| Abstract |
The food industry is in constant search of alternative sweeteners to cover the large-scale
demands for sugar. However, this extensive use of sugars in daily diet is correlated to health
issues, such as obesity, type II diabetes, etc. In an effort to replace carbohydrate sweeteners,
the manufactories oriented toward the use of artificial or natural nonnutritive sweeteners
like aspartame and steviol glucosides. Nowadays, these sweeteners have already proved to
have adverse side effects on health, such as dizziness, headaches, psychological problems, so
the interest shifted to natural non-carbohydrate sweeteners (terpenoids, flavonoids, and
proteins).
Most proteins are tasteless and flavorless; however, some proteins elicit a sweet taste or
have taste modifying properties response on the human palate. Six proteins, thaumatin,
brazzein, mabinlin, monellin, pentadin, and egg white lysozyme, were identified to elicit a
sweet taste. Furthermore, there are two more proteins Miraculin, and Neoculin, which are
taste-modifying proteins, converting sourness into sweetness. In general, their sweetening
effect is hundreds or thousands of times higher compared to sucrose. However, almost 50
years later, only thaumatin has an industrial application of these proteins, reasoning to the
difficult availability and the expensive production.
In the framework of this project, we focused on the comprehension of the chemical and
structural determinants that provide the sweetening effect on sweet taste proteins. We
perform a meta-analysis to identify amino acids and motifs critical for the elucidation of the
sweet taste. Our interest concentrated on the two sweet proteins, MNE and Mabinlin-II, and
along with the results from the structure-function analysis, we searched for putative
sweetener proteins from the sequence space. Subsequently, we found five putative
sequences namely according to their organism: BrasCret1, BrasCret2, BrasCret3, ArabisAlp,
and Cryza.M.
The first goal of the framework was the elucidation of the interaction of the targeted proteins
with the sweet taste receptor (T1R2-T1R3)through docking simulations. For the receptor was
used a model from the literature, as the protein was not crystallized, for the known sweet
proteins their crystal structures, and for the putative proteins’ hybrid models were
constructed. According to docking simulations, the proteins interacted with the receptor
adopting a wedge motif in their structure. Our results establish five interactions, namely
Arg177 of T1R3 and Asn152, Glu170, Asp173, and Asp 218 of T1R2, that could have a critical
role in the interaction, both with the characterized sweet proteins and also with the putative
ones.
In a step further, we express these seven proteins in the E.coli expression host. Contrasting
the results from the literature, we did not succeed express these proteins in high yields
(2,1mg/ L cultivation for Mabinlin-II and 0,72mg/ L for MNEI).
In the third part of this project, we implement a sweet taste assay using Drosophila
melanogaster. Drosophila can detect basic tastes comprising of sweet, bitter, and salty.
According to this assay, the third instar larvae indicate a preference for the sweet proteins
Thaumatin, MNEI, and Mabinlin-II under specific conditions.
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