| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2003: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
Most proteins are tasteless and flavorless, while some proteins elicit a sweet-taste response on the human palate. Six proteins, thaumatin, monellin, mabinlin, brazzein, egg lysozyme, and neoculin (previously considered as curculin) have been identified as sweet-tasting proteins. However, no common features among them have been observed. Herein, recent advances in the research of sweet-tasting proteins and the production of such proteins by biotechnological approaches are reviewed. Information on the structure-sweetness relationship for these proteins would help not only in the clarification of the mechanism of interaction of sweet-tasting proteins with their receptors, but also in the design of more effective low-calorie sweeteners.
Thaumatin is a sweet-tasting protein, and comprised of a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin II was elucidated, the
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nucleotide sequence of thaumatin I has been controversial. The author has cloned a novel thaumatin cDNA from the fruit of Thaumatococcus daniellii Benth. The amino acid sequence deduced from the novel cDNA was the same amino acid sequence as that of thaumatin I only except the residue at position 113 (Asp instead of Asn), indicating that the novel thaumatin cDNA is that for thaumatin I. This thaumatin I cDNA was transformed into Pichia pastoris X-33, and the recombinant thaumatin I expressed was purified and characterized. The threshold value of sweetness of the recombinant thaumatin I was the same as that of the plant thaumatin I, while several unexpected amino acid residues were attached to the N-terminal of the recombinant thaumatin I. These indicate that the N-terminal portion of thaumatin is not critical for the elicitation of sweetness.
Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the α-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P.pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P.pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P.pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.
Both thaumatin and hen egg lysozyme are sweet-tasting proteins with sweetness threshold values around 50nM and 7μM, respectively. Although it has been reported that some sweet-tasting proteins activate the T1R2+T1R3 sweet-taste receptor as well as low-molecular mass sweetener, a protein concentration that was extremely higher than the in vivo threshold value was necessary for the activation of the T1R2+T1R3 receptor. To elucidate this low sensitivity of sweet-tasting proteins for the T1R2+T1R3 receptor expressed in culture cells, the author examined the sweetness of thaumatin and lysozyme by both an in vitro cell-based assay and an in vivo sensory analysis. The author constructed Hek293 cells that stably expressed the human T1R2+T1R3 receptor and then analyzed their responses to thaumatin and lysozyme by monitoring the levels of intracellular cAMP. These sweet-tasting proteins as well as aspartame induced a decrease in the intracellular cAMP accumulation of the T1R2+T1R3-expressing cells, indicating that both sweet proteins activated the T1R2+T1R3 receptor. The author observed that the sweetness of sweet-tasting proteins is extremely sensitive to NaCl. Furthermore, from the results of both in vitro and in vivo experiments the author confirmed that the sweetness inhibitor lactisole significantly suppressed the sweetness of sweet-tasting proteins. Less
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