新型双功能谷胱甘肽合成酶的构效机制及应用基础研究

Glutathione (GSH) is an important bioactive compounds, and has been applied widely in food and pharmaceutical industries. However, GSH in the domestic market is totally supplied by foreign makers. Glutathione biosynthesis involves two sequential ATP-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS) and GSH synthetase (GS). In most organisms, the activity of γ-GCS is subjected to feedback inhibition seriously by GSH. Thus, use of these enzymes results in low intracellular level, yield, and productivity of GSH. In our previous research, we cloned several genes encoding the novel bifunctional glutathione synthetases from different microorganism, such as Streptococcus thermophilus, Actinobacillus succinogense and Actinobacillus pleuropneumonia. These enzymes possesse both the γ-GCS and GS activities, and are not sensitive to GSH inhibition. The preliminary results indicated that these enzymes have better performances and are advantageous in GSH biosynthesis. In this research, we will focus on the detailed enzymatic characteristics and structure-function relationship. It is expected to explain the enzymatic structure and catalytic mechanism. The effects of protein sequences and improtant active sites on the catalytic acitivity will also be studied to provide information on further enhancement of the enzymatic performance. Additionally, the bifunctional glutathione synthetases will be overexpressed in Escherichia coli and Pichia pastrois. We will optimize the new process of GSH production using these recombinant strains and enhance the yield and productivity of GSH significantly.

谷胱甘肽(Glutathione, GSH)是一种重要的生物活性物质，广泛应用于医药和食品领域，但目前国内GSH原料依赖进口，尚无高效生物合成工艺。GSH通常由γ-谷氨酰基半胱氨酸合成酶和谷胱甘肽合成酶分两步合成，受产物反馈抑制严重，大大影响生产水平和效率。本课题组前期从嗜热链球菌等多种微生物获得同时具有两种酶活性的新型双功能谷胱甘肽合成酶，且对GSH抑制不敏感，初步研究表明这些酶具有良好的酶学特性和应用前景。本课题拟对这些酶继续深入开展工作，通过详细的酶学特性和构效关系研究，阐释其结构和催化机理，辨析蛋白序列和重要氨基酸位点对催化活性影响，进一步构建高效谷胱甘肽合成酶；在大肠杆菌和毕赤酵母中过量表达该类谷胱甘肽合成酶，研究高谷胱甘肽含量对细胞代谢的影响，以葡萄糖代谢途径有效耦合GSH合成所需ATP的供给优化发酵过程，提高谷胱甘肽合成水平和生产强度。

谷胱甘肽（GSH）是生物体中含量最丰富的非蛋白小分子硫醇化合物之一。 谷胱甘肽可作为还原剂维持细胞内的氧化还原平衡，用于外界异物的解毒，清除羟基过氧化物、自由基和亲电试剂，参与DNA的合成与修复、蛋白质的合成等。目前，谷胱甘肽的生产方法主要为发酵法和酶法。谷胱甘肽通常由γ-谷氨酰基半胱氨酸合成酶和谷胱甘肽合成酶分两步合成，受产物反馈抑制严重，大大影响生产水平和效率。本项目针对谷胱甘肽双功能合成酶，通过在蛋白数据库中搜索和比对，在实验室已有的谷胱甘肽双功能合成酶库中增加了6种不同来源的谷胱甘肽双功能合成酶，并对这些酶的酶学性质进行了研究，确定了催化反应的最适条件。针对谷胱甘肽在发酵合成过程中得率较低的问题，通过对谷胱甘肽合成代谢途径关键降解基因以及L-半胱氨酸降解基因的敲除，显著减少谷胱甘肽合成后期的降解，同时结合发酵过程优化策略，显著提高了谷胱甘肽对于L-半胱氨酸的得率。与此同时，ATP供应不足是抑制谷胱甘肽高产的另一重要原因，ATP的昂贵价格，在工业生产中直接添加ATP成本太高。本项目通过对多聚磷酸激酶的筛选，得到了5种高效的多聚磷酸激酶，通过反应体系的系列优化，实现了多聚磷酸激酶与谷胱甘肽双功能合成酶耦合，一锅法体外催化高效合成谷胱甘肽。这些研究结果将为后续谷胱甘肽产业化生产奠定坚实的理论基础。