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.

通过微生物转氨酶（mTG）酶促交联酪蛋白被广泛用于食品工业中以改变乳蛋白的功能性质。在目前的科学文献中，相应的报告主要集中在应用和技术现象学方面。到目前为止，几乎没有研究由mTG处理产生的结构后果以及酶促反应的分子背景。在牛奶中，酪蛋白聚集成胶束。根据申请人目前的研究，该项目基于以下假设：酪蛋白酶促交联的选择性特别取决于酪蛋白胶束的结构。作为胶束内蛋白质-蛋白质相互作用的结果，应该存在用于通过mTG选择性修饰的明确定义的热点（即赖氨酸和精氨酸残基）。这反过来又是形成具有确定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）胶束内单个酪蛋白的酶促交联的反应位点将阐明，在蛋白质序列交联的哪些位置由mTG诱导，以及胶束的内部结构如何影响酶促反应。为此，将单个酪蛋白（α S1-、α S2、β-和κ-酪蛋白）、非胶束酪蛋白酸钠以及从生乳中分离的酪蛋白胶束与mTG一起孵育，并在酶促水解后使用LC/MS（肽图谱）鉴定mTG攻击的热点。两种策略，即标记的反应性谷氨酰胺或赖氨酸残基与丹磺酰尸胺或三甘氨酸，或直接分析的isopeptide containing肽，分别将遵循。(ii)mTG交联的酪蛋白胶束的功能。将研究mTG修饰的酪蛋白胶束的大小和稳定性以及mTG修饰的酪蛋白胶束与所选化合物的相互作用。它将被澄清，如何胶束内交联影响酪蛋白胶束在乙醇，EDTA和肽酶的存在下的稳定性。关于与客体分子（溶菌酶，磷酸丝氨酸和β-胡萝卜素）的相互作用的信息将提供有关胶束的内部结构的信息，并将显示有关使用酶交联酪蛋白胶束作为工具，用于封装和递送生物活性食品化合物的前景。该项目将有助于对酪蛋白胶束内mTG诱导的交联过程的基本理解，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，酶交联酪蛋白胶束的制备和使用的前景作为纳米胶囊的营养相关的化合物将显示。