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Studies on the taste stimulating ability of food proteins

食品蛋白质味觉刺激能力的研究

基本信息

批准号：
15380093
负责人：
KITABATAKE Naofumi
金额：
$ 4.42万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15380093/
关键词：
sweet-tasting protein thaumatin egg white lysozyme sweet receptor taste stimulation taste umami bitter taste 甘味 sweet protein thaumatin lysozyme site-directed mutagenesis Pichia pastoris リゾチーム塩基性タンパク質アルギニン残基等電点

项目摘要

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 … More 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

大多数蛋白质是无味和无味的，而一些蛋白质在人类味觉上引起甜味反应。已经鉴定出六种甜味蛋白，即奇异果甜蛋白、莫内林蛋白、马宾林蛋白、植物甜蛋白、卵溶菌酶和新oculin（以前被认为是仙茅蛋白）。然而，没有观察到它们之间的共同特征。本文综述了近年来甜味蛋白的研究进展以及利用生物技术生产甜味蛋白的方法。这些蛋白质的结构-甜度关系的信息不仅有助于澄清甜味蛋白质与其受体的相互作用机制，而且有助于设计更有效的低热量甜味剂。Thaumatin是一种甜味蛋白，由一些变体的混合物组成。主要的变体是thaacetin I和II。尽管已知索马甜蛋白I的氨基酸序列并且阐明了索马甜蛋白II的cDNA的核苷酸序列， ...更多信息索马甜I的核苷酸序列一直是有争议的。作者从丹尼奇异果果实中克隆了一个新的奇异果蛋白cDNA。从新cDNA推导的氨基酸序列与索马甜I的氨基酸序列相同，只是在113位的残基（Asp而不是Asn）不同，这表明新的索马甜cDNA是索马甜I的cDNA。将该cDNA转化毕赤酵母X-33，表达的重组索马甜蛋白I进行了纯化和鉴定。重组thaumatin I的甜度阈值与植物thaumatin I的甜度阈值相同，但在重组thaumatin I的N-末端连接了几个意想不到的氨基酸残基。Thaumatin是一种分子量为22-kDa的甜味蛋白，含有8个二硫键。当使用与来自酿酒酵母的α-因子信号序列和Kex 2蛋白酶切割位点融合的奇异果甜蛋白cDNA在巴斯德毕赤酵母中表达奇异果甜蛋白时，分泌的奇异果甜蛋白分子的N-末端序列未被正确加工。为了研究索马甜蛋白cDNA编码的N端前序列和C端前序列在索马甜蛋白加工中的作用以及在毕赤酵母中的生产效率，构建了4个具有不同前序列和前序列的表达质粒，并转化到毕赤酵母中。含有具有天然植物信号的前奇异果甜蛋白基因的转化子在培养基中分泌奇异果甜蛋白分子。正确处理分泌的索马甜蛋白分子的N-末端氨基酸序列。索马甜蛋白的产量不受其C-末端前序列的影响，并且该前序列在毕赤酵母中不被加工，表明前序列不是索马甜蛋白合成所必需的。索马甜蛋白和鸡蛋溶菌酶都是甜味蛋白，甜味阈值分别为50 nM和7μM。尽管已经报道了一些甜味蛋白质激活T1 R2 + T1 R3甜味受体以及低分子量甜味剂，但是对于T1 R2 + T1 R3受体的激活来说，极高于体内阈值的蛋白质浓度是必需的。为了阐明甜味蛋白对培养细胞中表达的T1 R2 + T1 R3受体的这种低敏感性，作者通过体外基于细胞的测定和体内感觉分析来检查索马甜和溶菌酶的甜度。作者构建了稳定表达人T1 R2 + T1 R3受体的Hek 293细胞，然后通过监测细胞内cAMP水平来分析它们对索马甜和溶菌酶的反应。这些甜味蛋白质以及甜菊糖诱导T1 R2 + T1 R3表达细胞的细胞内cAMP积累减少，表明这两种甜味蛋白质激活T1 R2 + T1 R3受体。作者观察到甜味蛋白的甜度对NaCl极其敏感。此外，从体外和体内实验的结果，作者证实，甜味抑制剂lactisole显着抑制甜味蛋白的甜味。少