Reaction sites and functionality of enzymatically cross-linked casein micelles

酶交联酪蛋白胶束的反应位点和功能

• 批准号: 271806234
• 负责人: Professor Dr. Thomas Henle
• 金额: --
• 依托单位: Professur für Lebensmittelchemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/271806234?language=en
• 关键词: Reaction; sites; functionality; enzymatically; cross

Enzymatic cross-linking of casein by microbial transglutaminase (mTG) is widely used in the food industry in order to modify the functional properties of the milk proteins. In the current scientific literature, corresponding reports mainly focus on applications and on technological-phenomenological aspects. The structural consequences resulting from mTG-treatment as well as the molecular background of the enzymatic reaction are hardly studied until now. In milk, caseins are aggregated as micelles. Based on current studies of the applicant, this project is based on the hypothesis that the selectivity of enzymatic cross-linking of casein particularly depends on the structure of the casein micelle. As a consequence of intramicellar protein-protein-interactions, well-defined hot spots (i.e. lysine and arginine residues) for selective modification by mTG should exist. This, in turn, is the basis for the formation of casein networks with defined functional properties. The project will follow two general aims: (i) Reaction sites of enzymatic crosslinking of individual caseins within micelles It will be clarified, at which positions within the protein sequence crosslinking is induced by mTG and, thus, how the internal structure of the micelle affects the enzymatic reaction. For this, individual caseins (alpha s1-, alphaS2, beta- und kappa-casein), non-micellar sodium caseinate as well as casein micelles isolated from raw milk will be incubated with mTG and the hot spots attacked by mTG will be identified after enzymatic hydrolysis using LC/MS (peptide mapping). Two strategies, namely labelling of reactive glutamine or lysine residues with dansylcadaverine or triglycine, or direct analysis of isopeptide-containg peptides, respectively, will be followed. (ii) Functionality of mTG-cross-linked casein micelles. Size and stability of mTG-modified casein micelles as well as interactions of mTG-modified casein micelles with selected compounds will be studied. It will be clarified, how intramicellar cross-linking affects the stability of casein micelles in the presence of ethanol, EDTA and peptidases. Information concerning interactions with guest molecules (lysozyme, phoshoserine and beta-carotene) will provide information about the internal structure of the micelles and will show perspectives concerning the use of enzymatically cross-linked casein micelles as tools for encapsulation and delivery of bioactive food compounds. The project will contribute to the fundamental understanding of the course of mTG-induced cross-linking within the casein micelle and will deliver general information concerning the structure of the casein micelle before and after mTG-treament. Furthermore, perspectives for the preparation and use of enzymatically cross-inked casein micelles as nanocapsules for nutritionally relevant compound will be shown.

通过微生物转谷氨酰胺酶 (mTG) 进行酪蛋白的酶交联广泛应用于食品工业，以改变乳蛋白的功能特性。在当前的科学文献中，相应的报道主要集中在应用和技术现象学方面。迄今为止，mTG 处理所产生的结构后果以及酶促反应的分子背景还很少被研究。在牛奶中，酪蛋白聚集成胶束。根据申请人目前的研究，该项目基于这样的假设：酪蛋白的酶促交联的选择性特别取决于酪蛋白胶束的结构。由于胶束内蛋白质-蛋白质相互作用，应该存在 mTG 选择性修饰的明确热点（即赖氨酸和精氨酸残基）。这反过来又是形成具有明确功能特性的酪蛋白网络的基础。该项目将遵循两个总体目标： (i) 胶束内单个酪蛋白酶促交联的反应位点 将阐明 mTG 诱导蛋白质序列交联的哪些位置，以及胶束的内部结构如何影响酶促反应。为此，将单独的酪蛋白（α s1-、αS2、β- 和 kappa-酪蛋白）、非胶束酪蛋白酸钠以及从原料奶中分离出的酪蛋白胶束与 mTG 一起孵育，然后识别出受 mTG 攻击的热点。使用 LC/MS（肽图谱）进行酶水解。将遵循两种策略，即分别用丹酰尸胺或三甘氨酸标记反应性谷氨酰胺或赖氨酸残基，或直接分析含有异肽的肽。 (ii) mTG 交联酪蛋白胶束的功能。将研究 mTG 修饰酪蛋白胶束的尺寸和稳定性以及 mTG 修饰酪蛋白胶束与选定化合物的相互作用。将阐明在乙醇、EDTA 和肽酶存在下胶束内交联如何影响酪蛋白胶束的稳定性。有关与客体分子（溶菌酶、磷酸丝氨酸和β-胡萝卜素）相互作用的信息将提供有关胶束内部结构的信息，并将展示有关使用酶交联酪蛋白胶束作为封装和递送生物活性食品化合物的工具的观点。该项目将有助于从根本上理解酪蛋白胶束内 mTG 诱导的交联过程，并将提供有关 mTG 处理前后酪蛋白胶束结构的一般信息。此外，还将展示酶交联酪蛋白胶束作为营养相关化合物的纳米胶囊的制备和使用的前景。