L-天冬酰胺酶能从根源上控制热加工食品中潜在致癌物丙烯酰胺的形成。前期研究发现，地衣芽孢杆菌L-天冬酰胺酶（BlAase）I型和II型存在底物专一性差异，且丙烯酰胺降解率明显不同，推测其应用效果差异可能与酶的底物识别与结合能力有关，但其底物特异性的分子机制尚不清晰。本研究拟通过制备BlAase I型、BlAase II型晶体及其与配体复合物晶体，解析其结构与功能，探究酶底物结合口袋的空间构型及关键位点的变化，阐明酶底物特异性分子机制；并通过两者结构比对，揭示其底物特异性差异的分子机制。在此基础上，针对BlAase I型底物特异性差的问题，通过分子对接、分子动力学模拟等方法，分析影响底物特异性关键位点，确定靶位点，并采用定点突变和迭代饱和突变技术，获得底物特异性显著提高的突变体，结合突变体功能变化分析其构效关系，揭示影响其底物特异性的分子机制,为L-天冬酰胺酶分子改造和酶学理论提供科学依据。

The formation of acrylamide that is a potential carcinogen from starch-based food can be controlled by L-asparaginase. Our previous studies revealed that there was significant difference in substrate specificity between L-asparaginase (BlAase) I and II from Bacillus licheniformis, as well as their degradation effect on acrylamide. Accordingly, it was supposed that the degradation effect of BlAase depended on their substrate specificity. However, the molecular mechanism of the degradation of acrylamide relating to the substrate specificity of BlAase has not yet been clarified. In this project, the crystals of BlAase I, BlAase II and their complexes with different ligands will be produced first. The function and structure of the crystals will then be explored, so as to clarify the changes in spatial configuration and key sites of enzyme substrate binding pockets. Next, the detailed molecular mechanism about substrate specificity of BlAase will be explored through structure comparison. Moreover, different methodologies, such as molecular docking and molecular dynamics simulation, will be employed to analyze the key site affecting enzyme substrate specificity and subsequently identify the targte site. Site-directed mutagenesis and iterative saturation mutagenesis technologies will also be utilzied to obtain higher substrate-specific mutants. Lastly, the factors influencing enzyme substrate specificity will be revealed through the analysis of structure-function relationship of mutants. Ovearall, the outcomes of this project can provide guidance about the molecular modification of BlAase, as well as enrich the fundamental theory of BlAase.