Synthesis and characterization of the sweet protein brazzein

Izawa, Hiroyuki; Ota, Masafumi; Kohmura, Masanori; Ariyoshi, Yasuo

doi:10.1002/(sici)1097-0282(199607)39:1<95::aid-bip10>3.0.co;2-b

Cited by 32 publications

(21 citation statements)

References 27 publications

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“…38−42 Our ratios of the recognition threshold values for the three brazzein forms and the control substance sucrose were consistent with those of Assadi-Porter et al 2 These authors observed, in line with our results, that the threshold for des-pyrE-bra brazzein produced using E. coli was 2 times lower than that of the natural brazzein, which is ∼80% pyrE-bra and ∼20% des-pyrE-bra. 5 Moreover, the sweetness potencies determined here correspond well with those previously indicated by Izawa et al, 14 ranging from 500 to 2000, and with those determined by Assadi-Porter et al, 2 being approximately 500 for natural brazzein and 1000 for des-pyrE-bra brazzein produced by E. coli relative to a sucrose solution of 20 g/L.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Brazzein combines a long history of human consumption, high sweet potency (from 500-to 2000-fold sweeter than a sucrose solution on a weight basis and from 9500-to 38 000-fold on a per-molecule basis), high water-solubility, and exceptional thermostability. 2,5,14,15 In addition, brazzein tastes purely sweet with no sourness, saltiness, or bitterness, making it a good alternative to artificial sweeteners. 5 Because of the difficulties in obtaining brazzein from its natural source, brazzein has been expressed using various recombinant expression systems.…”

Section: ■ Introductionmentioning

confidence: 99%

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Efficient Production and Characterization of the Sweet-Tasting Brazzein Secreted by the Yeast Pichia pastoris

Poirier

Roudnitzky

Brockhoff

et al. 2012

J. Agric. Food Chem.

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Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and (1)H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Efficient Production and Characterization of the Sweet-Tasting Brazzein Secreted by the Yeast Pichia pastoris

Poirier

Roudnitzky

Brockhoff

et al. 2012

J. Agric. Food Chem.

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“…Non-carbohydrate sweet-tasting substances include several chemically distinct groups of small molecule artificial sweeteners, such as sucralose, saccharin, cyclamate, and acesulfame K, as well as dipeptide sweeteners, such as aspartame and neotame. Sweet-tasting proteins have been found as naturally occurring molecules from plants (or rarely from animals) including thaumatin 1, monellin 2, mabinlin 3, brazzein 4; 5, eggwhite lysozyme 6, and curculin/neoculin 7. In addition, a pure sweet-antagonist protein, the glycoprotein RBP from chicken egg, has been reported to inhibit specifically the taste of sweet-protein but not carbohydrates or artificial sweeteners 8…”

Section: Introductionmentioning

confidence: 99%

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2–T1R3 Human Sweet Receptor

Assadi‐Porter

Maillet

Radek

et al. 2010

Journal of Molecular Biology

104

106

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The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N-and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9-19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9-19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct brazzein binding. Results from this study support a multipoint interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.

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“…The major form, with a pyroglutamic acid (pGlu) at its Nterminus, consisting of 54 amino acid residues, is 500-2000 times sweeter than sucrose. Nevertheless, the minor form (despGlu1), without the N-terminal pGlu, and thus bearing only 53 amino acid residues, is twice as sweet as the major form (Izawa et al, 1996).…”

Section: Introductionmentioning

confidence: 99%