its aggregation behavior

Cited by 25 publications

References 28 publications

Dissecting the Disulfide Linkage of the N-Terminal Domain of HMW 1Dx5 and Its Contributions to Dough Functionality

Dissecting the Disulfide Linkage of the N-Terminal Domain of HMW 1Dx5 and Its Contributions to Dough Functionality

Expression of a fungal manganese peroxidase in Escherichia coli: a comparison between the soluble and refolded enzymes

Characterization of a Novel Food Grade Emulsion Stabilized by the By- Product Proteins Extracted From the Head of Giant Freshwater Prawn (Macrobrachium rosenbergii)

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