地衣芽胞杆菌Tat信号肽介导PGase分泌表达机制探究

Protein-L-glutamine amidohydrolase (EC 3.5.1.44, PGase) has been proved to be useful as a new food-processing enzyme. However the preparation of the PGase is not high for industrial application. The project intends to develop a high-efficient process for preparation of the PGase based on a novel way. The following aspects will been comprehensively investigated. First, the amino acid composition and characteristic of the Tat-type signal peptides from all the secretion proteins will be analysised based on the genome of Bacillius licheniformis; Second, the optimal Tat-type signal peptides will be obtained based on which channeling the PGase secretion efficiency using the PGase as the reporter gene; Then, the mutation of the specific site amino acids of the selected optimal Tat-type signal peptides will be performed by site-directed mutation. The key amino acids and the structural features that related to the secretion efficiency will be studied; Finally, a novel recombinant strain and recombinant enzyme will be constructed using the candidate Tat-type signal peptides or its mutations based on the better Bacillus licheniformis cell host obtained before. The fermentation technology of a novel recombinant strain will be also studied. The research results may provide a new strategy to obtain the high secretion effiency of PGase and to develop a high-efficient and low-running cost process for preparation of the PGase based on a novel Bacillus licheniformis Tat-type secretion pathway.

现有研究结果揭示蛋白质谷氨酰胺氨基水解酶（EC 3.5.1.44，PGase)在食品加工中具有重要应用价值与意义，但现有制备此酶的效率不高。本项目拟基于地衣芽胞杆菌Tat分泌途径探索PGase的高效制备新途径。首先分析与遴选其包含的全部Tat分泌型蛋白的信号肽组成与特征；其次以PGase为靶向合成物，研究与分析全部Tat信号肽在介导PGase分泌中的作用与效率，获得最优引导PGase分泌的候选Tat信号肽；再通过定点突变研究并分析候选信号肽的组成特征及其功能相关关系；最后借助前期选育的地衣芽胞杆菌及其优选出的Tat信号肽或其突变体，构建重组菌，研究相应的发酵生产技术。期望建立起基于Tat分泌途径的地衣芽胞杆菌PGase高效表达并为实现PGase高效低成本规模化制备奠定基础。

蛋白质谷氨酰胺氨基水解酶（EC 3.5.1.44，PGase)，主要针对蛋白质分子中谷氨酰胺残基上的氨酰基，进行特异性脱氨基作用，将其转换为谷氨酸残基。.PGase在食品蛋白改性领域具有广泛的应用前景，可以作为脱酰胺剂作用于多种植物蛋白和酪蛋白及其相关食品。与其它脱酰胺作用的酶类相比较，PGase对小肽分子和蛋白质中的Gln残基都能够进行脱酰胺反应，特异性高，能明显提高蛋白质的功能性质，因而被认为是最有效的脱酰胺改性方法。正因这些优势使得PGase在食品加工方面有广泛的应用前景，但因其酶产量低、且应用方面成本高，目前其应用还仅停留在实验室阶段。故现有的PGase的制备方法还有待进一步提高。.本课题在全面分析地衣芽胞杆菌基因组信息的基础上，对其所包含的Tat分泌型蛋白质的信号肽组成与特征进行分析；进一步通过基因克隆与重组技术，以PGase为靶向合成物，研究与分析了Tat信号肽在介导PGase分泌中的作用与效率，获得了最优引导PGase分泌的候选Tat信号肽GlmU，其特征序列为MDNRDNGGQYMDKRFAVVLA；通过定点突变研究并分析了候选信号肽的组成特征及其功能相关关系，获得了最优介导PGase分泌的信号肽突变体GlmUII， 其特征序列为MDRRDNGGQYMDKRFAVVLA；借助前期选育的地衣芽胞杆菌优秀宿主CBBD302并基于Tat分泌途径及其优选出的Tat信号肽突变体GlmUII，构建了地衣芽胞杆菌重组菌，研究其相应的发酵生产技术，并初步建立了25-L发酵罐的发酵工艺，发酵酶活达到6.8 U/mL；进一步对其在大豆蛋白方面的应用进行了初步的应用评价，结果表明PGase在一定程度上提高了大豆蛋白的溶解性、乳化性及起泡性等功能性质。