利用实时荧光监控法研究超高静压激活γ-多聚谷氨酸水解酶的机理

Natural folate is a water soluble B-group vitamin. Accumulated investigation supports that "low" folate status enhances risk of cardiovascular disease and the well-known risk of neural tube defects in newborns. Vegetables are abundant in natural folate. Thus increasing consumption of vegetables is the best way to optimize folate status without health concerns. However, this approach is limited by inferior bioavailability of natural folate compared to folate supplements, and the polyglutamyl conjugate is regarded as the limiting factor. γ-glutamyl hydrolase (GGH) is a ubiquitous enzyme existing in vegetables. Previous study has shown that folate polyglutamates are hydrolyzed to be monoglutamate under high hydrostatic pressure (HHP) by enabling action of native GGH. 98% of polyglutamate were converted to monoglutamate under this technique. However, this could not be achieved under atmospheric pressure. The bioavailability of processed broccoli is 100% compared to unprocessed form, however, the mechanism of deglutamylation by GGH under HHP has not been investigated..In this project, enzyme properties will be characterized. Real-time fluorescence method will be incorporated for monitoring the conformation change of enzyme under pressure in situ. We have found no report about such method. Different pressure processing condition (pressure, duration, temperature and pH) will be investigated. Through this project, we will disclose structural and functional details of the pressure-induced dissociation of the trimer, tertiary and secondary structure. On the basis of fully understanding such mechanism, we hope to identify the optimized conformation that can be immobilized in future study to replace the HHP to hydrolyze the native folate at atmospheric pressure. To our best knowledge, we have not found report about this topic.Such research may lead to production of vegetables with extended shelf life and enhanced levels of highly bioavailable natural folate. This strategy should be a valuable and cost-effective intervention in fighting folate deficiency without health concern.

天然叶酸（TF）在预防婴儿出生缺陷和心血管疾病方面发挥巨大作用，是人体必需维生素。虽然蔬菜中TF含量丰富，但常因其结合多聚谷氨酸而吸收率极低。申请人前期研究发现，超高静压处理能激活常压下呈惰性的γ-多聚谷氨酸水解酶 (GGH)，使多聚谷氨酸彻底水解而释放出TF，并使其生物有效性提高100%，但GGH的作用机制不清楚。本项目以花椰菜中GGH为研究对象，采用实时荧光光谱监控技术（RT-FL）研究超高静压时（而不是高压处理后）GGH的结构变化、酶学特性、激酶因子的释放与酶催化活性之间的一一对应关系，阐明超高静压激活该酶的作用机理。RT-FL技术克服了传统方法中卸压后酶结构可逆反应所得到的错误结果，真实反映了高压下酶结构的变化。本项目不仅能为开展超高静压环境下酶或蛋白的研究搭建新的技术平台，而且为制备高活性GGH提供指导。达到提高TF人体吸收和蔬菜资源增值利用的目的。

基于常压及超高静压下谷氨酸水解酶催化反应机制不清晰的现状，本研究通过基因克隆表达得到三种花椰菜γ-谷氨酸盐水解酶（LeGGH1、LeGGH2和LeGGH3），SDS-PAGE初步鉴定表达酶，对三种LeGGHs融合蛋白进行测序分析；运用各项技术探讨酶学特性，阐明酶的构象特征；揭示三种LeGGHs在食品基质中催化能力的差异性。研究结果如下：.①三种蛋白的相对分子量均约为38 kDa，其中LeGGH1和LeGGH2具有活性，而LeGGH3无活性。.②根据氨基酸序列对比分析， LeGGH1、LeGGH2和LeGGH3分别由340、344、337个氨基酸残基组成，且都含有高度保守的半胱氨酸残基；LeGGH1与LeGGH2、LeGGH3 的同源度分别为73%和67%，LeGGH2与LeGGH3的同源度为79%；同时发现它们的序列存在一定差异，差异较大的几处发生在2 ~50及324 ~344氨基酸区段。.③根据二级结构，发现三种LeGGHs的有序结构含量大致相当，达61.5~63.3%，但LeGGH1与LeGGH2中α-螺旋含量明显高于LeGGH3，β-折叠含量则相反。LeGGH1和LeGGH3中色氨酸和酪氨酸残基荧光基团表面含量较高而容易受环境影响，其中LeGGH1在低pH和高盐酸胍的条件下荧光强度明显减弱和荧光光谱红移现象最明显；而LeGGH2中色氨酸和酪氨酸残基荧光基团倾向于分子内部，不易受外部环境的影响，这些结构差异变化与酶的催化活性密切相关。.④运用UPLC-MS/MS技术探讨LeGGH1、LeGGH2和LeGGH3在催化蔬菜中水解多聚谷氨酸叶酸的差异性。研究发现LeGGH1在复杂基质中仍然具有催化活力，且催化能力与催化单一底物相当；而 LeGGH2无催化能力。.⑤研究了超高静压处理及超高静压后储藏对蔬菜中的LeGGHs的影响。研究发现超高静压下GGH可以将多聚谷氨酸叶酸水解为短链形式。经过450MPa和600MPa处理后的蔬菜中的多聚谷氨酸叶酸冷藏储存2天后全部转化为单谷氨酸叶酸。这个研究说明了利用超高静压可以将天然叶酸转化为生物利用性更高的形式。.⑥研究了超高静压对LeGGH1催化反应的影响及机制。首先确立高静压下LeGGH1催化体系。100-300MPa可以显著提高酶的活力。随着压力的提高，酶的活力逐渐降低。高压导致表观Km降低，Vmax增大。活化体积增大。