Identification of sweet active site of sweet protein, thaumatin

甜蛋白索马甜甜味活性位点的鉴定

• 批准号: 06660157
• 负责人: KITABATAKE Naofumi
• 金额: $ 1.28万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660157/
• 关键词: thaumatin; sweet; taste; sweet protein; food protein

Thaumatin was purified from the crude sample with ion-exchange chromatography and G-75 Sephadex chromatography. The obtained thaumatin showed a single band on PAGE and SDS-PAGE.The arginine residue of thaumatin was chemically modified with 1,2 cyclohexandione, the lysine residue with pyridoxal phosphate, and the tyrosine residue with sodium iode. From these experiments it has been clarified that one or two residues of each residue are realted to express sweet activity of thaumatin. In order to identify these residues the modified thaumatin was digested with pepsin and fractionated by HPLC.The peptide fragment containing the modified amino acid residue was obtained and applied to protein sequencer. To identify the active site of thaumatin, the sweet reception mechanism should be known. Then, isolation of receptor taste cells and thaumatin-receptor protein has been tried. Effects of pI on the sweet taste expression have been also examined

采用离子交换层析和G-75 Sephadex层析对粗样品进行了纯化。在PAGE和SDS-PAGE上显示单峰。用1,2环己二酮对索玛汀精氨酸残基进行化学修饰，用磷酸吡哆醛对赖氨酸残基进行化学修饰，用碘化钠对酪氨酸残基进行化学修饰。从这些实验中，已经明确了每个残基的一个或两个残基与表达thumatin的甜味活性有关。用胃蛋白酶酶切和高效液相色谱法对其进行分离鉴定。获得了含有修饰氨基酸残基的肽片段，并应用于蛋白质测序仪。为了确定thumatin的活性位点，必须了解甜味接收机制。然后，尝试分离受体味觉细胞和thaumatin受体蛋白。还研究了pI对甜味表达的影响

项目成果

Kinekawa, Y. and Kitabatake, N: "Turbidity and Rheological Properties of Gels and Sols by Heating Process Whey Protein" Bioscience Biotechnology and Biochemistry. 59. 834-840 (1995)

Kinekawa, Y. 和 Kitabatake, N：“乳清蛋白加热过程中凝胶和溶胶的浊度和流变特性”生物科学生物技术和生物化学。

Kinekawa, Y.and Kitabatake N.: "Turbidity and Rheological Properties of Gels and Sols by Heating Process Whey Protein" Bioscience, Biotechnology and Biochemistry. 59. 834 (1995)

Kinekawa, Y. 和 Kitabatake N.：“乳清蛋白加热过程中凝胶和溶胶的浊度和流变特性”生物科学、生物技术和生物化学。

Kitabatake, N.and Kinekawa, Y.: "Turbidity Measurement of Heated Egg Proteins Using a Microplate System" Food Chemistry. 54. 201 (1995)

Kitabatake, N. 和 Kinekawa, Y.：“使用微孔板系统测量加热鸡蛋蛋白质的浊度”食品化学。

Kitabatake, N. and Kinekawa, Y: "Turbidity Measurement of Heated Egg Proteins Using a Microplate System" Food Chemistry. 54. 201-203 (1995)

Kitabatake, N. 和 Kinekawa, Y：“使用微孔板系统测量加热鸡蛋蛋白质的浊度”食品化学。

Kitabatake, N. and Kinekaw, Y: "Turbidity Measurement of Heated Egq Proteins Using a Microplate System" Food Chemistry. 54. 201-203 (1995)

Kitabatake, N. 和 Kinekaw, Y：“使用微孔板系统测量加热的 Egq 蛋白的浊度”食品化学。