Correlation of structure and properties of protein crystals in consideration of downstream processing and formulation

考虑下游加工和配方的蛋白质晶体结构和性质的相关性

• 批准号: 315462951
• 负责人: Dr. Rebekka Biedendieck
• 金额: --
• 依托单位: Institut für Partikeltechnik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/315462951?language=en
• 关键词: Correlation; structure; properties; protein; crystals

Because of their high volumetric catalytic activity and their high chemical and thermal resistance, enzymes in protein crystals are an excellent choice for application as immobilized biocatalysts. Since enzyme activity, product release rate as well as mechanical rigidity are dependent on crystal shape and size, their molecular basis shall be investigated. For this, the complete process chain consisting of the genetic modification, production, purification, crystallization, cross-linking and mechanical characterization of the model proteins halohydrin dehalogenase HheG and Penicillin G acylase PGA has been established in the first funding period. Three new structures of PGAs exhibiting different thermostabilities have been solved. For both model proteins, many genetic variants have been generated, successfully produced and tested towards their activity and thermal stability. The most promising candidates have been selected for the generation of native and cross-linked protein crystals in order to investigate their mechanical and catalytic properties. Currently, the particle structure of the protein crystals is modelled by Discrete Element Method to emulate their properties. Main focus of the second funding period will be the systematic modification of the proteins by genetic and chemical methods for an efficient cross-linking of the resulting protein crystals. In this respect, HheG will be used for a detailed investigation of the impact of cross-linking on the mechanical rigidity of the crystals, whereas the different PGAs will be used to test the transfer of gained knowledge from one to another protein. Next to this systematic engineering of enzymes, the scale up of the whole process chain will be established in order to determine the mechanical behavior of cross-linked protein crystals in model processes. Additionally, statistical and numerical methods will be used to explain observed changes of mechanical behavior based on protein interactions within the crystal as well as cross-linking sites. In summary, structural principles of crystal formation and cross-linking will be elucidated with the aim to obtain thermally and mechanically stable protein crystals for biotechnological application based on systematic genetic modification of the proteins.

由于其高体积催化活性和高耐化学性和耐热性，蛋白质晶体中的酶是作为固定化生物催化剂应用的极好选择。由于酶活性、产物释放速率以及机械刚度取决于晶体形状和大小，因此应研究其分子基础。为此，在第一个供资期内建立了完整的工艺链，包括模型蛋白卤代醇脱卤酶HheG和青霉素G酰化酶PGA的遗传修饰、生产、纯化、结晶、交联和机械表征。解决了三种具有不同热稳定性的PGA的新结构。对于这两种模型蛋白，已经产生了许多遗传变体，成功地生产并测试了它们的活性和热稳定性。最有前途的候选人已被选定为生成天然和交联的蛋白质晶体，以研究其机械和催化性能。目前，蛋白质晶体的颗粒结构是由离散元方法模拟其性能。第二个资助期的主要重点将是通过遗传和化学方法对蛋白质进行系统性修饰，以使所得蛋白质晶体有效交联。在这方面，HheG将用于详细研究交联对晶体机械刚度的影响，而不同的PGA将用于测试从一种蛋白质到另一种蛋白质的知识转移。除了酶的系统工程之外，还将建立整个工艺链的放大，以确定模型工艺中交联蛋白质晶体的机械行为。此外，统计和数值方法将被用来解释观察到的机械行为的变化的基础上的蛋白质相互作用的晶体以及交联位点。总之，将阐明晶体形成和交联的结构原理，目的是获得热稳定和机械稳定的蛋白质晶体，用于基于蛋白质的系统遗传修饰的生物技术应用。