蒜氨酸酶的微生物资源分布趋向及关键酶分子催化机制研究

大蒜素独特的生理活性使其具有抗菌、保护心血管、抑制肿瘤等多种医疗作用。针对化学合成污染或植物蒜氨酸酶催化合成大蒜素的酶提取率低等问题，本项目拟围绕微生物来源的蒜氨酸酶这一科学问题进行深入系统研究。以分析不同作物根系土壤的微生物群落结构和产酶微生物为起点，系统研究：（1）产蒜氨酸酶的微生物资源分布趋向与特点，阐述其环境因子关联性；（2）分离获得系列产酶微生物，构建产蒜氨酸酶微生物资源库；（3）结合基因组分析、粗酶液纯化及简并引物扩增等手段，对关键酶进行序列分析鉴定并预测其在微生物中的生理功能和代谢途径；（4）优化异源表达体系获得高表达蛋白，通过生物信息学手段对蒜氨酸酶的序列和结构进行分析，结合定点突变等手段解析其分子催化机制。预期研究结果不仅为大蒜素的生物合成提供丰富的微生物酶资源库，还能为蒜氨酸酶的定向改造和应用提供坚实的理论支撑。

The unique physiological activities of allicin confer it various medical functions, such as antibiosis, cardiovascular protection, tumor inhibition, etc. Due to the contamination of allicin chemosynthesis or the low extraction efficiency of alliinase from plants in its enzymatic synthesis, our project intends to focus on the key science problem— allinase for systematic and in depth research. Starting with a the analysis of microbial community structure and enzyme-producing microorganisms in rhizosphere soils of different crops, to systematically study the following content: (1) The distribution trend and characteristics of alliinase-producing microbial resources will be explored, with its correlation of environmental factors to be investigated; (2) A series of enzyme-producing microorganisms will be isolated, for the bank of alliinase-producing microbial resources to be constructed; (3) The methods, such as genome analysis, crude enzyme purification, degenerate primer amplification, etc., will be combined to carry out the sequence analysis and identification of key enzyme, as well as to predict its physiological functions and metabolic pathways in microorganisms; (4) The sequence and structure of alliinase will be analyzed by bioinformatics tools and the optimization of the heterologous expression system will be implemented to get the highly expressed protein, the molecular catalytic mechanism of which will be analyzed by site-directed mutagenesis. It is expected that our research results will not only provide a rich pool of microbial enzymes for the biosynthesis of allicin, but also a solid theoretical support for the directional modification and application of alliinase.