TGase催化玉米蛋白的糖基化及对蛋白加工特性和生理功能特性的影响

Corn proteins have unbalanced amino acid composition and poor solubility in water. These properties of corn proteins limit their application in food industry. However, corn proteins have special amino acid composition, and have special functional amino acid sequences among polypeptide chain of corn proteins. So, corn proteins have potential advantages as raw-material in functional food production. In this project, based on properties of corn proteins special amino acid composition and higher glutamine content, corn proteins will be glycosylated by transglutaminase, the structure of corn proteins will be changed, new glyco-proteins will be produced and corn proteins functionality will be released. The system of reactants was established and the effect of glyco-proteins structure to the processing properties and bioactivity will be analyzed. An affinity chromatography method, based on special affinity between lectins and glyco-proteins, will be applied to separate the new glycosylated corn proteins, in order to discover and clarify the effects of glycosylation on structures of corn proteins. The structural characteristics of glycosylated corn proteins will be analyzed and compared to those of corn proteins without glycosylation with advanced spectroscopy, and other means, Processing characteristics of glycosylated corn proteins, such as solubility, viscosity, emulsifying property, foamability, and functionality such as antioxidative activity and facilitating alcohol metabolic and hepatoprotective activity will be analyzed by the methods of physical properties of food and biological sciences. Finally, the relationship between glycosylated corn protein structure and processing characteristics and functionality will be further analyzed which will provide theoretical basis for novel applications of corn protein.

针对玉米蛋白氨基酸组成不平衡、疏水性强，加工应用受限的问题，根据玉米蛋白多肽链中存在具有抗氧化、促酒精代谢等多肽功能序列，且含有高比例谷氨酰胺残基的特点，用谷氨酰胺转胺酶分别催化玉米醇溶蛋白和玉米谷蛋白与多种氨基糖发生糖基化反应，建立反应体系，用亲和色谱法分离糖基化玉米蛋白，用光谱学等手段，对糖蛋白进行结构特征分析，并与玉米蛋白进行比较，探索酶法糖基化反应的糖基化度与玉米蛋白结构变化的关系；用食品物性学相关手段分析糖基化玉米蛋白的溶解性、黏度、乳化性和起泡性等加工特性；用生物学相关手段分析糖基化玉米蛋白的抗氧化活性、促酒精代谢和保护肝脏活性等生理功能特性，探索玉米蛋白结构对其加工特性和生理功能特性的影响，这对阐明酶法糖基化反应使蛋白加工特性和生理功能特性得到较大改善的机理有重要意义，为发现和开拓玉米蛋白的新用途提供理论依据。

玉米蛋白氨基酸组成不平衡、疏水性强等特性限制了其在食品工业中的应用。本项目利用谷氨酰胺转胺酶分别催化玉米醇溶蛋白和谷蛋白与三种氨基糖发生糖基化反应，建立了反应体系，完成了糖基化修饰玉米醇溶蛋白和谷蛋白的分离纯化、结构特征、溶解性等加工特性及抗氧化活性和促酒精代谢活性的研究。获得了如下重要结果：.1、建立了氨基葡萄糖、氨基半乳糖和壳寡糖糖基化修饰玉米醇溶蛋白和谷蛋白的反应体系。.2、采用SDS-PAGE和HPLC法确认在转谷氨酰胺酶的催化下，氨基糖与玉米醇溶蛋白及谷蛋白共价结合形成了糖蛋白。.3、与玉米蛋白和交联玉米蛋白相比，多羟基糖基的导入使糖基化玉米蛋白的表面疏水性显著降低，表明其水溶性显著改善，进而改善了玉米蛋白的起泡性、体外消化性和表观黏度等加工特性，而乳化性、持水性和吸油性等性质的改善程度因蛋白及氨基糖种类的不同而不同。.4、采用HitrapTM Con A-Sepharose4B亲和层析色谱柱和SephadexG-25凝胶层析色谱柱顺次对糖基化玉米蛋白进行分离纯化，获得纯度较高的糖蛋白。在此基础上，对糖蛋白的结构性质进行分析。与玉米蛋白相比，糖基化修饰玉米蛋白分子中的赖氨酸和精氨酸含量降低，说明酶法糖基化反应包括一定程度的交联反应和非酶法糖基化反应；通过红外光谱分析，糖基化蛋白的吡喃环羟基特征吸收峰强度增加，表明糖基引入蛋白分子中；通过表面巯基和游离巯基、圆二色光谱、表面形貌及热学性质研究，确定糖基化修饰玉米蛋白游离巯基含量降低而二硫键含量增加，无规卷曲的含量减少而α-螺旋含量增加，变性温度增加，表面形态更加致密，说明糖基化修饰使玉米蛋白的分子排布更加有序，热稳定性增加。.5、与玉米蛋白及交联玉米蛋白相比，多羟基糖基的导入使玉米蛋白成为良好的氢供体及电子供体，使其体外抗氧化活性显著增强，包括DPPH自由基清除活性、羟基自由基清除活性、超氧阴离子自由基清除活性、还原力和亚铁离子螯合能力，且抗氧化活性呈剂量依赖关系。.6、当白酒灌胃剂量为3.5 mL/kg时，在灌胃10 min后，小鼠血液中乙醇浓度达到最大值，为56.74±6.09 mg/100m。在此基础上，确定氨基半乳糖糖基化修饰玉米醇溶蛋白和壳寡糖糖基化修饰玉米醇溶蛋白的促酒精代谢有效剂量分别为0.6mg/g小鼠体重和2.5mg/g小鼠体重。.本项目研究成果为发现和开拓玉米蛋白的新用途提供理论依据。