基于肌原纤维蛋白结构探讨磷酸盐对谷氨酰胺转氨酶催化交联的作用机理

Transglutaminase (TGase) has been applied commercially to improve the quality of meat products. The efficiency of TGase cross-linking is influenced by the structure of substrate protein. Our previous study showed that the cross-linking pattern of TGase varied due to structural changes of myofibrillar protein (MP) induced by oxidation. Phosphate, which is commonly used for improving the quality of meat products, can alter both structure and oxidation pattern of MP. However, the mechanism of these changes on TGase cross-linking is unclear. Cys707, Cys697, Lys83 and Gln are specifically labeled by fluorescent probe and fluorochrome in this study. Changes of these active amino acid residues in natural and oxidized myosin are tracked to study effects of phosphate on the cross-linking pattern of TGase through determination of fluorescence and amino acid sequences. Effects of phosphate-mediated actomyosin dissociation in natural and oxidized MP on the cross-linking pattern of TGase are revealed by RP-HPLC and MALDI-TOF/TOF MS. Gelling ability, emulsifying property and water holding capacity are used to analyze effects of cross-linking pattern on MP functionality. Then, the correlations of protein structure under the action of phosphate, cross-linking pattern of TGase and protein functionality are studied to illuminate structural factors influencing TGase-catalyzed cross-linking and product quality. The findings of this study will provide theoretical foundation for improving the quality of meat products with TGase treatment.

谷氨酰胺转氨酶（TGase）已广泛用于改善肉制品品质，其交联效果受底物蛋白结构影响。申请者前期研究表明，氧化引起的肌原纤维蛋白（MP）结构变化会改变TGase交联模式。而磷酸盐作为肉制品常用添加剂在改变MP结构同时还可改变蛋白氧化模式，这些变化对TGase交联的影响机理尚不清楚。本项目拟利用荧光探针/染料特异标记活性SH1、SH2、RLR和Gln残基，通过荧光检测和氨基酸测序等方法追踪分析磷酸盐引起的天然和氧化肌球蛋白中活性氨基酸变化对交联模式影响。利用反相HPLC和MALDI-TOF/TOF质谱等方法分析磷酸盐引起的天然和氧化MP中肌动球蛋白解离对交联模式影响。通过测定凝胶性、乳化性和持水性，分析交联模式变化对蛋白功能性质影响。综合探讨磷酸盐作用下蛋白结构、TGase交联模式和蛋白功能性质之间相关性，揭示影响TGase交联和产品品质的结构因素，以期对TGase改善肉制品品质提供理论依据。

TSPP能够改变肌球蛋白的二级结构和表面疏水性，修饰活性氨基酸残基，在一定程度上抑制肌球蛋白的氧化，但会促进TGase交联。TGase的交联程度及其对肌球蛋白α-螺旋含量的影响随着氧化程度增加呈现先增高后降低的趋势。RLR残基是TGase交联的关键性底物。参与TGase交联的活性Gln残基含量受氧化程度(特别是重度氧化)的影响。未氧化样品的主要交联位点为S1和HMM，随着氧化程度增加，S2的交联程度逐渐增加，TSPP处理后S2交联程度的增加速度较慢。TSPP、氧化和TGase三者共同作用可显著提高蛋白的凝胶性能，但单独观察由TGase交联引起的凝胶性能提高发现，相对于-TSPP条件，+TSPP条件下蛋白氧化对其具有更强的抑制作用。相关性分析结果显示，在-TSPP和+TSPP条件下，氧化引起的肌球蛋白羰基、自由氨基、α-螺旋变化及SH1、SH2封闭与凝胶性能显著相关(P<0.05)；对于-TSPP样品，TGase交联引起的α-螺旋和表面疏水性结构的改变与凝胶性能显著相关(P<0.05)，而对于+TSPP样品，只有表面疏水性的改变与之显著相关(P<0.05)；对于+TSPP肌动球蛋白，氧化引起的Rod和LMM变化与TGase交联程度显著相关(P<0.05)，氧化引起的LMM变化与TGase交联后凝胶弹性显著相关(P<0.05)，氧化引起的S1和HMM变化与交联后凝胶粘度显著相关(P<0.05)。研究结果为深入揭示影响TGase交联模式和蛋白功能性质的结构因素，为利用TGase改善肉制品品质提供了新思路和理论指导。