Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design

Leone, Serena; Pica, Andrea; Merlino, Antonello; Sannino, Filomena; Temussi, Piero Andrea; Picone, Delia

doi:10.1038/srep34045

Cited by 39 publications

(35 citation statements)

References 63 publications

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“…Analysis of variance results (Table ) showed that the sweeteners did not differ for almost every parameter extracted from the t – I curves ( I start , I end , I max , t start , t SPl , t EPl ) ( p > 0.05), except for t end ( p ≪ 0.01), which ranged between 42 and 50 s, for xylitol, sucrose, and sorbitol, without significant differences among these sweeteners, to 66 s, for MNEI. These results indicate that the sweetness imparted by MNEI ended almost 20 s later compared to the other sweeteners in stirred yogurts, hinting to a different mechanism of interaction between the protein and sweet taste receptors, already underlined in previous studies (Di Monaco et al, ; Leone et al, ; Miele, Cabisidan, Galiñanes Plaza, et al, ).…”

Section: Resultssupporting

confidence: 61%

Temporal sweetness profile of the emerging sweetener MNEI in stirred yogurt

Miele

Leone

Cabisidan³

et al. 2019

Journal of Sensory Studies

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Yogurt is widely consumed by people under‐controlled dietary regimes. The addition of hypocaloric sweeteners to the yogurt is typically used to make them more palatable, but this may affect the fermentation phase and/or its physical and sensory characteristics. The aim of this work was to test the possible application of an emerging sweetener, MNEI, a variant of a sweet protein, monellin. In particular, its sweetness profile was compared with that of other natural sweeteners, sucrose, xylitol, and sorbitol, in yogurt. First, determinations of iso‐sweet concentration of MNEI and of those sweeteners were performed, both for set and stirred yogurts. Then, the sweetness temporal profiles of all the sweeteners were evaluated as the mean of time–intensity data (t–I). In set yogurt, the iso‐sweet concentrations of xylitol and sorbitol, but not of MNEI, were found, whereas, in stirred yogurts, the concentrations were determined for all the sweeteners. t–I results showed that sweeteners had similar behaviors, except for the end time of sweetness perception: in particular, in stirred yogurt, MNEI resulted in a more lingering sweet aftertaste. These results suggest new applications of the sweet protein MNEI in food preparations, although the combination with other natural sweeteners could be explored. Practical applications Yogurts are ideal products to develop potential applications of MNEI, a protein that has been demonstrated stable to acidic environments, and to our knowledge, this is the first report of the use of a sweet protein to sweeten viscous food preparations. Preliminary experimental results suggest new possible applications of sweet protein MNEI in food samples. In particular, it could be used to sweeten‐stirred yogurt.

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Section: Resultssupporting

confidence: 61%

Temporal sweetness profile of the emerging sweetener MNEI in stirred yogurt

Miele

Leone

Cabisidan³

et al. 2019

Journal of Sensory Studies

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“…High thermal stability makes both protein storage and handling easier and allows some high‐temperature reactions. A wide number of experimental and theoretical approaches have been developed aimed at engineering proteins endowed with a remarkable thermal stability . However, a full comprehension of the basic principles that govern protein thermal stability represents one of the main goals of structural biology.…”

Section: Discussionmentioning

confidence: 99%

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold

et al. 2019

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The definition of the structural basis of protein thermostability represents a major topic in structural biology and protein chemistry. We have recently observed that proteins isolated from thermophilic organisms show a better adherence to the fundamental rules of protein topology previously unveiled by Baker and coworkers (Koga et al. Nature. 2012; 491: 222–227). Here, we explored the possibility that ad hoc modifications of a natural protein following these rules could represent an efficient tool to stabilize its structure. Hence, we here designed and characterized novel variants of Escherichia coli thioredoxin (EcTrx) using a repertoire of biophysical/structural techniques. Trx chimeric variants were prepared by replacing the loop of EcTrx with the corresponding ones present in the Trxs isolated from Sulfolobus solfataricus and Sulfolobus tokodaii that show a better adherence to the topological rules. Interestingly, although the loop sequences of these proteins did not display any significant similarity, their insertion in EcTrx induced a remarkable stabilization of the protein (≥10 °C). The crystallographic structure of one of these variants corroborates the hypothesis that the optimization of the loop size is the driving force of the observed stabilization. The remarkable stabilization of the two novel chimeric Trxs, generated by applying the topological rules, represents the proof of concept that these rules may be used to stabilize natural proteins through the ad hoc optimization of the loop size. Based on the present results, we propose a novel protocol of protein stabilization that can be potentially applied to other proteins.

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“…Most mutational studies have aimed to shed light on the interaction of monellin and the sweet taste receptor though some also intend to increase the stability of the protein. Mutation E23Q increased the sweet taste of monellin 50 . A variant of the E23Q mutant with increased stability was constructed by Leone et al .…”

Section: Discussionmentioning

confidence: 98%

“…Sweet assessment studies have shown that the substitution of arginine 39 with glutamic acid or aspartic acid produced mutants with detection thresholds at 13 000 nM and 89 000 nM, respectively 49 . According to the “wedge model” R39 together with D7, R88 and R65 are crucial to retain sweetness 50 . Our findings correlates well with the wedge model in which arginine 39 interacts with the sweet taste receptor while serine 76 is situated far away from the interaction site 32 .…”

Section: Discussionmentioning

confidence: 99%

Protein stabilization with retained function of monellin using a split GFP system

Weiffert

Linse

2018

Sci Rep

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Sweet proteins are an unexploited resource in the search for non-carbohydrate sweeteners mainly due to their low stability towards heating. Variants of the sweet protein monellin, with increased stability, were derived by an in vivo screening method based on the thermodynamic linkage between fragment complementation and protein stability. This approach depends on the correlation between mutational effects on the affinity between protein fragments and the stability of the intact protein. By linking the two fragments of monellin to the split GFP (green fluorescent protein) system, reconstitution of GFP was promoted and moderately fluorescent colonies were obtained. Two separate random libraries were produced for the monellin chains and the mutant clones were ranked based on fluorescence intensity. Mutants with increased affinity between the fragments, and subsequently increased stability, caused increased fluorescence intensity of split GFP. Single chain monellin variants of the top-ranked mutants for each chain, S76Y in the A-chain and W3C + R39G in the B-chain and all combinations thereof, were expressed and the increase in stability was verified by temperature denaturation studies using circular dichroism spectroscopy. Functionality studies showed that mutant S76Y has retained sweetness and has potential use within the food industry.

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Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design

Cited by 39 publications

References 63 publications

Temporal sweetness profile of the emerging sweetener MNEI in stirred yogurt

Temporal sweetness profile of the emerging sweetener MNEI in stirred yogurt

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold

Protein stabilization with retained function of monellin using a split GFP system

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